Posts Tagged ‘arrhythmia’

Genetic Analysis of Atrial Fibrillation

Author and Curator: Larry H Bernstein, MD, FCAP  


Curator: Aviva-Lev Ari, PhD, RN

This article is a followup of the wonderful study of the effect of oxidation of a methionine residue in calcium dependent-calmodulin kinase Ox-CaMKII on stabilizing the atrial cardiomyocyte, giving protection from atrial fibrillation.  It is also not so distant from the work reviewed, mostly on the ventricular myocyte and the calcium signaling by initiation of the ryanodyne receptor (RyR2) in calcium sparks and the CaMKII d isoenzyme.

We refer to the following related articles published in pharmaceutical Intelligence:

Oxidized Calcium Calmodulin Kinase and Atrial Fibrillation
Author: Larry H. Bernstein, MD, FCAP and Curator: Aviva Lev-Ari, PhD, RN

Jmjd3 and Cardiovascular Differentiation of Embryonic Stem Cells

Author: Larry H. Bernstein, MD, FCAP and Curator: Aviva Lev-Ari, PhD, RN


Contributions to cardiomyocyte interactions and signaling
Author and Curator: Larry H Bernstein, MD, FCAP  and Curator: Aviva Lev-Ari, PhD, RN

Cardiac Contractility & Myocardium Performance: Therapeutic Implications for Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses
Editor: Justin Pearlman, MD, PhD, FACC, Author and Curator: Larry H Bernstein, MD, FCAP, and Article Curator: Aviva Lev-Ari, PhD, RN

Part I. Identification of Biomarkers that are Related to the Actin Cytoskeleton
Curator and Writer: Larry H Bernstein, MD, FCAP

Part II: Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility
Larry H. Bernstein, MD, FCAP, Stephen Williams, PhD and Aviva Lev-Ari, PhD, RN

Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets
Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN

Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
Aviva Lev-Ari, PhD, RN

Part VII: Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmias and Non-ischemic Heart Failure – Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

Part VIII: Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

Part IX: Calcium-Channel Blockers, Calcium Release-related Contractile Dysfunction (Ryanopathy) and Calcium as Neurotransmitter Sensor
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

Part X: Synaptotagmin functions as a Calcium Sensor: How Calcium Ions Regulate the fusion of vesicles with cell membranes during Neurotransmission
Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

The material presented is very focused, and cannot be found elsewhere in Pharmaceutical Intelligence with respedt to genetics and heart disease.  However, there are other articles that may be of interest to the reader.

Volume Three: Etiologies of Cardiovascular Diseases – Epigenetics, Genetics & Genomics

Curators: Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

PART 3.  Determinants of Cardiovascular Diseases: Genetics, Heredity and Genomics Discoveries

3.2 Leading DIAGNOSES of Cardiovascular Diseases covered in Circulation: Cardiovascular Genetics, 3/2010 – 3/2013

The Diagnoses covered include the following – relevant to this discussion

  • MicroRNA in Serum as Bimarker for Cardiovascular Pathologies: acute myocardial infarction, viral myocarditis, diastolic dysfunction, and acute heart failure
  • Genomics of Ventricular arrhythmias, A-Fib, Right Ventricular Dysplasia, Cardiomyopathy
  • Heredity of Cardiovascular Disorders Inheritance

3.2.1: Heredity of Cardiovascular Disorders Inheritance

The implications of heredity extend beyond serving as a platform for genetic analysis, influencing diagnosis,

  1. prognostication, and
  2. treatment of both index cases and relatives, and
  3. enabling rational targeting of genotyping resources.

This review covers acquisition of a family history, evaluation of heritability and inheritance patterns, and the impact of inheritance on subsequent components of the clinical pathway.

SOURCE:   Circulation: Cardiovascular Genetics.2011; 4: 701-709.  http://dx.doi.org/10.1161/CIRCGENETICS.110.959379

3.2.2: Myocardial Damage MicroRNA in Serum as Biomarker for Cardiovascular Pathologies: acute myocardial infarction, viral myocarditis,  diastolic dysfunction, and acute heart failure

Increased MicroRNA-1 and MicroRNA-133a Levels in Serum of Patients With Cardiovascular Disease Indicate Myocardial Damage
Y Kuwabara, Koh Ono, T Horie, H Nishi, K Nagao, et al.
SOURCE:  Circulation: Cardiovascular Genetics. 2011; 4: 446-454   http://dx.doi.org/10.1161/CIRCGENETICS.110.958975 Circulating MicroRNA-208b and MicroRNA-499 Reflect Myocardial Damage in Cardiovascular Disease

MF Corsten, R Dennert, S Jochems, T Kuznetsova, Y Devaux, et al.
SOURCE: Circulation: Cardiovascular Genetics. 2010; 3: 499-506.  http://dx.doi.org/10.1161/CIRCGENETICS.110.957415 Large-Scale Candidate Gene Analysis in Whites and African Americans Identifies IL6R Polymorphism in Relation to Atrial Fibrillation

The National Heart, Lung, and Blood Institute’s Candidate Gene Association Resource (CARe) Project
RB Schnabel, KF Kerr, SA Lubitz, EL Alkylbekova, et al.
SOURCE:  Circulation: Cardiovascular Genetics.2011; 4: 557-564   http://dx.doi.org/10.1161/CIRCGENETICS.110.959197

 Weighted Gene Coexpression Network Analysis of Human Left Atrial Tissue Identifies Gene Modules Associated With Atrial Fibrillation

N Tan, MK Chung, JD Smith, J Hsu, D Serre, DW Newton, L Castel, E Soltesz, G Pettersson, AM Gillinov, DR Van Wagoner and J Barnard
From the Cleveland Clinic Lerner College of Medicine (N.T.), Department of Cardiovascular Medicine (M.K.C., D.W.N.), and Department of Thoracic & Cardiovascular Surgery (E.S., G.P., A.M.G.); and Department of Cellular & Molecular Medicine (J.D.S., J.H.), Genomic Medicine Institute (D.S.), Department of Molecular Cardiology (L.C.), and Department of Quantitative Health Sciences (J.B.), Cleveland Clinic Lerner Research Institute, Cleveland, OH
Circ Cardiovasc Genet. 2013;6:362-371; http://dx.doi.org/10.1161/CIRCGENETICS.113.000133
http://circgenetics.ahajournals.org/content/6/4/362   The online-only Data Supplement is available at http://circgenetics.ahajournals.org/lookup/suppl/doi:10.1161/CIRCGENETICS.113.000133/-/DC1

Background—Genetic mechanisms of atrial fibrillation (AF) remain incompletely understood. Previous differential expression studies in AF were limited by small sample size and provided limited understanding of global gene networks, prompting the need for larger-scale, network-based analyses.

Methods and Results—Left atrial tissues from Cleveland Clinic patients who underwent cardiac surgery were assayed using Illumina Human HT-12 mRNA microarrays. The data set included 3 groups based on cardiovascular comorbidities: mitral valve (MV) disease without coronary artery disease (n=64), coronary artery disease without MV disease (n=57), and lone AF (n=35). Weighted gene coexpression network analysis was performed in the MV group to detect modules of correlated genes. Module preservation was assessed in the other 2 groups. Module eigengenes were regressed on AF severity or atrial rhythm at surgery. Modules whose eigengenes correlated with either AF phenotype were analyzed for gene content. A total of 14 modules were detected in the MV group; all were preserved in the other 2 groups. One module (124 genes) was associated with AF severity and atrial rhythm across all groups. Its top hub gene, RCAN1, is implicated in calcineurin-dependent signaling and cardiac hypertrophy. Another module (679 genes) was associated with atrial rhythm in the MV and coronary artery disease groups. It was enriched with cell signaling genes and contained cardiovascular developmental genes including TBX5.

Conclusions—Our network-based approach found 2 modules strongly associated with AF. Further analysis of these modules may yield insight into AF pathogenesis by providing novel targets for functional studies. (Circ Cardiovasc Genet. 2013;6:362-371.)

Key Words: arrhythmias, cardiac • atrial fibrillation • bioinformatics • gene coexpression • gene regulatory networks • genetics • microarrays


trial fibrillation (AF) is the most common sustained car­diac arrhythmia, with a prevalence of ≈1% to 2% in the general population.1,2 Although AF may be an isolated con­dition (lone AF [LAF]), it often occurs concomitantly with other cardiovascular diseases, such as coronary artery disease (CAD) and valvular heart disease.1 In addition, stroke risk is increased 5-fold among patients with AF, and ischemic strokes attributed to AF are more likely to be fatal.1 Current antiarrhythmic drug therapies are limited in terms of efficacy and safety.1,3,4 Thus, there is a need to develop better risk pre­diction tools as well as mechanistically targeted therapies for AF. Such developments can only come about through a clearer understanding of its pathogenesis.

Family history is an established risk factor for AF. A Danish Twin Registry study estimated AF heritability at 62%, indicating a significant genetic component.5 Substantial progress has been made to elucidate this genetic basis. For example, genome-wide association studies (GWASs) have identified several susceptibil­ity loci and candidate genes linked with AF. Initial studies per­formed in European populations found 3 AF-associated genomic loci.6–9 Of these, the most significant single-nucleotide polymor-phisms (SNPs) mapped to an intergenic region of chromosome 4q25. The closest gene in this region, PITX2, is crucial in left-right asymmetrical development of the heart and thus seems promising as a major player in initiating AF.10,11 A large-scale GWAS meta-analysis discovered 6 additional susceptibility loci, implicating genes involved in cardiopulmonary development, ion transport, and cellular structural integrity.12

Differential expression studies have also provided insight into the pathogenesis of AF. A study by Barth et al13 found that about two-thirds of the genes expressed in the right atrial appendage were downregulated during permanent AF, and that many of these genes were involved in calcium-dependent signaling pathways. In addition, ventricular-predominant genes were upregulated in right atrial appendages of sub­jects with AF.13 Another study showed that inflammatory and transcription-related gene expression was increased in right atrial appendages of subjects with AF versus controls.14 These results highlight the adaptive responses to AF-induced stress and ischemia taking place within the atria.

Despite these advances, much remains to be discovered about the genetic mechanisms of AF. The AF-associated SNPs found thus far only explain a fraction of its heritability15; furthermore, the means by which the putative candidate genes cause AF have not been fully established.9,15,16 Additionally, previous dif­ferential expression studies in human tissue were limited to the right atrial appendage, had small sample sizes, and provided little understanding of global gene interactions.13,14 Weighted gene coexpression network analysis (WGCNA) is a technique to construct gene modules within a network based on correla­tions in gene expression (ie, coexpression).17,18 WGCNA has been used to study genetically complex diseases, such as meta­bolic syndrome,19 schizophrenia,20 and heart failure.21 Here, we obtained mRNA expression profiles from human left atrial appendage tissue and implemented WGCNA to identify gene modules associated with AF phenotypes.


Subject Recruitment

From 2001 to 2008, patients undergoing cardiac surgery at the Cleveland Clinic were prospectively screened and recruited. Informed consent for research use of discarded atrial tissues was ob­tained from each patient by a study coordinator during the presur­gical visit. Demographic and clinical data were obtained from the Cardiovascular Surgery Information Registry and by chart review. Use of human atrial tissues was approved by the Institutional Review Board of the Cleveland Clinic.

Table S1: Clinical definitions of cardiovascular phenotype groups

Criterion Type Mitral Valve (MV) Disease Coronary Artery Disease (CAD) Lone Atrial Fibrillation (LAF)
Inclusion Criteria Surgical indication – Surgical indication – History of atrial fibrillation
mitral valve repair or replacement coronary artery bypass graft
Surgical indication
– MAZE procedure
Preserved ejection fraction (≥50%)
Exclusion Criteria Significant coronary artery disease: Significant mitral valve disease: Significant
coronary artery
– Significant (≥50%) stenosis – Documented echocardiography disease:
 in at least finding of – Significant
one coronary artery  mitral regurgitation (≥3) or (≥50%) stenosis in
via cardiac catheterization mitral stenosis at least one
– History of revascularization – History of mitral valve coronary artery via
(percutaneous coronary intervention or coronary artery bypass graft surgery)  repair or replacement cardiac catheterization
– History of revascularization
(percutaneous coronary intervention or coronary artery bypass graft surgery)
Significant valvular heart disease:
-Documented echocardiography finding of valvular regurgitation (≥3) or stenosis
-History of valve repair or replacement

RNA Microarray Isolation and Profiling

Left atria appendage specimens were dissected during cardiac surgery and stored frozen at −80°C. Total RNA was extracted using the Trizol technique. RNA samples were processed by the Cleveland Clinic Genomics Core. For each sample, 250-ng RNA was reverse tran­scribed into cRNA and biotin-UTP labeled using the TotalPrep RNA Amplification Kit (Ambion, Austin, TX). cRNA was quantified using a Nanodrop spectrophotometer, and cRNA size distribution was as­sessed on a 1% agarose gel. cRNA was hybridized to Illumina Human HT-12 Expression BeadChip arrays (v.3). Arrays were scanned using a BeadArray reader.

Expression Data Preprocessing

Raw expression data were extracted using the beadarray package in R, and bead-level data were averaged after log base-2 transformation. Background correction was performed by fitting a normal-gamma deconvolution model using the NormalGamma R package.22 Quantile normalization and batch effect adjustment with the ComBat method were performed using R.23 Probes that were not detected (at a P<0.05 threshold) in all samples as well as probes with relatively lower vari­ances (interquartile range ≤log2[1.2]) were excluded.

The WGCNA approach requires that genes be represented as sin­gular nodes in such a network. However, a small proportion of the genes in our data have multiple probe mappings. To facilitate the representation of singular genes within the network, a probe must be selected to represent its associated gene. Hence, for genes that mapped to multiple probes, the probe with the highest mean expres­sion level was selected for analysis (which often selects the splice isoform with the highest expression and signal-to-noise ratio), result­ing in a total of 6168 genes.

Defining Training and Test Sets

Currently, no large external mRNA microarray data from human left atrial tissues are publicly available. To facilitate internal validation of results, we divided our data set into 3 groups based on cardiovascular comorbidities: mitral valve (MV) disease without CAD (MV group; n=64), CAD without MV disease (CAD group; n=57), and LAF (LAF group; n=35). LAF was defined as the presence of AF without concomitant structural heart disease, according to the guidelines set by the European Society of Cardiology.1 The MV group, which was the largest and had the most power for detecting significant modules, served as the training set for module derivation, whereas the other 2 groups were designated test sets for module reproducibility. To mini­mize the effect of population stratification, the data set was limited to white subjects. Differences in clinical characteristics among the groups were assessed using Kruskal–Wallis rank-sum tests for con­tinuous variables and Pearson x2 test for categorical variables.

Weight Gene Coexpression Network Analysis

WGCNA is a systems-biology method to identify and characterize gene modules whose members share strong coexpression. We applied previously validated methodology in this analysis.17 Briefly, pair-wise gene (Pearson) correlations were calculated using the MV group data set. A weighted adjacency matrix was then constructed. I is a soft-thresholding pa­rameter that provides emphasis on stronger correlations over weaker and less meaningful ones while preserving the continuous nature of gene–gene relationships. I=3 was selected in this analysis based on the criterion outlined by Zhang and Horvath17 (see the online-only Data Supplement).

Next, the topological overlap–based dissimilarity matrix was com­puted from the weighted adjacency matrix. The topological overlap, developed by Ravasz et al,24 reflects the relative interconnectedness (ie, shared neighbors) between 2 genes.17 Hence, construction of the net­work dendrogram based on this dissimilarity measure allows for the identification of gene modules whose members share strong intercon-nectivity patterns. The WGCNA cutreeDynamic R function was used to identify a suitable cut height for module identification via an adap­tive cut height selection approach.18 Gene modules, defined as branches of the network dendrogram, were assigned colors for visualization.

Network Preservation Analysis

Module preservation between the MV and CAD groups as well as the MV and LAF groups was assessed using network preservation statis­tics as described in Langfelder et al.25 Module density–based statistics (to assess whether genes in each module remain highly connected in the test set) and connectivity-based statistics (to assess whether con­nectivity patterns between genes in the test set remain similar com­pared with the training set) were considered in this analysis.25 In each comparison, a Z statistic representing a weighted summary of module density and connectivity measures was computed for every module (Zsummary). The Zsummary score was used to evaluate module preserva­tion, with values ≥8 indicating strong preservation, as proposed by Langfelder et al.25 The WGCNA R function network preservation was used to implement this analysis.25

Table S2: Network preservation analysis between the MV and CAD groups – size and Zsummary scores of gene modules detected.

Module Module Size


Black 275 15.52
Blue 964 44.79
Brown 817 12.80
Cyan 119 13.42
Green 349 14.27
Green-Yellow 215 19.31
Magenta 239 15.38
Midnight-Blue 83 15.92
Pink 252 23.31
Purple 224 16.96
Red 278 17.30
Salmon 124 13.84
Tan 679 28.48
Turquoise 1512 44.03

Table S3: Network preservation analysis between the MV and LAF groups – size and Zsummary scores of gene modules detected

Module Module Size ZSummary
Black 275 13.14
Blue 964 39.26
Brown 817 14.98
Cyan 119 11.46
Green 349 14.91
Green-Yellow 215 20.99
Magenta 239 18.58
Midnight-Blue 83 13.87
Pink 252 19.10
Purple 224 8.80
Red 278 16.62
Salmon 124 11.57
Tan 679 28.61
Turquoise 1512 42.07

Clinical Significance of Preserved Modules

Principal component analysis of the expression data for each gene module was performed. The first principal component of each mod­ule, designated the eigengene, was identified for the 3 cardiovascular disease groups; this served as a summary expression measure that explained the largest proportion of the variance of the module.26 Multivariate linear regression was performed with the module ei-gengenes as the outcome variables and AF severity (no AF, parox­ysmal AF, persistent AF, permanent AF) as the predictor of interest (adjusting for age and sex). A similar regression analysis was per­formed with atrial rhythm at surgery (no AF history, AF history in sinus rhythm, AF history in AF rhythm) as the predictor of interest. The false discovery rate method was used to adjust for multiple com­parisons. Modules whose eigengenes associated with AF severity and atrial rhythm were identified for further analysis.

In addition, hierarchical clustering of module eigengenes and se­lected clinical traits (age, sex, hypertension, cholesterol, left atrial size, AF state, and atrial rhythm) was used to identify additional module–trait associations. Clusters of eigengenes/traits were detected based on a dissimilarity measure D, as given by

D=1−cor(Vi,Vj),i≠j                                                                              (3)

where V=the eigengene or clinical trait.

Enrichment Analysis

Gene modules significantly associated with AF severity and atrial rhythm were submitted to Ingenuity Pathway Analysis (IPA) to determine enrichment for functional/disease categories. IPA is an application of gene set over-representation analysis; for each dis-ease/functional category annotation, a P value is calculated (using Fisher exact test) by comparing the number of genes from the mod­ule of interest that participate in the said category against the total number of participating genes in the background set.27 All 6168 genes in the current data set served as the background set for the enrichment analysis.

Hub Gene Analysis

Hub genes are defined as genes that have high intramodular connectivity17,20

Alternatively, they may also be defined as genes with high module membership21,25

Both definitions were used to identify the hub genes of modules associated with AF phenotype.

To confirm that the hub genes identified were themselves associ­ated with AF phenotype, the expression data of the top 10 hub genes (by intramodular connectivity) were regressed on atrial rhythm (ad­justing for age and sex). In addition, eigengenes of AF-associated modules were regressed on their respective (top 10) hub gene expres­sion profiles, and the model R2 indices were computed.

Membership of AF-Associated Candidate Genes From Previous Studies

Previous GWAS studies identified multiple AF-associated SNPs.8,9,12,15,28 We selected candidate genes closest to or containing these SNPs and identified their module locations as well as their clos­est within-module partners (absolute Pearson correlations).

Sensitivity Analysis of Soft-Thresholding Parameter

To verify that the key results obtained from the above analysis were robust with respect to the chosen soft-thresholding parameter (I=3), we repeated the module identification process using I=5. The eigen-genes of the detected modules were computed and regressed on atrial rhythm (adjusting for age and sex). Modules significantly associated with atrial rhythm in ≥2 groups of data set were compared with the AF phenotype–associated modules from the original analysis.


Subject Characteristics

Table 1 describes the clinical characteristics of the cardiac surgery patients who were recruited for the study. Subjects in the LAF group were generally younger and less likely to be a current smoker (P=2.0×10−4 and 0.032, respectively). Subjects in the MV group had lower body mass indices (P=2.7×10−6), and a larger proportion had paroxysmal AF compared with the other 2 groups (P=0.033).

Table 1. Clinical Characteristics of Study Subjects


MV Group (n=64)

CAD Group (n=57)

LAF Group (n=35)

P Value*

Age, median y (1st–3rd quartiles)

60 (51.75–67.25)

64 (58.00–70.00)

56 (45.50–60.50)


Sex, female (%) 19 (29.7) 6 (10.5)

7 (20.0)


BMI, median (1st–3rd quartiles)

25.97 (24.27–28.66)

29.01 (27.06–32.11)

29.71 (26.72–35.10)


Current smoker (%) 29 (45.3) 35 (61.4)

12 (21.1)


Hypertension (%) 21 (32.8) 39 (68.4)

16 (45.7)


AF severity (%)
No AF 7 (10.9) 7 (12.3)

0 (0.0)


Paroxysmal 19 (29.7) 10 (17.5)

7 (20.0)

Persistent 30 (46.9) 26 (45.6)

15 (42.9)

Permanent 8 (12.5) 14 (24.6)

13 (37.1)

Atrial rhythm at surgery (%)
No AF history in sinus rhythm 7 (10.9) 7 (12.3)

0 (0)


AF history in sinus rhythm 28 (43.8) 16 (28.1)

11 (31.4)

AF History in AF rhythm 29 (45.3) 34 (59.6)

24 (68.6)

Gene Coexpression Network Construction and Module Identificationsee document at  http://circgenetics.ahajournals.org/content/6/4/362

A total of 14 modules were detected using the MV group data set (Figure 1), with module sizes ranging from 83 genes to 1512 genes; 38 genes did not share similar coexpression with the other genes in the network and were therefore not included in any of the identified modules

Figure 1. Network dendrogram (top) and colors of identified modules (bottom).

Figure 1. Network dendrogram (top) and colors of identified modules (bottom). The dendrogram was constructed using the topological overlap matrix as the similarity measure. Modules corresponded to branches of the dendrogram and were assigned colors for visualization.

Network Preservation Analysis Revealed Strong Preservation of All Modules Between the Training and Test Sets

All 14 modules showed strong preservation across the CAD and LAF groups in both comparisons, with Z [summary]  scores of >10 in most modules (Figure 2). No major deviations in the Z [summary] score distributions for the 2 comparisons were noted, indicating that modules were preserved to a similar extent across the 2 groups

Figure 2. Preservation of mod-ules between mitral valve (MV) and coronary artery disease

Figure 2. Preservation of mod­ules between mitral valve (MV) and coronary artery disease (CAD) groups (left), and MV and lone atrial fibrillation (LAF) groups (right). A Zsummary sta­tistic was computed for each module as an overall measure of its preservation relating to density and connectivity. All modules showed strong pres­ervation in both comparisons with Zsummary scores >8 (red dot­ted line).

Regression Analysis of Module Eigengene Profiles Identified 2 Modules Associated With AF Severity and Atrial Rhythm

Table IV in the online-only Data Supplement summarizes the proportion of variance explained by the first 3 principal components for each module. On average, the first principal component (ie, the eigengene) explained ≈18% of the total variance of its associated module. For each group, the mod­ule eigengenes were extracted and regressed on AF severity (with age and sex as covariates). The salmon module (124 genes) eigengene was strongly associated with AF severity in the MV and CAD groups (P=1.7×10−6 and 5.2×10−4, respec­tively); this association was less significant in the LAF group (P=9.0×10−2). Eigengene levels increased with worsening AF severity across all 3 groups, with the greatest stepwise change taking place between the paroxysmal AF and per­sistent AF categories (Figure 3A). When the module eigen-genes were regressed on atrial rhythm, the salmon module eigengene showed significant association in all groups (MV: P=1.1×10−14; CAD: P=1.36×10−6; LAF: P=2.1×10−4). Eigen-gene levels were higher in the AF history in AF rhythm cat­egory (Figure 3B).

Table S4: Proportion of variance explained by the principal components for each module.












20.5% 22.2% 20.1% 21.8% 21.4% 22.8% 19.6%


4.1% 3.6% 4.8% 5.7% 4.5% 5.9% 3.9%


3.4% 3.1% 3.8% 4.4% 3.9% 3.7% 3.7%



12.5% 18.6% 7.1% 16.8% 12.2% 20.3% 12.8%


6.0% 5.5% 5.0% 7.0% 5.5% 6.1% 6.4%


4.9% 4.1% 4.4% 6.5% 4.8% 4.4% 4.8%



14.0% 16.6% 11.7% 14.3% 14.7% 20.8% 20.2%


8.9% 8.5% 7.6% 9.3% 7.3% 11.1% 6.9%


6.5% 6.3% 5.5% 8.2% 6.1% 5.3% 6.2%



Midnight- Blue









28.5% 22.6% 18.7% 20.5% 22.3% 19.0% 25.8%


4.6% 6.0% 4.7% 4.1% 6.9% 4.0% 3.5%


4.2% 4.2% 4.2% 3.5% 4.0% 3.6% 3.3%



23.4% 17.1% 15.5% 15.0% 18.0% 14.6% 18.2%


7.4% 8.6% 6.0% 6.4% 7.2% 5.8% 6.6%


5.1% 5.4% 5.3% 5.4% 6.2% 5.1% 4.5%



23.5% 18.4% 12.0% 15.9% 16.9% 13.7% 16.5%


7.9% 8.5% 9.8% 9.4% 9.5% 9.1% 9.6%


6.7% 7.0% 6.6% 6.0% 6.9% 6.8% 6.3%

Figure 3. Boxplots of salmon module eigengene expression levels with respect to atrial fibrillation (AF) severity (A) and atrial rhythm (B).

Figure 3. Boxplots of salmon module eigengene expression levels with respect to atrial fibrillation (AF) severity (A) and atrial rhythm (B).
A, Eigengene expression correlated positively with AF severity, with the largest stepwise increase between the paroxysmal AF and per­manent AF categories. B, Eigengene expression was highest in the AF history in AF rhythm category in all 3 groups. CAD indicates coro­nary artery disease; LAF, lone AF; and MV, mitral valve.

The regression analysis also revealed statistically significant associations between the tan module (679 genes) eigengene and atrial rhythm in the MV and CAD groups (P=5.8×10−4 and 3.4×10−2, respectively). Eigengene levels were lower in the AF history in AF rhythm category compared with the AF history in sinus rhythm category (Figure 4); this trend was also observed in the LAF group, albeit with weaker statistical evidence (P=0.15).

Figure 4. Boxplots of tan module eigengene expression levels with respect to atrial rhythm.

Figure 4. Boxplots of tan module eigengene expression levels with respect to atrial rhythm.
Eigengene expression levels were lower in the atrial fibrillation (AF) history in AF rhythm category compared with the AF history in sinus rhythm category. CAD indicates coronary artery disease; LAF, lone AF; and MV, mitral valve

Hierarchical Clustering of Eigengene Profiles With Clinical Traits

Hierarchical clustering was performed to identify relation­ships between gene modules and selected clinical traits. The salmon module clustered with AF severity and atrial rhythm; in addition, left atrial size was found in the same cluster, sug­gesting a possible relationship between salmon module gene expression and atrial remodeling (Figure 5A). Although the tan module was in a separate cluster from the salmon module, it was negatively correlated with both atrial rhythm and AF severity (Figure 5B).

Figure 5. Dendrogram (A) and correlation heatmap (B) of module eigengenes and clinical traits.

Figure 5. Dendrogram (A) and correlation heatmap (B) of module eigengenes and clinical traits

A, The salmon module eigengene but not the tan module eigengene clustered with atrial fibrillation (AF) severity, atrial rhythm, and left atrial size. B, AF severity and atrial rhythm at surgery correlated positively with the salmon module eigengene and negatively with the tan module eigengene. Arhythm indicates atrial rhythm at surgery; Chol, cholesterol; HTN, hypertension; and LASize, left atrial size.

IPA Enrichment Analysis of Salmon and Tan Modules

The salmon module was enriched in genes involved in cardio­vascular function and development (smallest P=4.4×10−4) and organ morphology (smallest P=4.4×10−4). In addition, the top disease categories identified included endocrine system disor­ders (smallest P=4.4×10−4) and cardiovascular disease (small­est P=2.59×10−3).

The tan module was enriched in genes involved in cell-to-cell signaling and interaction (smallest P=8.9×10−4) and cell death and survival (smallest P=1.5×10−3). Enriched disease categories included cancer (smallest P=2.2×10−4) and cardio­vascular disease (smallest P=4.5×10−4).

see document at  http://circgenetics.ahajournals.org/content/6/4/362

Hub Gene Analysis of Salmon and Tan Modules

We identified hub genes in the 2 modules based on intramod-ular connectivity and module membership. For the salmon module, the gene RCAN1 exhibited the highest intramodular connectivity and module membership. The top 10 hub genes (by intramodular connectivity) were significantly associated with atrial rhythm, with false discovery rate–adjusted P values ranging from 1.5×10−5 to 4.2×10−12. These hub genes accounted for 95% of the variation in the salmon module eigengene.

In the tan module, the top hub gene was CPEB3. The top 10 hub genes (by intramodular connectivity) correlated with atrial rhythm as well, although the statistical associations in the lower-ranked hub genes were relatively weaker (false discovery rate–adjusted P values ranging from 1.1×10−1 to 3.4×10−4). These hub genes explained 94% of the total varia­tion in the tan module eigengene.

The names and connectivity measures of the hub genes found in both modules are presented in Table 2.

Table 2. Top 10 Hub Genes in the Salmon (Left) and Tan (Right) Modules as Defined by Intramodular Connectivity and Module Membership

Salmon Module

Tan Module









RCAN1 8.2






DNAJA4 7.7






PDE8B 7.7












PTPN4 6.7






SORBS2 6.0






ADCY6 5.7






FHL2 5.7






BVES 5.4






TMEM173 5.3







A visualiza­tion of the salmon module is shown using the Cytoscape tool (Figure 6). A full list of the genes in the salmon and tan mod­ules is provided in the online-only Data Supplement.

Figure 6. Cytoscape visualization of genes in the salmon module.
Nodes representing genes with high intramodu-lar connectivities, such as RCAN1 and DNAJA4, appear larger in the network. Strong connections are visualized with darker lines, whereas weak connections appear more translucent

Figure 6. Cytoscape visualization of genes in the salmon module.

Membership of AF-Associated Candidate Genes From Previous Studies

The tan module contained MYOZ1, which was identified as a candidate gene from the recent AF meta-analysis. PITX2 was located in the green module (n=349), and ZFHX3 was located in the turquoise module (n=1512). The locations of other can­didate genes (and their closest partners) are reported in the online-only Data Supplement.

Sensitivity Analysis of Key Results

We repeated the WGCNA module identification approach using a different soft-thresholding parameter (β=5). One mod­ule (n=121) was found to be strongly associated with atrial rhythm at surgery across all 3 groups of data set, whereas another module (n=244) was associated with atrial rhythm at surgery in the MV and CAD groups. The first module over­lapped significantly with the salmon module in terms of gene membership, whereas most of the second modules’ genes were contained within the tan module. The top hub genes found in the salmon and tan modules remained present and highly connected in the 2 new modules identified with the dif­ferent soft-thresholding parameter.


To our knowledge, our study is the first implementation of an unbiased, network-based analysis in a large sample of human left atrial appendage gene expression profiles. We found 2 modules associated with AF severity and atrial rhythm in 2 to 3 of our cardiovascular comorbidity groups. Functional analy­ses revealed significant enrichment of cardiovascular-related categories for both modules. In addition, several of the hub genes identified are implicated in cardiovascular disease and may play a role in AF initiation and progression.

In our study, WGCNA was used to construct modules based on gene coexpression, thereby reducing the net-work’s dimensionality to a smaller set of elements.17,21 Relating modulewise changes to phenotypic traits allowed statistically significant associations to be detected at a lower false discovery rate compared with traditional differential expression studies. Furthermore, shared functions and path­ways among genes in the modules could be inferred via enrichment analyses.

We divided our data set into 3 groups to verify the repro­ducibility of the modules identified by WGCNA; 14 modules were identified in the MV group in our gene network. All were strongly preserved in the CAD and LAF groups, suggesting that gene coexpression patterns are robust and reproducible despite differences in cardiovascular comorbidities.

The use of module eigengene profiles as representative summary measures has been validated in a number of studies.20,26 Additionally, we found that the eigengenes accounted for a significant proportion (average 18%) of gene expression variability in their respective modules. Regression analysis of the module eigengenes found 2 modules associated with AF severity and atrial rhythm in ≥2 groups of data set. The association between the salmon module eigengene and AF severity was statistically weaker in the LAF group (adjusted P=9.0×10−2). This was probably because of its significantly smaller sample size compared with the MV and CAD groups. Despite this weaker association, the relationship between the salmon module eigengene and AF severity remained consistent among the 3 groups (Figure 3A). Similarly, the lack of statistical significance for the association between the tan module eigengene and atrial rhythm at surgery in the LAF group was likely driven by the smaller sample size and (by definition) lack of samples in the no AF category.

A major part of our analysis focused on the identifica­tion of module hub genes. Hubs are connected with a large number of nodes; disruption of hubs therefore leads to wide­spread changes within the network. This concept has powerful applications in the study of biology, genetics, and disease.29,30 Although mutations of peripheral genes can certainly lead to disease, gene network changes are more likely to be motivated by changes in hub genes, making them more biologically inter­esting targets for further study.17,29,31 Indeed,

  • the hub genes of the salmon and tan modules accounted for the vast majority of the variation in their respective module eigengenes, signaling their importance in driving gene module behavior.

The hub genes identified in the salmon and tan modules were significantly associated with AF phenotype overall. It was noted that this association was statistically weaker for the lower-ranked hub genes in the tan module. This highlights an important aspect and strength of WGCNA—to be able to capture module-wide changes with respect to disease despite potentially weaker associations among individual genes.

The implementation of WGCNA necessitated the selection of a soft-thresholding parameter 13. Unlike hard-thresholding (where gene correlations below a certain value are shrunk to zero), the soft-thresholding approach gives greater weight to stronger correlations while maintaining the continuous nature of gene–gene relationships. We selected a 13 value of 3 based on the criteria outlined by Zhang and Horvath.17 His team and other investigators have demonstrated that module identifica­tion is robust with respect to the 13 parameter.17,19–21 In our data, we were also able to reproduce the key findings reported with a different, larger 13 value, thereby verifying the stability of our results relating to 13.

The salmon module (124 genes) was associated with both AF phenotypes; furthermore, IPA analysis of its gene con­tents suggested enrichment in cardiovascular development as well as disease. Its eigengene increased with worsening AF severity, with the largest stepwise change occurring between the paroxysmal AF and persistent AF categories (Figure 3). Hence,

  • the gene expression changes within the salmon mod­ule may reflect the later stages of AF pathophysiology.

The top hub gene of the salmon module was RCAN1 (reg­ulator of calcineurin 1). Calcineurin is a cytoplasmic Ca2+/ calmodulin-dependent protein phosphatase that stimulates cardiac hypertrophy via its interactions with NFAT and L-type Ca2+ channels.32,33 RCAN1 is known to inhibit calcineurin and its associated pathways.32,34 However, some data suggest that RCAN1 may instead function as a calcineurin activator when highly expressed and consequently potentiate hypertrophic signaling.35 Thus,

  • perturbations in RCAN1 levels (attribut­able to genetic variants or mutations) may cause an aberrant switching in function, which in turn triggers atrial remodeling and arrhythmogenesis.

Other hub genes found in the salmon module are also involved in cardiovascular development and function and may be potential targets for further study.

  • DNAJA4 (DnaJ homolog, subfamily A, member 4) regulates the trafficking and matu­ration of KCNH2 potassium channels, which have a promi­nent role in cardiac repolarization and are implicated in the long-QT syndromes.36

FHL2 (four-and-a-half LIM domain protein 2) interacts with numerous cellular components, including

  1. actin cytoskeleton,
  2. transcription machinery, and
  3. ion channels.37

FHL2 was shown to enhance the hypertrophic effects of isoproterenol, indicating that

  • FHL2 may modulate the effect of environmental stress on cardiomyocyte growth.38
  • FHL2 also interacts with several potassium channels in the heart, such as KCNQ1, KCNE1, and KCNA5.37,39

Additionally, blood vessel epicardial substance (BVES) and other members of its family were shown to be highly expressed in cardiac pacemaker cells. BVES knockout mice exhibited sinus nodal dysfunction, suggesting that BVES regulates the development of the cardiac pacemaking and conduction system40 and may therefore be involved in the early phase of AF development.

The tan module (679 genes) eigengene was negatively correlated with atrial rhythm in the MV and CAD groups (Figure 4); this may indicate a general decrease in gene expres­sion of its members in fibrillating atrial tissue. IPA analysis revealed enrichment in genes involved in cell signaling as well as apoptosis. The top-ranked hub gene, cytoplasmic polyade-nylation element binding protein 3 (CPEB3), regulates mRNA translation and has been associated with synaptic plasticity and memory formation.41 The role of CPEB3 in the heart is currently unknown, so further exploration via animal model studies may be warranted.

Natriuretic peptide-precursor B (NPPB), another highly interconnected hub gene, produces a precursor peptide of brain natriuretic peptide, which

  • regulates blood pressure through natriuresis and vasodilation.42

(NPPB) gene variants have been linked with diabetes mellitus, although associations with cardiac phenotypes are less clear.42 TBX5 and GATA4, which play important roles in the embryonic heart development,43 were members of the tan module. Although not hub genes, they may also contribute toward developmental sus­ceptibility of AF. In addition, TBX5 was previously reported to be near an SNP associated with PR interval and AF in separate large-scale GWAS studies.12,28 MYOZ1, another candidate gene identified in the recent AF GWAS meta-analysis, was found to be a member as well; it associates with proteins found in the Z-disc of skeletal and cardiac muscle and may suppress calcineurin-dependent hypertrophic signaling.12

Some, but not all, of the candidate genes found in previous GWAS studies were located in the AF-associated modules. One possible explanation for this could be the difference in sample sizes. The meta-analysis involved thousands of indi­viduals, whereas the current study had <100 in each group of data set, which limited the power to detect significant differ­ences between levels of AF phenotype even with the module-wise approach. Additionally, transcription factors like PITX2 are most highly expressed during the fetal phase of develop­ment. Perturbations in these genes (attributable to genetic variants or mutations) may therefore initiate the development of AF at this stage and play no significant role in adults (when we obtained their tissue samples).

Limitations in Study

We noted several limitations in this study. First, no human left atrial mRNA data set of adequate size currently exists publicly. Hence, we were unable to validate our results with an external, independent data set. However, the network pres­ervation assessment performed within our data set showed strong preservation in all modules, indicating that our findings are robust and reproducible.

Although the module eigengenes captured a significant pro­portion of module variance, a large fraction of variability did remain unaccounted for, which may limit their use as repre­sentative summary measures.

We extracted RNA from human left atrial appendage tis­sue, which consists primarily of cardiomyocytes and fibro­blasts. Atrial fibrosis is known to occur with AF-associated remodeling.44 As such, the cardiomyocyte to fibroblast ratio is likely to change with different levels of AF severity, which in turn influences the amount of RNA extracted from each cell type. Hence, true differences in gene expression (and coexpression) within cardiomyocytes may be confounded by changes in cellular composition attributable to atrial remod­eling. Also, there may be significant regional heterogeneity in the left atrium with respect to structure, cellular composi­tion, and gene expression,45 which may limit the generaliz-ability of our results to other parts of the left atrium.

All subjects in the study were whites to minimize the effects of population stratification. However, it is recognized that the genetic basis of AF may differ among ethnic groups.9 Thus, our results may not be generalizable to other ethnicities.

Finally, it is possible for genes to be involved in multiple processes and functions that require different sets of genes. However, WGCNA does not allow for overlapping modules to be formed. Thus,

  • this limits the method’s ability to character­ize such gene interactions.


In summary, we constructed a weighted gene coexpression network based on RNA expression data from the largest collection of human left atrial appendage tissue specimens to date. We identified 2 gene modules significantly associated with AF severity or atrial rhythm at surgery. Hub genes within these modules may be involved in the initiation or progression of AF and may therefore be candidates for functional stud­ies.


1. European Heart Rhythm Association, European Association for Cardio-Thoracic Surgery, Camm AJ, Kirchhof P, Lip GY, Schotten U, et al. Guidelines for the management of atrial fibrillation: the task force for the management of atrial fibrillation of the European Society of Cardiology (ESC). Eur Heart J. 2010;31:2369–2429.

2. Lemmens R, Hermans S, Nuyens D, Thijs V. Genetics of atrial fibrilla­tion and possible implications for ischemic stroke. Stroke Res Treat. 2011;2011:208694.

3. Wann LS, Curtis AB, January CT, Ellenbogen KA, Lowe JE, Estes NA III, et al; ACCF/AHA/HRS. 2011 ACCF/AHA/HRS focused update on the management of patients with atrial fibrillation (Updating the 2006 Guideline): a report of the American College of Cardiology Foundation/ American Heart Association Task Force on Practice Guidelines. J Am Coll Cardiol. 2011;57:223–242.

4. Dobrev D, Carlsson L, Nattel S. Novel molecular targets for atrial fibrilla­tion therapy. Nat Rev Drug Discov. 2012;11:275–291.

5. Christophersen IE, Ravn LS, Budtz-Joergensen E, Skytthe A, Haunsoe S, Svendsen JH, et al. Familial aggregation of atrial fibrillation: a study in Danish twins. Circ Arrhythm Electrophysiol. 2009;2:378–383.

6. Gudbjartsson DF, Arnar DO, Helgadottir A, Gretarsdottir S, Holm H, Sig-urdsson A, et al. Variants conferring risk of atrial fibrillation on chromo­some 4q25. Nature. 2007;448:353–357.

7. Ellinor PT, Lunetta KL, Glazer NL, Pfeufer A, Alonso A, Chung MK, et al. Common variants in KCNN3 are associated with lone atrial fibrillation. Nat Genet. 2010;42:240–244.

8. Benjamin EJ, Rice KM, Arking DE, Pfeufer A, van Noord C, Smith AV, et al. Variants in ZFHX3 are associated with atrial fibrillation in individuals of European ancestry. Nat Genet. 2009;41:879–881.

9. Sinner MF, Ellinor PT, Meitinger T, Benjamin EJ, Kääb S. Genome-wide association studies of atrial fibrillation: past, present, and future. Cardio-vasc Res. 2011;89:701–709.

10. Clauss S, Kääb S. Is Pitx2 growing up? Circ Cardiovasc Genet. 2011;4:105–107.

11. Kirchhof P, Kahr PC, Kaese S, Piccini I, Vokshi I, Scheld HH, et al. PITX2c is expressed in the adult left atrium, and reducing Pitx2c expres­sion promotes atrial fibrillation inducibility and complex changes in gene expression. Circ Cardiovasc Genet. 2011;4:123–133.

12. Ellinor PT, Lunetta KL, Albert CM, Glazer NL, Ritchie MD, Smith AV, et al. Meta-analysis identifies six new susceptibility loci for atrial fibrillation. Nat Genet. 2012;44:670–675.

13. Barth AS, Merk S, Arnoldi E, Zwermann L, Kloos P, Gebauer M, et al. Reprogramming of the human atrial transcriptome in permanent atrial fi­brillation: expression of a ventricular-like genomic signature. Circ Res. 2005;96:1022–1029.

Continues to 45.  see



Atrial fibrillation is the most common sustained cardiac arrhythmias in the United States. The genetic and molecular mecha­nisms governing its initiation and progression are complex, and our understanding of these mechanisms remains incomplete despite recent advances via genome-wide association studies, animal model experiments, and differential expression studies. In this study, we used weighted gene coexpression network analysis to identify gene modules significantly associated with atrial fibrillation in a large sample of human left atrial appendage tissues. We further identified highly interconnected genes (ie, hub genes) within these gene modules that may be novel candidates for functional studies. The discovery of the atrial fibrillation-associated gene modules and their corresponding hub genes provide novel insight into the gene network changes that occur with atrial fibrillation, and closer study of these findings can lead to more effective targeted therapies for disease management.

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Oxidized Calcium Calmodulin Kinase and Atrial Fibrillation

Author: Larry H. Bernstein, MD, FCAP


Curator: Aviva Lev-Ari, PhD, RN



This is a review of a recent work from the laboratory of Mark E. Anderson and associates at the University of Iowa.  WE have covered the role of CaMKII in calcium signaling and myocardiocyte contraction, as well as signaling in smooth muscle, skeletal muscle, and nerve transmission.  There are tissue specific modus operandi, partly related to the ryanogen receptor, and also related to tissue specific isoenzymes of CaMKII.  There is much ground that has been traversed in exploring these mechanisms, most recently, the discoverey of hormone triggering by the release from vesicles at the nerve muscle junction, and much remains open to investigation.  The recently published work by Mark E. Anderson and associates in Mannheim and Heidelberg, Germany, clarifies the relationship between the oxidized form of CaMKII and the triggering of atrial fibrillation. The following studies show:

  1. Ang II infusion increased the susceptibility of mice to AF induction by rapid right atrial pacing and established a framework for us to test the hypothesized role of ox-CaMKII in promoting AF. ox-CaMKII is critical for AF.
  2. Estalished a critical role of ox-CaMKII in promoting AF
  3. Ang II induced increases in ROS production seen in WT atria were absent in atria from MsrA TG mice suggesting that MsrA sensitive targets represent an important component of Ang II mediated atrial oxidation.
  4. The protection from AF in MsrA TG mice appeared to be independent of pressor effects that are critical for the proarrhythmic actions.
  5. These findings suggest that NADPH oxidase dependent ROS and elevated ox-CaMKII drive Ang II  -pacing-induced AF and that
  6. targeted antioxidant therapy, by MsrA over-expression, can reduce or prevent AF in Ang -II-infused mice.  
  7. Atrial myocytes from Ang II treated WT mice showed a significant (p<0.05) increase in spontaneous Ca2+ sparks compared to atrial myocytes from saline treated control mice
  8. In contrast to findings in WT mice, the atrial myocytes isolated from Ang II treated MM-VV mice did not show an increase in Ca2+ sparks compared to saline treated MM-VV mice
  9. These data to suggest that  in ox–the proarrhythmic effects of Ang I I infusion depend upon an increaseCaMKII, sarcoplasmic reticulum Ca2+ leak and DADs.
  10. Enhanced CaMKII-mediated phosphorylation of serine 2814 on RyR2 is associated with an increased susceptibility to acquired arrhythmias, including AF
  11. Proarrhythmic actions of ox-CaMKII require access to RyR2 serine 2814.
  12. Mutant S2814A knock-in mice (lacking serine 2814) were highly resistant to Ang II mediated AF
  13. AC3-I mice with transgenic myocardial expression of a CaMKII inhibitory peptide were also resistant to the proarrhythmic effects of Ang II infusion on pacing-induced AF
  14. S2814A, AC3-I and WT mice, all developed similar BP increases and cardiac hypertrophy in response to Ang II, indicating that these mice were not resistant to the hemodynamic effects of Ang II, but were nevertheless protected from AF.
  15. selectively targeted antioxidant therapies could be effective in preventing or reducing AF 
  16. half of patients enrolled in the Mode Selection Trial (MOST) with sinus node dysfunction had a history of AF
  17. Ang II and diabetes-induced CaMKII oxidation caused sinus node dysfunction by increased pacemaker cell death and fibrosis
  18.  ox-CaMKII increases susceptibility for AF via increased diastolic sarcoplasmic reticulum Ca2+ release
  19. clinical association between sinus node dysfunction and AF might have a mechanistic basis because sinus node dysfunction and AF are downstream consequences of elevated ox-CaMKII.

We refer to the following related articles published in pharmaceutical Intelligence:

Contributions to cardiomyocyte interactions and signaling
Author and Curator: Larry H Bernstein, MD, FCAP  and Curator: Aviva Lev-Ari, PhD, RN

Cardiac Contractility & Myocardium Performance: Therapeutic Implications for Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses
Editor: Justin Pearlman, MD, PhD, FACC, Author and Curator: Larry H Bernstein, MD, FCAP, and Article Curator: Aviva Lev-Ari, PhD, RN

Part I. Identification of Biomarkers that are Related to the Actin Cytoskeleton
Curator and Writer: Larry H Bernstein, MD, FCAP

Part II: Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility
Larry H. Bernstein, MD, FCAP, Stephen Williams, PhD and Aviva Lev-Ari, PhD, RN

Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets
Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN

Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD
Aviva Lev-Ari, PhD, RN

Part VII: Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmias and Non-ischemic Heart Failure – Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

Part VIII: Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

Part IX: Calcium-Channel Blockers, Calcium Release-related Contractile Dysfunction (Ryanopathy) and Calcium as Neurotransmitter Sensor
Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

Part X: Synaptotagmin functions as a Calcium Sensor: How Calcium Ions Regulate the fusion of vesicles with cell membranes during Neurotransmission
Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

Oxidized CaMKII Triggers Atrial Fibrillation

Running title: Purohit et al.; oxCaMKII and AF

Anil Purohit, Adam G. Rokita, Xiaoqun Guan, Biyi Chen, Olha M. Koval, Niels Voigt, Stefan Neef, Thomas Sowa, Zhan Gao, Elizabeth D. Luczak, Hrafnhildur Stefansdottir, Andrew C. Behunin, Na Li, Ramzi N. El Accaoui, Baoli Yang, Paari Dominic Swaminathan, Robert M. Weiss, Xander H. T. Wehrens, Long-Sheng Song, Dobromir Dobrev, Lars S. Maier and Mark E. Anderson

1Dept of Internal Medicine, Division of Cardiovascular Medicine and Cardiovascular Research Center, Carver College of Medicine, University of Iowa, Iowa City, IA; 2Institute of Pharmacology, Faculty of Medicine, University Duisburg-Essen, Essen, Germany, and Division of Experimental Cardiology, Medical Faculty Mannheim, University of Heidelberg, Mannheim, Germany; 3Cardiology and Pneumology, German Heart Center, University Hospital Goettingen, Goettingen, Germany; 4Dept of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX; 5Dept of Obstetrics and Gynecology; 6Dept of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA
Circulation Sept 12, 2013;



Journal Subject Codes: Basic science research:[132] Arrhythmias – basic studies, Etiology:[5] Arrhythmias, clinical electrophysiology, drugs


Background—Atrial fibrillation is a growing public health problem without adequate therapies. Angiotensin II (Ang II) and reactive oxygen species (ROS) are validated risk factors for atrial fibrillation (AF) in patients, but the molecular pathway(s) connecting ROS and AF is unknown. The Ca2+/calmodulin-dependent protein kinase II (CaMKII) has recently emerged as a ROS activated proarrhythmic signal, so we hypothesized that oxidized CaMKII􀄯(ox-CaMKII) could contribute to AF.  Methods and Results—We found ox-CaMKII was increased in atria from AF patients compared to patients in sinus rhythm and from mice infused with Ang II compared with saline. Ang II treated mice had increased susceptibility to AF compared to saline treated WT mice, establishing Ang II as a risk factor for AF in mice. Knock in mice lacking critical oxidation sites in CaMKIId (MM-VV) and mice with myocardial-restricted transgenic over-expression of methionine sulfoxide reductase A (MsrA TG), an enzyme that reduces ox-CaMKII, were resistant to AF induction after Ang II infusion. Conclusions—Our studies suggest that CaMKII is a molecular signal that couples increased ROS with AF and that therapeutic strategies to decrease ox-CaMKII may prevent or reduce AF.

Key words: atrial fibrillation, calcium/calmodulin-dependent protein kinase II, angiotensin II, reactive oxygen species, arrhythmia (mechanisms)


Atrial fibrillation (AF) is the most common sustained  arrhythmia. AF produces lifestyle-limiting symptoms and increases the risk of stroke and death,1 but current therapies have limited efficacy. The renin-angiotensin-system is upregulated in cardiovascular disease and elevated Angiotensin II (Ang II) favors AF.2,3 Ang II activates NADPH oxidase, leading to increased ROS and fibrillating atria are marked by increased reactive oxygen species (ROS).4,5 We recently identified the multifunctional Ca2+ and calmodulin-dependent protein kinase II (CaMKII) as a ROS sensor6 and proarrhythmic signal.7 Oxidation of critical methionines (281/282) in the CaMKII regulatory domain lock CaMKII into a constitutively active, Ca2+ and calmodulinin-dependent conformation that is associated with cardiovascular disease.8 Based on this information, we asked if oxidized CaMKII (ox-CaMKII) could be a biomarker and proarrhythmic signal for connecting increased atrial ROS to AF. We found that ox-CaMKII was increased in atrial tissue from patients with AF compared to patients in sinus rhythm, and in atrial tissue from Ang II-infused, compared to saline-infused, mice. We used a validated mouse model of AF induction by rapid right atrial pacing9,10 and found that mice with prior Ang II infusion were at significantly higher risk of AF compared to vehicle-infused mice. We tested AF induction in Ang II and vehicle-infused mice with genetically engineered resistance to CaMKII oxidation by knock-in replacement of methionines 281/282 with valines in CaMKIId (MM-VV), the isoform associated with cardiovascular disease11-14 or by myocardial-targeted antioxidant therapy by transgenic over-expression of methionine sulfoxide reductase A (MsrA), an enzyme that reduces ox-CaMKII.15,16  Collectively, our results support a view that Ang II promotes AF induction by increasing ROS, ox-CaMKII, CaMKII activity, sarcoplasmic reticulum Ca2+ leak and delayed after-depolarizations (DADs). Our findings provide novel insights into a ROS and Ang II-dependent mechanism of AF by linking oxidative stress to dysfunctional intracellular Ca2+ signaling via ox-CaMKII and identify a potential new approach for treating AF by targeted antioxidant therapy.


Human samples and immunodetection of ox-CaMKII.

The human samples were provided by the Georg-August-University Goettingen and the University of Heidelberg after approval by the local ethics committee of the Georg-August-University Göttingen and the Medical Faculty Mannheim, University of Heidelberg (#2011-216N-MA). 

Right atrial appendage tissue samples were obtained from patients undergoing thoracotomy with sinus rhythm or with AF (Table 1) as published previously.17 For immunostaining experiments a total of 9 samples were studied including 5 patients with sinus rhythm and 4 patients with AF ( Table 1A). For immunob lotting a total of 51 samples were studied including 25 patients with SR and 26 patients with AF (Table 1B). The pat ei nt charts were reviewed by the authors to obtain relevant clinical information.

Mouse Models and Experimental Methods

All mice used in the study were available to us in C57Bl6 background. All experiments were performed in male mice 8-12 weeks of age. In total we studied 262 mice. Numbers for each experimental group are provided in the figures or figure legends. See Supplemental Material for detailed methods.


Data are presented as mean ± SEM. P values were assessed with a Student’s t-test (2-tailed), ANOVA or two-way ANOVA, as appropriate, for continuous data. The effect of Ang II compared to saline on ox-CaMKII, CaMKII, and ox-CaMKII/CaMKII ratio was tested within each mouse genotype (strain) and compared among the four genotypes using the two-way analysis of variance (ANOVA). The factors that were tested in the ANOVA model were genotype (WT, MM-VV, p47-/- and MsrA TG), treatment (Ang II versus saline), and genotype treatment interaction effect. A significant genotype treatment interaction (*) indicated that the effect of Ang II (versus saline) differed significantly among the strains. Post hoc comparisons after ANOVA were performed using the Bonferroni test. Discrete variables were analyzed by Fisher’s exact test.


Oxidized CaMKII is increased in AF

Patients with AF have increased atrial CaMKII activity18,19 and high circulating levels of serum markers for oxidative stre ss. 4, 5 We first obtained right atrial tissue from patients undergoing cardiac surgery (Table 1) and measured ox-CaMKII using a validated antiserum against oxidized Met 281/282 in the CaMKII regulatory domains.6 These pilot immunofluorescence studies on atrial tissue samples made available upon consent by patients with AF or normal sinus rhythm (Table 1A) showed significantly (p<0.05) higher (~2.5 fold) ox-CaMKII levels in patients with AF (Figure 1A and B). Based on these initial findings, we measured ox-CaMKII in atrial tissue from a larger cohort of patients (Table 1B; for complete gels see supplementary Figure 1) in sinus rhythm (N = 25) or AF (N = 26) using Western blots, and confirmed that AF patients have significantly elevated expression of ox-CaMKII, while there was no difference in total CaMKII (Figure 1C-F). The patient characteristics in the two groups (Table 1) were similar in terms of age, presence of hypertension, diabetes and left ventricular ejection fraction, recognized risk factors for AF.20 The subgroup of AF patients that were not treated with angiotensin converting enzyme inhibitor (ACE-i) or angiotensin receptor blockers (ARB) showed the highest levels of ox-CaMKII and total CaMKII (Supplementary Figure 1A and B). Taken together, these findings showed a positive association between AF and increased expression of atrial ox-CaMKII and a loss of this association in AF patients treated with ACE-i or ARBs.

Ang II treatment enhances AF susceptibility


To test the hypothesis that ox-CaMKII contributes to AF we developed a mouse model of AF by infusing wild type (WT) mice with Ang II (2000 ng/kg/min) or an equal volume of normal saline via osmotic mini-pumps for three weeks. We previously established that this dose of Ang II caused a significant increase in atrial ox-CaMKII7 and resulted in serum Ang II levels similar to those measured in heart failure patients.21
In order to test if Ang II treatment can promote AF we performed burst pacing in the right atrium of anesthetized mice, using an established method ( Figure 2A-C). 10 Mice treated wit Ang II showed significantly higher AF induction rates compared to saline treated mice (64% [9/14] versus 18% [2/14], p=0.018 Fisher’s exact test) (Figure 2D). Ang II is known to contribute to hypertension, left ventricular hypertrophy and heart failure, all established clinical risk factors for AF.20 Therefore, we measured blood pressure (BP) by tail-cuff and assessed left ventricular size and systolic function by echocardiography. As expected, Ang II treatment significantly increased systolic BP (Figure 2E; p<0.01) and left ventricular mass (Figure 2F; p<0.001). Ang II treated mice maintained a normal left ventricular ejection fraction, similar to saline-infused control mice (Figure 2G). These data showed that Ang II infusion increased the susceptibility of mice to AF induction by rapid right atrial pacing and established a framework for us to test the hypothesized role of ox-CaMKII in promoting AF. ox-CaMKII is critical for AF.
In order to test if ox-CaMKII was required for AF induction in our model we used oxidation resistant knock in MM-VV mice (Supplementary Figure 2).22 CaMKII with the MM-VV mutation is resistant to oxidative activation but retains normal Ca2+ and calmodulin dependent activation and is capable of transitioning into a Ca2+ and calmodulin independent enzyme after threonine 287 autophosphorylation.6 The MM-VV mice were significantly resistant to AF induction after Ang II infusion, compared to WT controls (Figure 3A), suggesting that ox-CaMKII is required for increased AF susceptibility in Ang II infused mice. WT mice treated with Ang II showed significantly higher (~2.7 fold; 95% confidential interval, CI: 1.4, 5.1) ) levels of mice. When indexed to total CaMKII levels (Supplementary Figure 3A and B) this increase in ox-CaMKII was much higher (~14. 2 fold; 95% confidential interval, CI: 1.4, 5.1)  in Ang II treated WT mice (figure 4C).  The residual increase in ox–CaMKII in the -MM-VV mice likely results from expression of atrial ox-CaMKII compared to saline treated mice. As expected, Ang II infusion increased ox-CaMKII less in -MM-VV (~2.1 fold; 95% CI: 1.1, 4.0) than in control WT.  ox-CaMKII was much higher (~14.2 fold; 95% CI: 5.9, 34.5) in Ang II treated WT mice.
CaMKIILI, a myocardial CaMKII isoform not affected by the MM-VV mutation.23 However, despite the greater increase in ox-CaMKII in WT compared to MM-VV mice, Ang II-related ROS production was increased in both WT and MM-VV mice to a similar degree (Supplementary Figure 4). Interestingly, Ang II treated WT mice showed a significant decrease in total CaMKII levels (Supplementary Figure 3A and B) suggesting feedback inhibition of total CaMKII expression.
Atrial lysates from MM-VV mice showed significantly less Ca2+ and calmodulin-independent activity after Ang II treatment, but retained WT level CaMKII activity increases in response to isoproterenol (Supplementary Figure 2A). At 8 weeks MM-VV mice had body weight (Supplementary Figure 2B) and BP (Figure 3B) that were similar to WT mice, suggesting CaMKIIį methionine 281/282 oxidation did not affect basal BP or developmentally appropriate growth. CaMKII is known to regulate the chronotropic response to stress and mice with CaMKII inhibition have a smaller increase in heart rate with isoproterenol treatment compared to controls.24 Isolated Langendorff-perfused hearts from WT and MM-VV mice had similar resting heart rates (Supplementary Figure 2C) and comparable heart rate increases after isoproterenol treatment (Supplementary Figure 2D), suggesting that CaMKII dependent physiological heart rate increases do not require CaMKIIį methionine oxidation. L-type Ca2+ currents were similar in MM-VV and WT mice, and L-type Ca2+ current facilitation, a CaMKII-dependent phenotype, was also preserved in MM-VV mice.25,26 KN-93, a small molecule CaMKII inhibitor,27 significantly reduced facilitation in WT and -MM-VV mice (Supplementary Figure 5). MM-VV mice and WT controls showed similar increases in systolic BP (Figure 3B) and heart weight (Figure 3C) or left ventricular mass estimated by echocardiography after Ang II infusion ( Supplementary Figure 6), suggesting that -ox-CaMK IIį is dispensable for hypertensive and myocardial hypertrophic actions of Ang II. Taken together, these findings indicate loss of methionines 281/282 in CaMKIIį selectively reduce the pro-arrhythmic actions of Ang II in a pacing-induced model of AF.

NADPH oxidase and MsrA regulate ox-CaMKII and AF susceptibility.

  •  Ang II increases intracellular ROS in myocardium by activating NADPH oxidase and
  • p47-/-mice28, lacking functional NADPH oxidase, are resistant to Ang II dependent increases in ROS and ox-CaMKII.6
  • Atrial lysates from Ang II treated p47-/- mice did not show an increase in ox-CaMKII (Figure 4), and
  • the p47-/- mice were also resistant to Ang II-mediated increases in AF
However, there were similar increases in BP (Figure 3B) effects of Ang II. This was observed with MsrA TG and WT mice (Figure 3A), showing similar increases in BP (Figure 3B), overall heart weight (Figure 3C) and estimated left ventricular mass (Supplementary Figure 6) after Ang II treatment compared to WT controls. ox-CaMKII is reduced by MsrA15 and transgenic mice with myocardial-delimited MsrA overexpression (MsrA TG) have increased atrial MsrA protein (Supplementary Figure 3C) and
  • are resistant to ROS induced myocardial injury.16

We found that Ang II treated MsrA TG mice showed decreased AF induction compared to Ang II-treated WT mice (Figure 3A) and

  • had similar atrial ox-CaMKII expression compared to saline treated controls (Figure 4).
  • Ang II induced increases in ROS production seen in WT atria were absent in atria from MsrA TG mice (Supplementary Figure 4),
suggesting that MsrA sensitive targets represent an important component of Ang II mediated atrial oxidation. The protection from AF in MsrA TG mice appeared to be independent of pressor effects that are critical for the proarrhythmic actions. Taken together, these findings suggest that
  • NADPH oxidase dependent ROS and elevated ox-CaMKII drive Ang II  -pacing-induced AF and that
  • targeted antioxidant therapy, by MsrA over-expression, can reduce or prevent AF in Ang -II-infused mice.

Ang II increases Ca2+ sparks and triggered action potentials

CaMKII contributes to increased sarcoplasmic reticulum Ca2+ leak in mice with a RyR2 mutation modeled after a human arrhythmia syndrome, catecholaminergic polymorphic ventricular tachycardia,9 in a goat model of AF and in atrial myocytes isolated from patients with AF.18,29 Atrial myocytes from patients with AF
  • show increased CaMKII activity and increased CaMKII-dependent ryanodine receptor phosphorylation at serine 2814.29
  •  CaMKII inhibition with KN-93 reduced the open probability of single RyR2 channels and
  • prevented the increased frequency of sarcoplasmic reticulum Ca2+ sparks in atrial myocardium biopsied from AF patients.18,29
Based on this knowledge, we asked if increased RyR2 Ca2+ leak also contributed to the mechanism of AF in WT Ang II infused mice and measured diastolic Ca2+ sparks, a marker of RyR2 Ca2+ leak.30
  • Atrial myocytes from Ang II treated WT mice showed a significant (p<0.05) increase in spontaneous Ca2+ sparks compared to atrial myocytes from saline treated control mice (Figure 5A and B).
Other Ca2+ spark parameters and sarcoplasmic reticulum Ca2+ content were not different between the saline and Ang II treated WT mice (Supplementary Figure 7). In contrast to findings in WT mice,
  • the atrial myocytes isolated from Ang II treated MM-VV mice did not show an increase in Ca2+ sparks compared to saline treated MM-VV mice (Figure 5A and B).
  • A significantly greater proportion of atrial myocytes isolated from Ang II treated WT mice showed DADs, compared to atrial myocytes from saline treated mice (Figure 5C and D, p=0.03; Fisher’s exact test).
  • atrial myocytes from Ang II infused MM-VV mice did not show a significant increase in DADs compared to the atrial myocytes from saline treated MM-VV mice.

We interpret these data to suggest that the proarrhythmic effects of Ang I I infusion depend upon an increase in ox–CaMKII, sarcoplasmic reticulum Ca2+ leak and DADs.

Mice with CaMKII-resistant RyR2 are protected from AF after Ang II infusion

Enhanced CaMKII-mediated phosphorylation of serine 2814 on RyR2 is associated with an increased susceptibility to acquired arrhythmias, including AF.31 Based on our findings

  • that atrial myocytes from Ang II infused WT mice developed more Ca2+ sparks than atrial myocytes from saline-infused mice,

we hypothesized that the proarrhythmic actions of ox-CaMKII require access to RyR2 serine 2814. We tested this hypothesis by treating mutant S2814A knock-in mice (lacking serine 2814)9 with Ang II or saline and performing right atrial burst pacing.

  • The S2814A mice were highly resistant to Ang II mediated AF (Figure 6A). Similarly,
  • AC3-I mice with transgenic myocardial expression of a CaMKII inhibitory peptide32 were also resistant to the proarrhythmic effects of Ang II infusion on pacing-induced AF (Figure 6A). S2814A,

AC3-I and WT mice, all developed similar BP increases (Figure 6B) and cardiac hypertrophy (Figure 6C) in response to Ang II, indicating that

  • these mice were not resistant to the hemodynamic effects of Ang II, but were nevertheless protected from AF.


AF usually develops in patients with underlying structural heart disease, such as left ventricular hypertrophy, coronary artery disease, valve disease and congestive heart failure.20 Elevated ROS is a common feature of these conditions.33 The dose of Ang II used in our model produces a fourfold increase in plasma Ang II compared to saline controls,7 similar to increases in Ang II observed in heart failure patients evidence of elevated ROS in structural heart disease, clinical trials with antioxidants have generally been unsatisfactory.34-36 One potential obstacle to developing effective antioxidant therapies is lack of detailed understanding of molecul ra pathways that are affected by ROS. The renin-angiotensin-system is one of the best understood pathways that contributes to ROS production in AF patients.37 In the current study, we created a model of AF by infusing mice with Ang II for three weeks and assembled a cohort of genetically altered mice to rigorously test a novel molecular pathway that links oxidative stress to AF (Figure 7). Our current study provides strong evidence that CaMKII is a critical ROS sensor for transducing increased ROS into enhanced AF susceptibility in mice and suggests that atrial ox-CaMKII could contribute to AF in patients.

CaMKII and increased ROS are now widely recognized to contribute to cardiac arrhythmias.8,38,39 Recent studies suggest that patients with persistent AF have elevated markers of oxidative stress in serum4 and depleted levels of atrial glutathione.40 Under increased oxidative stress CaMKII is activated by oxidation of methionines (M281/282),6 which lock it into a constitutively active conformation, suggesting a possible role for ox-CaMKII as a ROS activated proarrhythmic signal in AF.39 Our laboratory recently demonstrated that

  • ox-CaMKII plays a major role in sinus node dysfunction,7,22
  • adverse post-myocardial infarct remodeling6 and
  • cardiac rupture16.

In the current study, we investigated the role of ox-CaMKII in AF. Human atria (Figure 1) and Ang II treated WT mouse atria showed significantly elevated ox-CaMKII (Figure 4).

  • Atrial myocytes from Ang II treated WT mice had a higher frequency of spontaneous Ca2+ sparks and DADs compared to controls (Figure 5).

Based on these findings we hypothesized that oxidation of methionines 281/282 on CaMKII į causes diastolic sarcoplasmic reticulum Ca2+ leak and DADs, both cellular AF triggers. However, resistant to oxidative activation,22

  • Ang II, the myocardial CaMKII a recently developed knock-in mouse (MM-VV) where CaMKII isoform implicated in myocardial disease,1,2 13 treatment
  • did not increase Ca2+ and calmodulin independent CaMKII activity (Supplementary Figure 2A), Ca2+ sparks (Figure 5A and B), DADs (Figure 5C and D) or enhance AF susceptibility in MM-VV mice (Figure 3A).

It is important to note that the MM-VV mutant form of CaMKIIį selectively ablates the response to oxidation while retaining other aspects of CaMKII molecular physiology, such as

  • activation by Ca2+ and calmodulin and
  • constitutive activation by threonine 287 autophosphorylation.6

Thus, the residual AF observed in Ang II infused MM-VV mice could be a result of non-oxidation-dependent mechanisms for CaMKIIį activation in our model. We found that atrial tissue from AF patients treated with ACE-i or ARBs did not show elevated ox-CaMKII, suggesting that Ang II stimulation oxidizes CaMKII in human atria and that ox-CaMKII independent pathways are operative in AF patients. AF in patients is more complex than AF in our Ang II infused mice. In particular, patients present with variable chronicity, tissue and structural changes. In contrast the triggers for our mice are uniform (i.e. Ang II infusion and rapid right atrial pacing) and result in a similar, modest degree of hypertrophy. We interpret the data showing that an increase in ox-CaMKII in AF patients is reduced or eliminated by clinical antagonist drugs that reduce Ang II signaling to validate our findings in mice that Ang II increases ox-CaMKII. However, we suppose that the presence of AF in patients on ACE-i or ARBs means that other pathways also result in AF. Our sample is not powered to ask if AF resistance to Ang II antagonist drugs represents later stage disease, but this is our hypothesis. Furthermore, CaMKII can be activated independently of oxidation, although oxidation appears to be the primay r pathway for activating CaMKII during Ang II infusion. Thus, it is unknown if CaMKII is also important for AF progression in the group of patients treated by Ang II antagonist drugs who exhibit normal levels of ox -CaMKII.

Although we did not see higher total CaMKII in AF patients (as compared with patients in sinus rhythm), the sub-group of AF patients who were not treated with ACE-i or ARBs did show significantly elevated CaMKII levels, supporting prior studies that reported elevated CaMKII activity in AF18,19.  In contrast to the situation in patients, total CaMKII expression was reduced in mice after sub-acute Ang II infusion. While the mechanism(s) for the variable response of CaMKII expression in mice and patients is unclear, the change in expression in mice and in humans in response to manipulation of the Ang II pathway supports the idea that CaMKII is a fundamental component of Ang II signaling. The relatively small number of patient samples is not powered for analysis of AF subtypes, but human AF may transition from paroxysmal to persistent and permanent (chronic) forms.41 In contrast, our mouse model is simpler because it is triggered by a single upstream event (i.e. Ang II infusion) and elicited in a highly controlled environment by rapid atrial pacing. The resistance of MM-VV mice to AF provides new evidence that oxidative activation of CaMKII delta (d) is important for initiation of AF, while the finding that ox-CaMKII is elevated in atrial tissue from AF patients and particularly in AF patients naive to Ang II antagonist therapies suggests this pathway may also participate in human AF.

Thus, our findings in MM-VV mice provide strong, mechanistic evidence that ox-CaMKII plays a critical role in proarrhythmic responses to Ang II. Our studies showed that mice deficient in NADPH oxidase (p47-/-) and mice expressing increased MsrA are also resistant to AF (Figure 3A), suggesting that

  • selectively targeted antioxidant therapies could be effective in preventing or reducing AF.
  • Half of patients enrolled in the Mode Selection Trial (MOST) with sinus node dysfunction had a history of AF48,

but a clear mechanistic link between increased risk of AF and sinus node dysfunction is unknown. In recent studies we showed that Ang II and diabetes-induced CaMKII oxidation caused sinus node dysfunction by increased pacemaker cell death and fibrosis,7 while MM-VV mice are resistant to sinus node dysfunction evoked by hyperglycemia.22 Here we provide evidence that

  • ox-CaMKII increases susceptibility for AF via increased diastolic sarcoplasmic reticulum Ca2+ release, showing that
  • the proarrhythmic actions of ox-CaMKII may occur in cardiomyocytes by increasing sarcoplasmic reticulum Ca2+ leak or by enhanced cell death.

Our findings suggest that the clinical association between sinus node dysfunction and AF might have a mechanistic basis because sinus node dysfunction and AF are downstream consequences of elevated ox-CaMKII.

Selected References

1. Benjamin EJ, Wolf PA, D’Agostino RB, Silbershatz H, Kannel WB, Levy D. Impact of atrial fibrillation on the risk of death: the Framingham Heart Study. Circulation. 1998;98:946-952.
2. Khatib R, Joseph P, Briel M, Yusuf S, Healey J. Blockade of the renin-angiotensinaldosterone system (RAAS) for primary prevention of non-valvular atrial fibrillation: A systematic review and meta analysis of randomized controlled trials. Int J Cardiol. 2013;165:17-24.

4. Shimano M, Shibata R, Inden Y, Yoshida N, Uchikawa T, Tsuji Y, Murohara T. Reactive oxidative metabolites are associated with atrial conduction disturbance in patients with atrial
fibrillation. Heart Rhythm. 2009;6:935-940.
5. Neuman RB, Bloom HL, Shukrullah I, Darrow LA, Kleinbaum D, Jones DP, Dudley SC. Oxidative stress markers are associated with persistent atrial fibrillation. Clin Chem.
 6. Erickson JR, Joiner M-LA, Guan X, Kutschke W, Yang J, Oddis CV, Bartlett RK, Lowe JS, O’Donnell SE, Aykin-Burns N, Zimmerman MC, Zimmerman K, Ham A-JL, Weiss RM, Spitz DR, Shea MA, Colbran RJ, Mohler PJ, Anderson ME. A dynamic pathway for calciumin-dependent activation of CaMKII by methionine oxidation. Cell. 2008;133:462-474.

7. Swaminathan PD, Purohit A, Soni S, Voigt N, Singh MV, Glukhov AV, Gao Z, He BJ, Luczak ED, Joiner M-LA, Kutschke W, Yang J, Donahue JK, Weiss RM, Grumbach IM, Ogawa M, Chen P-S, Efimov I, Dobrev D, Mohler PJ, Hund TJ, Anderson ME. Oxidized CaMKII
causes cardiac sinus node dysfunction in mice. J Clin Invest. 2011;121:3277-3288.

8. Erickson JR, He BJ, Grumbach IM, Anderson ME. CaMKII in the cardiovascular system: sensing redox states. Physiol Rev. 2011;91:889-915.
9. Chelu MG, Sarma S, Sood S, Wang S, van Oort RJ, Skapura DG, Li N, Santonastasi M, Müller FU, Schmitz W, Schotten U, Anderson ME, Valderrábano M, Dobrev D, Wehrens XHT. Calmodulin kinase II-mediated sarcoplasmic reticulum Ca2+ leak promotes atrial fibrillation in mice. J Clin Invest. 2009;119:1940-1951.
15. Moskovitz J, Bar-Noy S, Williams WM, Requena J, Berlett BS, Stadtman ER. Methionine sulfoxide reductase (MsrA) is a regulator of antioxidant defense and lifespan in mammals. Proc Natl Acad Sci USA. 2001;98:12920-12925.
16. He BJ, Joiner M-LA, Singh MV, Luczak ED, Swaminathan PD, Koval OM, Kutschke W, Allamargot C, Yang J, Guan X, Zimmerman K, Grumbach IM, Weiss RM, Spitz DR, Sigmund CD, Blankesteijn WM, Heymans S, Mohler PJ, Anderson ME. Oxidation of CaMKII determines the cardiotoxic effects of aldosterone. Nat Med. 2011;17:1610-1618.
18. Neef S, Dybkova N, Sossalla S, Ort KR, Fluschnik N, Neumann K, Seipelt R, Schöndube FA, Hasenfuss G, Maier LS. CaMKII-dependent diastolic SR Ca2+ leak and elevated diastolic Ca2+ levels in right atrial myocardium of patients with atrial fibrillation. Circ Res. 2010;106:1134-1144.

19. Tessier S, Karczewski P, Krause EG, Pansard Y, Acar C, Lang-Lazdunski M, Mercadier JJ, Hatem SN. Regulation of the transient outward K+ current by Ca2+/calmodulin-dependent protein kinases II in human atrial myocytes. Circ Res. 1999;85:810-819.
22. Luo M, Guan X, Luczak ED, Lang D, Kutschke W, Gao Z, Yang J, Glynn P, Sossalla S, Swaminathan PD, Weiss RM, Yang B, Rokita AG, Maier LS, Efimov IR, Hund TJ, Anderson ME. Diabetes increases mortality after myocardial infarction by oxidizing CaMKII. J Clin Invest. 2013;123:1262-1274.
24. Wu Y, Gao Z, Chen B, Koval OM, Singh MV, Guan X, Hund TJ, Kutschke W, Sarma S, Grumbach IM, Wehrens XHT, Mohler PJ, Song L-S, Anderson ME. Calmodulin kinase II is required for fight or flight sinoatrial node physiology. Proc Natl Acad Sci USA. 2009;106:5972-5977.
25. Dzhura I, Wu Y, Colbran RJ, Balser JR, Anderson ME. Calmodulin kinase determines calcium-dependent facilitation of L-type calcium channels. Nat Cell Biol. 2000;2:173-177.
26. Koval OM, Guan X, Wu Y, Joiner ML, Gao Z, Chen B, Grumbach IM, Luczak ED, Colbran RJ, Song LS, Hund TJ, Mohler PJ, Anderson ME. CaV1.2 -subunit coordinates CaMKII triggered cardiomyocyte death and afterdepolarizations. Proc Natl Acad Sci USA. 2010;107:4996–5000.
44. Anderson ME. Multiple downstream proarrhythmic targets for calmodulin kinase II: moving beyond an ion channel-centric focus. Cardiovasc Res. 2007;73:657-666.

46. Chang HY, Lin YJ, Lo LW, Chang SL, Hu YF, Li CH, Chao TF, Yin WH, Chen SA. Sinus node dysfunction in atrial fibrillation patients: the evidence of regional atrial substrate remodelling. Europace. 2013;15:205-211.
47. Lee JMS, Kalman JM. Sinus node dysfunction and atrial fibrillation: two sides of the same coin? Europace. 2013;15:161-162.

Table 1. Summary of patient characteristics.
A. Patient characteristics for immunofluorescence studies in Figure 1A and B. B. Patient characteristics for immunoblotting experiments in Figure 1C-F.

Figures and/or Legends

The source of all the figures is from the circulation article – including supplementary.  Obtaining the images and presenting them in a cropped form was difficult.



Figure 1. ox-CaMKII is increased in atria from patients with Atrial Fibrillation (AF).
A. Representative immunofluorescence images using antiserum against ox-CaMKII in fixed sections of right atrial tissue from patients with sinus rhythm (SR) or AF. B. Image  quantification showing significantly higher ox-CaMKII in patients with AF compared to SR (*p<0.05, Student’s t-test). C. Representative immunoblots with ox-CaMKII antiserum in right atrial tissue homogenates from patients in SR or AF. D. Quantification of immunoblots showing significantly higher ox-CaMKII expression in patients with AF compared to SR (*p<0.05, Student’s t-test). The % value indicates the mean ox-CaMKII/GAPDH ratio as normalized to the mean ox-CaMKII/GAPDH ratio in the SR group. E. CaMKII antiserum in right atrial tissue homogenates from patients in SR or AF. F. Quantification of immunoblots showing similar total CaMKII expression in patients with AF and SR (p=0.3, Student’s t-tes )t . The % value indicates the mean CaMKII/GAPDH ratio as normalized to the me na CaMKII/GAPDH ratio in the SR group. The numerals shown in the bars indicate the sample size in each group, here and in subsequent figures.

Figure 2. Ang II treatment increases AF inducibility in WT mice.
A. Representative atrial (A-EGM) and ventricular (V-EGM) intracardiac electrograms and lead II surface ECG immediately after burst pacing show AF or SR in WT mice treated with Ang II or saline for 3 weeks. B. Contrasting R-R interval variability in AF and SR (C). Blue bars indicate calculated values from lead II ECGs shown in panel A. D. Higher AF inducibility in the Ang II treatment group (*p<0.05, Fisher’s exact test). E. Increase in systolic blood pressure (sBP) in WT mice after 3 

Figure 3. CaMKII oxidation is critical to Ang II mediated AF.
A. MM-VV, p47-/- and MsrA TG mice were resistant to Ang II mediated AF (*p<0.05 versus Ang II treated MM-VV, p47-/- and MsrA TG mice, Fisher’s exact test). B. All mice in panel A (WT, MM-VV, p47-/- and MsrA TG) showed a pressor response to Ang II. C. Ang II treatment induced cardiac hypertrophy as assessed by heart weight normalized to body weight (all comparisons versus saline controls from each genotype after 3 weeks of Ang II treatment(p< 0.05) (**p<0.01, Student’s t-test). The numerals shown in the graph indicate the number of mice in each group. F. Significantly higher echocardiographically estimated left ventricular (LV) mass in Ang II treated mice compared to saline controls (***p<0.001, Student’s t-test). G. Similar LV ejection fraction (LVEF) in Ang II and saline treated mice.  (** p<0.01 and ***p<0.001, Student’s t-test).

Figure 4. – ox-CaMKII in atria after Ang II or saline treatment
A. Atrial lys ate immunoblots from WT, MM-VV, p47 -/- and MsrA TG mice treated with Ang II or saline for 3 weeks and probed with an antiserum for ox-CaMKII. For quantification, ox-CaMKII bands were normalized to the total protein loading as assessed with Coomassie staining of the membrane. B. Increase in ox-CaMKII with Ang II treatment expressed as relative to the saline treated group. From each genotype 4 saline treated mice were used as controls. *p<0.05, for WT Ang II versus WT saline (*), in all other genotypes Ang II versus saline p>0.05; in addition, p=0.02 for WT Ang II versus MsrA TG Ang II and p=0.05 for MM-VV Ang II versus MsrA TG Ang II. C. Fold change in ox-CaMKII (over total CaMKII) in Ang II as relative to saline treated mice of the same genotype. From each genotype 4 saline treated mice were used as controls. ***p<0.001 versus WT saline, *p<0.05 versus MM-VV saline, #p<0.05 versus MsrA TG saline. WT Ang II versus p47-/- Ang II, P = 0.001, WT Ang II versus MsrA TG Ang II, P<0.0001, MM-VV Ang II versus MsrA TG Ang II, P=0.001. Data were analyzed using two-way ANOVA (for treatment and genotype) with Bonferroni post-hoc comparisons.

Figure 5. Ang II promotes Ca2+ sparks and DADs.
A. Representative examples of Ca2+ sparks in atrial myocytes from Ang II and saline treated WT and MM-VV mice. B. Summary of Ca2+ spark frequency data in atrial myocytes from Ang II treated mice compared to saline treated mice (*p<0.05 versus saline; Student’s t-test); WT saline (N=23 cells from 5 mice), WT Ang II (N=30 cells from 4 mice), MM-VV saline (N=36 cells from 4 mice) and MM-VV Ang II (N=28 cells from 4 mice). C. Examples of stimulated action potentials and a spontaneous, DAD triggered action potential. D. Higher incidence of DADs in atrial myocytes from Ang II treated WT mice ( *p<0.05 versus saline, Fisher’s exact test) but not in Ang II treated MM-VV mice compared to saline controls. Numerals show cells with DADs/total cells studied for each group.

Figure 6. CaMKII activation and RyR2 serine 2814 are required for AF in Ang II infused mice.
A. AC3-I and S2814A mice were treated with Ang II for 3 weeks and then burst paced to induce AF. AC3-I and S2814A mice were resistant to Ang II mediated AF promotion compared to WT Ang II treated mice (*p<0.05 versus all, Fisher’s Exact test, N=number of mice tested in each group). B. AC3-I and S2814A mice show similar systolic blood pressure (sBP) elevation after treatment with Ang II. Final sBP measurements were performed on three consecutive days prior to AF induction as shown in panel A. The numerals in the graph indicate the number of mice in each group. C. Ang II treatment causes similar cardiac hypertrophy in AC3-I and S2814A mice compared to saline controls (***p<0.001 versus AC3-I saline and **p=0.01 versus S2814A saline).

Figure 7. Schematic to illustrate the proposed mechanism of AF in Ang II infused mice.
Ang II binding activates NADPH oxidase (NOX) to increase reactive oxygen species (ROS), leading to oxidation of methionines 281/282 in CaMKII (ox-CaMKII). Elevated ox-CaMKII phosphorylates serine 2814 on RyR2, causing enhanced diastolic Ca2+ leak that promotes AF triggering DADs. Genetically modified mice were used to test key steps of the proposed pathway.

Additional Comments

This paper might be considered and compared with other papers in this series.

I Contributions to cardiomyocyte interactions and signaling

Author and Curator: Larry H Bernstein, MD, FCAP and  Curator: Aviva Lev-Ari, PhD, RN
This is a review of left ventricular cardiac hypertrophy and interaction with heparin-binding EGF,  based on work in the laboratory of Richard Lee, at Brigham and Women Hospital, Harvard Medical School, and MIT, titled…

Cardiomyocyte hypertrophy and degradation of connexin43 through spatially restricted autocrine/paracrine heparin-binding EGF

J Yoshioka, RN Prince, H Huang, SB Perkins, FU Cruz, C MacGillivray, DA Lauffenburger, and RT Lee *Cardiovascular Division, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA; and Biological Engineering Division, MIT, Cambridge, MA
PNAS 2005; 302(30):10622-10627.  http://pnas.org/cgi/doi/10.1073/pnas.0501198102

Growth factor signaling can affect tissue remodeling through autocrine/paracrine mechanisms. Recent evidence indicates that EGF receptor transactivation by heparin-binding EGF (HB-EGF) contributes to hypertrophic signaling in cardiomyocytes. Here, we show that HB-EGF operates in a spatially restricted circuit in the extracellular space within the myocardium, revealing the critical nature of the local microenvironment in intercellular signaling. This highly localized microenvironment of HB-EGF signaling demonstrated with 3D morphology, consistent with predictions from a computational model of EGF signaling. HB-EGF secretion by a given cardiomyocyte in mouse left ventricles led to cellular hypertrophy and reduced expression of connexin43 in the overexpressing cell and in immediately adjacent cells but not in cells farther away.

!!.  Ca2+/calmodulin δ Dependent Protein Kinase Modulates Cardiac Ryanodine Receptor Phosphorylation and Sarcoplasmic Reticulum Ca2+ Leak in Heart Failure.

Xun Ai, JW Curran, TR Shannon, DM Bers and SM Pogwizd.   
Circ Res. 2005;97:1314-1322  http://dx.doi.org/10.1161/01.RES.0000194329.41863.89

This contribution is unique in establishing a relationship between Ca2+ sparks in abnormal release from sarcoplasmic reticulum via the ryanodine receptor (RyR2) in contractile dysfunction and arrhythmogenesis in heart failure.  This is based on decreased transient amplitude and SR Ca2+ load with increased Na+/Ca++ exchange, and in nonischemic heart failure in a rabbit model.  In this case – with HF, expression of RyR2 and FK-506 binding protein 12.6 (FKBP12.6) were reduced, whereas inositol trisphosphate receptor (type 2) and Ca/calmodulin–dependent protein kinase II (CaMKII) expression were increased 50% to 100%.  In this study, the arrhythmogenesis appears to be ventricular.

Contractile dysfunction in HF is caused by diminished sarcoplasmic reticulum (SR) Ca load that could arise from enhanced activity of Na/Ca exchange (NCX), reduced SR Ca ATPase (SERCA) function, and increased diastolic SR Ca leak via ryanodine receptors (RyR), all of which we have demon¬strated to occur in our arrhythmogenic rabbit model of nonis-chemic HF. HF is also associated with a nearly 50% incidence of sudden cardiac death from ventricular tachycardia (VT) that degenerates to ventricular fibrillation (VF). In 3D cardiac mapping studies in our HF rabbit model, we showed that spontaneously occurring VT initiates by nonreentrant mechanisms associated with delayed afterdepolarizations. These arise from spontaneous SR Ca release that activates a transient inward current (Iti) carried primarily by NCX.2 Thus abnormal SR Ca release via RyR may contribute to both contractile dysfunction and arrhythmogenesis.

Abnormal release of Ca from sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) may contribute to contractile dysfunction and arrhythmogenesis in heart failure (HF). We previously demonstrated decreased Ca transient amplitude and SR Ca load associated with increased Na/Ca exchanger expression and enhanced diastolic SR Ca leak in an arrhythmogenic rabbit model of nonischemic HF. Here we assessed expression and phosphorylation status of key Ca handling proteins and measured SR Ca leak in control and HF rabbit myocytes. With HF, expression of RyR2 and FK-506 binding protein 12.6 (FKBP12.6) were reduced, whereas inositol trisphosphate receptor (type 2) and Ca/calmodulin–dependent protein kinase II (CaMKII) expression were increased 50% to 100%. The RyR2 complex included more CaMKII (which was more activated) but less calmodulin, FKBP12.6, and phosphatases 1 and 2A. The RyR2 was more highly phosphorylated by both protein kinase A (PKA) and CaMKII. Total phospholamban phosphorylation was unaltered, although it was reduced at the PKA site and increased at the CaMKII site. SR Ca leak in intact HF myocytes (which is higher than in control) was reduced by inhibition of CaMKII but was unaltered by PKA inhibition. CaMKII inhibition also increased SR Ca content in HF myocytes. Our results suggest that CaMKII-dependent phosphorylation of RyR2 is involved in enhanced SR diastolic Ca leak and reduced SR Ca load in HF, and may thus contribute to arrhythmias and contractile dysfunction in HF. (Circ Res. 2005;97:1314-1322.)

Key Words: ryanodine receptor -CaMKII -phosphorylation -heart failure -arrhythmia

III.  The Fire From Within: The Biggest Ca2+ Channel Erupts and Dribbles  – Mark E. Anderson

Circ Res. 2005;97:1213-1215  http://dx.doi.org/10.1161/01.RES.0000196744.62327.36

Mark E. Andserson makes the point that CaMKII(δ) is the biggest calcium signaling channel, and it is pluripotent in the heart muscle.

The multifunctional Ca2+ and calmodulin (CaM)-dependent protein kinase II (CaMKII) is a serine threonine kinase that is abundant in heart where it phosphorylates Ca2+i homeostatic proteins. It seems likely that CaMKII plays an important role in cardiac physiology because these target proteins significantly overlap with the more extensively studied serine threonine kinase, protein kinase A (PKA), which is a key arbiter of catecholamine responses in heart. However, the physiological functions of CaMKII remain poorly understood, whereas the potential role of CaMKII in signaling myocardial dysfunction and arrhythmias has become an area of intense focus. CaMKII activity and expression are upregulated in failing human hearts and in many animal models of structural heart disease. CaMKII inhibitory drugs can pre-vent cardiac arrhythmias and suppress afterdepolarizations that are a probable proximate focal cause of arrhythmias in heart failure.

Cardiac contraction is initiated when Ca2+ current (ICa), through sarcolemmal L-type Ca2+ channels (LTCC), triggers RyR opening by a Ca2+-induced Ca2+ release (CICR) mechanism. LTCCs “face off” with RyRs across a highly ordered cytoplasmic cleft that delineates a kind of Ca2+ furnace during each CICR-initiated heart beat (Figure). CICR has an obvious need to function reliably, so it is astounding to consider how this feed forward process is intrinsically unstable. The increased instability of CICR in heart failure is directly relevant to arrhythmias initiated by afterdepolarizations. RyRs partly rely on a collaboration of Ca2+-sensing proteins in the SR lumen to grade their opening probability and the amount of SR Ca2+ release to a given ICa stimulus.

LTCCs and RyRs form the protein machinery for initiating contraction in cardiac and skeletal muscle, but in cardiac muscle communication between these proteins occurs without a requirement for physical contact. PKA is preassociated with LTCCs and RyRs, and PKA-dependent phosphorylation increases LTCC8 and RyR9opening. The resultant increase in Ca2+i is an important reason for the positive inotropic response to cathecholamines. The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated by increased Ca2+I, and so catecholamine stimulation activatesCaMKII in addition to PKA. In contrast to PKA, which is tightly linked to inotropy, CaMKII inhibition does not cause a reduction in fractional shortening during acute cate-cholamine stimulation in mice.

The key clinical phenotypes of contractile dysfunction and electrical instability in heart failure involve problems with Ca2+i homeostasis. Broad changes in Ca2+I-handling proteins can occur in various heart failure models, but in general heart failure is marked by a reduction in the capacity for SR Ca2+ uptake, enhanced activity of the sarcolemmal Na+-Ca2+ exchanger, and reduction in CICR-coordinated SR Ca2+ release. On the other hand, the opening probability of individual LTCCs is increased in human heart failure.

The Marks group pioneered the concept that RyRs are hyperphosphorylated by PKA in patients with heart failure and showed that successful therapies, ranging from beta blockers to left ventricular assist devices, reduce RyR phosphorylation in step with improved mechanical function. They have developed a large body of evidence in patients and in animal models that PKA phosphorylation of Ser2809 on cardiac RyRs destabilizes binding of FK12.6 to RyRs and promotes increased RyR opening that causes an insidious Ca2+ leak. This leak is potentially problematic because it can reduce SR Ca2+ content (to depress inotropy), engage pathological Ca2+-dependent transcriptional programs (to promote myocyte hypertrophy), and activate arrhythmia-initiating af-terdepolarizations (to cause sudden death).


CIRCULATIONAHA_oxCaMKII_AF_AR_Image_535                  CIRCULATIONAHA_oxCaMKII_AF_AR_Image_534

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The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets

Author and Curator: Larry H Bernstein, MD, FCAP

Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC


Curator: Aviva Lev-Ari, PhD, RN


TAC – Transverse Aortic Constriction, AP, action potential; ARVD2, arrhythmogenic right ventricular cardiomyopathy type 2; CaMKII, Ca2+/calmodulim-dependent protein kinase II; CICR, Ca2+ induced Ca2+ release;CM, calmodulin; CPVT, catecholaminergic polymorphic ventricular tachycardia;  ECC, excitation–contraction coupling; FKBP12/12.6, FK506 binding protein; HF, heart failure; LCC, L-type Ca2+ channel;  P-1 or P-2, phosphatase inhibitor type-1 or type-2; PKA, protein kinase A; PLB, phosphoplamban; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A; RyR1/2, ryanodine receptor type-1/type-2; SCD, sudden cardiac death; SERCA, sarcoplasmic reticulum Ca2+ ATPase; SL, sarcolemma; SR, sarcoplasmic reticulum.

This is the Part IV of a series on the cytoskeleton and structural shared thematics in cellular movement and cellular dynamics. The last two are specific to the heart, and the third was renal tubular caicium exchange and the effects of Na+ and hormones.

In Part I, Identification of Biomarkers that are Related to the Actin Cytoskeleton


The prior articles discussed common management motifs across cell-types that are essential for cell division, embryogenesis, cancer metastasis, osteogenesis, musculoskeletal function, vascular compliance, and cardiac contractility.   This second article concentrates on specific functionalities for cardiac contractility based on Ca++ signaling in excitation-contraction coupling, addressing modifications specific to cardiac muscle and not to skeletal muscle.  In Part I there was discussion of the importance of Ca2+ signaling on innate immune system, and the roles of calcium in immunology will be further expanded in a third article of the series.

The Series consists of the following articles:

Part I: Identification of Biomarkers that are Related to the Actin Cytoskeleton

Larry H Bernstein, MD, FCAP


Part II: Role of Calcium, the Actin Skeleton, and Lipid Structures in Signaling and Cell Motility

Larry H. Bernstein, MD, FCAP, Stephen Williams, PhD and Aviva Lev-Ari, PhD, RN


Part III: Renal Distal Tubular Ca2+ Exchange Mechanism in Health and Disease

Larry H. Bernstein, MD, FCAP, Stephen J. Williams, PhD
 and Aviva Lev-Ari, PhD, RN


Part IV: The Centrality of Ca(2+) Signaling and Cytoskeleton Involving Calmodulin Kinases and Ryanodine Receptors in Cardiac Failure, Arterial Smooth Muscle, Post-ischemic Arrhythmia, Similarities and Differences, and Pharmaceutical Targets

Larry H Bernstein, MD, FCAP, Justin Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN


Part V: Ca2+-Stimulated Exocytosis:  The Role of Calmodulin and Protein Kinase C in Ca2+ Regulation of Hormone and Neurotransmitter

Larry H Bernstein, MD, FCAP
Aviva Lev-Ari, PhD, RN


Part VI: Calcium Cycling (ATPase Pump) in Cardiac Gene Therapy: Inhalable Gene Therapy for Pulmonary Arterial Hypertension and Percutaneous Intra-coronary Artery Infusion for Heart Failure: Contributions by Roger J. Hajjar, MD

Aviva Lev-Ari, PhD, RN


Part VII: Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmias and Non-ischemic Heart Failure – Therapeutic Implications for Cardiomyocyte Ryanopathy (Calcium Release-related Contractile Dysfunction) and Catecholamine Responses

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN


Part VIII: Disruption of Calcium Homeostasis: Cardiomyocytes and Vascular Smooth Muscle Cells: The Cardiac and Cardiovascular Calcium Signaling Mechanism

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN


Part IXCalcium-Channel Blockers, Calcium Release-related Contractile Dysfunction (Ryanopathy) and Calcium as Neurotransmitter Sensor

Justin Pearlman, MD, PhD, FACC, Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

Part X: Synaptotagmin functions as a Calcium Sensor: How Calcium Ions Regulate the fusion of vesicles with cell membranes during Neurotransmission

Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN


Part XI: Sensors and Signaling in Oxidative Stress

Larry H. Bernstein, MD, FCAP


Part XII: Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))

Larry H Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN


Observations of Tissues Dependent on Electrical Impulses and Differences in Calcium-Efflux Mechanisms

Voice of Justin Pearlman

Skeletal muscles are named for muscle bundles attached to skeleton elements, including head and neck,  thorax, and the long bones of limbs, but the same structural and neuronally controlled muscle type is also in the abdomenal wall and the scalp, face, and eyes (for eye motion), each serving the function of movement on demand. The skeletal element these muscles attach to are tendons (fibrous tissue), often anchored to bone before and after an articulation (joint). There are several features that distinguish skeletal muscle from smooth muscle and from myocardium (heart muscle). Skeletal muscles are striated. They have fast-twitch and slow-twitch fibers in various proportions. They are under voluntary neural control, not autonomic (involuntary).

In distinction, smooth muscles line arterial blood vessels, lymphatics, the urinary bladder, the gastrointestinal tract, the respiratory tract, and also the uterus, the pili of the skin (goose bumps), and are in the eyes to control pupil diameter and lens focus. They are controlled by autonomic innervation.

The myocardium, or heart muscle, is distinct in many ways. The heart muscle has a unique architecture with Z-bands. The heart muscle a syncytium of cardiac muscle made of cardiomyocytes, which means instead of a bundle of separate cells each distinctly bounded by a cell membrane, the entire heart muscle can be viewed as a single multinucleated cell (or merger of cells). Skeletal muscle has multinucleated cells also from the merger of multiple blast cells, but unlike the heart there are distinct cell boundaries between skeletal myocytes, known as myofibers. The heart has fiber layers with different orientations (spiral clockwise and counterclockwise arrangement of muscle fibers) that result in multiple types of motion, but technically all of the heart muscle fibers are part of a single conglomerate cell.   The motions of the heart include: translation, tilting, shortening, thickening, narrowing, twisting, rotating, lengthening and widening. The heart cell contracts and has innervation  to the AV node and the SA node, with both sympathetic and parasymptathetic innervation.

All three types of muscle apply a basic Motif of proteins that change length in response to a calcium signal. The calcium is stored is sacks called the sarcoplasmic reticulum. The calcium is released into the main fluid of the cell (the cytoplasm), where it controls different functions. Even in skeletal muscle there is a difference between thigh and thorax, and we know from comparative ornithology that the enzymology and energy metabolism of  the wings of birds that soar, hawks and eagles, differs from the chicken, or the turkey.

Key features are illustrated below.

Figure 1….. skeletal muscle vs heart  calcium channels.

receptors voltage gated Ca(2) channel

receptors voltage gated Ca(2) channel

We see in Figure 1 that both the skeletal muscle and the cardiomyocyte have a Ryanodyne receptor that is the flow device for carrying the Ca(2+) ions from the sarcoplasm into the cytoplasm.  In the skeletal muscle there is a dihydropyridine receptor.   The heart muscle is voltage gated.  The interaction with calmodulin (not shown) via Calcium/calmodulin-dependent  Protein Kinase Type II delta = CaMKI, II – IV.  CaMKII has isoforms a, b, c, d – and CaMKIId has two splice variants (cytoplasmic and nuclear).  These will be discussed fully in the fifth of the series.   Take note of the fact the CaMKII isoform is found only in the heart.  So we have here molecules with similar structure, but not completely homologous.  Structure and function have made small, requiring significant adaptations.

Figure 2.   A cardiomycyte structure with the sarcomere and calcium efflux into the cytoplasn, and with the mitochondrion available for Ca(2+) exchange with the cytoplasm, and with Ca(2+), Na(+) and K(+) channels contiguous with the extracellular space.



The arterial endothelium is functionally protected by eNOS converting arginine to citrulline.  This does not occur with adult form of urea cycle (Krebs Henseleit) disorder, as there is no substrate.  iNOS, a nitric oxide isoform present in macrophages that invade through intercellular spaces into the underlying matrix. A large study presented at the European Society of Cardiology (ESC) 2013 Congress has indicated that there is not a relationship of tight control of type 2 diabetes and cardiovascular events, even though we know that there is a relationship between diabetes and

  1. insulin resistance
  2. endothelial activation
  3. inflammatory markers
  4. homocysteine

Adipokines interact in type 2 diabetes with inflammatory cytokines for development of insulin resistance, and these are markers of arterial vascular disease.  But the association of diabetes with heart disease, long considered valid, has come into some dispute.  Recently, saxagliptin was associated with a significant 27% increased risk of hospitalizations for heart failure in the  Saxagliptin Assessment of Vascular Outcomes Recorded in Patients with Diabetes Mellitus (SAVOR-TIMI 53) study, a component of the prespecified secondary end point. In the Examination of Cardiovascular Outcomes with Alogliptin versus Standard of Care in Patients with Type 2 Diabetes Mellitus and Acute Coronary Syndrome (EXAMINE) study, there was no increased risk of heart failure with alogliptin.  While saxagliptin and alogliptin significantly reduced glycated hemoglobin levels, there was some debate about the role of the drugs, which are dipeptidyl peptidase-4 (DPP-4) inhibitors, in clinical practice.  There is some disappointment with respect to the diabetes issue, but that might be remedied by improvement based on the appropriate combination of biomarkers for prediction asnd monitoring at the earliest onset.  Dr William White said alogliptin lowers the glycemic index significantly, and such reductions can reduce the risk of microvascular complications. We know from the prior literature that it might take five years-plus before we determine a microvascular benefit.  A serious problem in the validity of the results was that statistically, saxagliptin met the primary end point of noninferiority, with the drug no worse than placebo. Glycated hemoglobin levels were reduced with saxagliptin, down from 8.0% at baseline to 7.7% at the end of the trial (p<0.001 vs placebo). In addition, more patients in the saxagliptin arm had glycated hemoglobin levels reduced to less than 7.0%.  The relevant question is what the effect was for patients who achieved a glycated Hb of < 7.7%, which makes the p-value meaningless for an 0.3% change overall.

Implications of ca(2+) handling dysfunction

A. if the dysfuction is in smooth muscle  – effect on arterial elasticity

B. if the dysfunction is in cardiomyocytes – Ventricular contractility & arrhythmias

We now review the calcium cycling of smooth muscle based on extracted work at MIT and Harvard Medical School, and at the University of Iowa.   The work focuses on the disordered Ca(2+) signaling that plays a large role in the development of “arterial stiffness”, not disregarding the competing roles of endothelial nitric oxide and the inflammatory cell mediated oxidative stress related iNOS in the arterial circulation, and the preference for stress points at the junction of arteries.   Disordered Ca(2+) in vascular smooth muscle leads to ischemic arterial disease, vascular rigidity from loss of flexibility, which can lead to ischemic myocardial damage.

Calcium Cycling in Synthetic and Contractile Phasic or Tonic Vascular Smooth Muscle Cells

L Lipskaia, I Limon, R Bobe and R Hajjar.

Chapter 2.  Intech Open. @2012.  http://dx.doi.org/10.5772/48240

Calcium ions (Ca2+) are present in low concentrations in the cytosol (~100 nM) and in high concentrations (in mM range) in both the extracellular medium and intracellular stores (mainly sarco/endo/plasmic reticulum, SR). This differential allows the calcium ion to be a ubiquitous 2nd messenger that carries information essential for cellular functions as diverse as contraction, metabolism, apoptosis, proliferation and/or hypertrophic growth. The mechanisms responsible for generating a Ca2+ signal greatly differ from one cell type to another. In the different types of vascular smooth muscle cells (VSMC), enormous variations do exist with regard to the mechanisms responsible for generating Ca2+ signal. In each VSMC phenotype (synthetic/proliferating1 and contractile2 [1], tonic or phasic), the Ca2+ signaling system is adapted to its particular function and is due to the specific patterns of expression and regulation of Ca2+ handling molecules (Figure 1).

1Synthetic VSMCs have a fibroblast appearance, proliferate readily, and synthesize increased levels of various extracellular matrix components, particularly fibronectin, collagen types I and III, and tropoelastin [1].

2Contractile VSMCs have a muscle-like or spindle-shaped appearance and well-developed contractile apparatus resulting from the expression and intracellular accumulation of thick and thin muscle filaments [1].

in contractile VSMCs, the initiation of contractile events is driven by membrane depolarization; and the principal entry-point for extracellular Ca2+ is the voltage-operated L-type calcium channel (LTCC). In contrast, in synthetic/proliferating VSMCs, the principal way-in for extracellular Ca2+ is the store-operated calcium (SOC) channel. Whatever the cell type, the calcium signal consists of limited elevations of cytosolic free calcium ions in time and space. The calcium pump, sarco/endoplasmic reticulum Ca2+ ATPase (SERCA), has a critical role in determining the frequency of SR Ca2+ release by controlling the velocity of Ca2+ upload into the sarcoplasmic reticulum (SR) and the Ca2+ sensitivity of SR calcium channels, Ryanodin Receptor, RyR and Inositol tri-Phosphate Receptor, IP3R.

Figure 1. Schematic representation of Calcium Cycling in Contractile and Proliferating VSMCs.

Schematic representation of Calcium Cycling in Contractile and Proliferating VSMCs

Schematic representation of Calcium Cycling in Contractile and Proliferating VSMCs

Left panel: schematic representation of calcium cycling in quiescent /contractile VSMCs. Contractile response is initiated by extracellular Ca2* influx due to activation of Receptor Operated Ca2* channels (through phosphoinositol-coupled receptor) or to activation of L-Type Calcium channels (through an increase in luminal pressure). Small increase of cytosolic due IP3 binding to IP3R (puff) or RyR activation by LTCC or ROC-dependent Ca2* influx leads to large SR Ca2* release due to the activation of IP3R or RyR clusters (“Ca2*-induced Ca2*release” phenomenon). Cytosolic Ca2* is rapidly reduced by SR calcium pumps (both SERCA2a and SERCA2b are expressed in quiescent VSMCs), maintaining high concentration of cytosolic Ca2* and setting the sensitivity of RyR or IP3R for the next spike. Contraction of VSMCs occurs during oscillatory Ca2* transient. Middle panel: schematic representation of atherosclerotic vessel wall. Contractile VSMC are located in the media layer, synthetic VSMC are located in sub-endothelial intima. Right panel: schematic representation of calcium cycling in quiescent /contractile VSMCs. Agonist binding to phosphoinositol-coupled receptor leads to the activation of IP3R resulting in large increase in cytosolic Ca2*. Calcium is weakly reduced by SR calcium pumps (only SERCA2b, having low turnover and low affinity to Ca2* is expressed). Store depletion leads to translocation of SR Ca2* sensor STIM1 towards PM, resulting in extracellular Ca2* influx though opening of Store Operated Channel (CRAC). Resulted steady state Ca2* transient is critical for activation of proliferation-related transcription factors ‘NFAT). Abbreviations: PLC – phospholipase C; PM – plasma membrane; PP2B – Ca2*/calmodulin-activated protein phosphatase 2B (calcineurin); ROC- receptor activated channel; IP3 – inositol-1,4,5-trisphosphate, IP3R – inositol-1,4,5-trisphosphate receptor; RyR – ryanodine receptor; NFAT – nuclear factor of activated T-lymphocytes; VSMC – vascular smooth muscle cells; SERCA – sarco(endo)plasmic reticulum Ca2* ATPase; SR – sarcoplasmic reticulum.

General aspects of calcium cycling and signaling in vascular smooth muscle cells

Besides maintaining vascular tone in mature vessels, VSMCs also preserve blood vessel integrity. VSMCs are instrumental for vascular remodeling and repair via proliferation and migration. Interestingly, Ca2* plays a central role in both physiological processes. In VSMCs, calcium signaling involves a cross-regulation of Ca2* influx, sarcolemmal membrane signaling molecules and Ca2* release and uptake from the sarco/endo/plasmic reticulum and mitochondria, which plays a central role in both vascular tone and integrity.

Calcium handling by the plasma membrane’s calcium channels and pumps

Membrane depolarization is believed to be a key process for the activation of calcium events in mature VSMCs. Thus, much attention has been given to uncovering the various mechanisms responsible for triggering this depolarization. Increased intra-vascular pressure of resistance arteries stimulates gradual membrane depolarization in VSMCs, increasing the probability of opening L-type high voltage-gated Ca2* channels (Cav1.2) (LTCC). Alternatively, the calcium-dependent contractile response can be induced through the activation of specific membrane receptors coupled to phospholipase C (PLC) isoforms3. The various isoforms of transient receptor potential (TRP) ion channel family, particularly TRPC3, TRPC6 and TRPC7 possibly activated directly by diacyl glycerol (DAG), can also contribute to initial plasma membrane Ca2* influx and subsequent membrane depolarization.

Among voltage-insensitive calcium influx pathways, the store-operated Ca2* channels (SOC), maintain a long-term cellular Ca2* signal. They are activated upon a decrease of internal store Ca2* concentration resulting from a Ca2* release via the opening of SR Ca2* release channels.   SOC has two essential regulatory components, the SR/ER located Ca2* sensor STIM1 (stromal interaction molecule) and the Ca2* channels Orai. Upon decrease of [Ca2*] in the reticulum (<500µM), Ca2* dissociates from STIM1; then STIM1 molecules oligomerize and translocate to specialized cortical reticulum compartments adjacent to the plasma membrane. There, the STIM1 cytosolic activating domains bind to and cluster the Orai proteins into an opened archaic Ca2* channel known as Ca2*-release activated Ca2* channel (CRAC).

  • All isoforms of PLC, catalyze the hydrolysis of phosphatidylinositol4,5-biphosphate (PIP2) to produce the intracellular messengers IP3 increase and diacylglycerol (DAG); both of which promote cytosolic Ca2* rise through activation of plasma membrane or sarcoplasmic reticulum calcium channels.
  •  The CRAC is responsible for the “2h cytosolic Ca2* increase” required to induce VSMCs proliferation.

The calcium signal is terminated by membrane hyper-polarization and cytosolic Ca2+ removal. First, calcium sparks resulting from the opening of sub-plasmalemmal clusters of RyR activate large-conductance Ca2+ sensitive K+ (BK) channels. Then, the resulting spontaneous transient outward currents (STOC) hyperpolarize the membrane and decrease the open probability of L-type Ca2+ channels. Cytosolic calcium is extruded at the level of plasma membrane by plasma membrane Ca2+ ATPase (PMCA) and the Na+/Ca2+ exchanger (NCX). The principal amount of cytosolic Ca2+ (> 70%) is re-uploaded to the internal store.

Calcium handling by the sarco/endoplasmic reticulum’s calcium channels and pumps

The initial entry of Ca2+ through plasma membrane channels triggers large Ca2+ release from the internal store via the process of Ca2+-induced Ca2+-release (CICR). The mechanism responsible for initiating Ca2+ release depends on Ca2+ sensitive SR calcium channels, the ryanodin receptor (RyR)5 or the IP3 receptor (IP3R). Indeed, IP3R and RyR are highly sensitive to cytosolic Ca2+ concentrations and when cytosolic Ca2+ concentration ranges from nM to µM, they open up. On the contrary, a higher cytosolic Ca2+ concentration (from µM to mM) closes them. In other words, cytosolic Ca2+ increase first exerts a positive feedback and facilitates SR channels opening whereas a further increase has an opposite effect and actually inhibits the SR channels opening. Importantly enough to be mentioned, RyR phosphorylation by the second messenger cyclic ADP ribose (cADPR) and protein kinase A (PKA) enhances Ca2+ sensitivity, the phosphorylation induced by the protein kinase C (PKC) decreases RyR sensitivity to Ca2+.

Sarco/Endoplasmic Ca2+ATPases (SERCA), the only calcium transporters expressed within sarco/endoplasmic reticulum (SR), serve to actively return calcium into this organelle. In mammals, three SERCA genes ATP2A1, ATP2A2 and ATP2A3 coding for SERCA1, SERCA2 and SERCA3 isoforms respectively have been identified [35]. Each gene gives rise to a different SERCA isoform through alternative splicing (Figure 2); they all have discrete tissue distributions and unique regulatory properties, providing a potential focal point within the cell for the integration of diverse stimuli to adjust and fine-tune calcium homeostasis in the SR/ER. In VSMCs, SERCA2a and the ubiquitous SERCA2b isoforms are expressed; besides vascular smooth muscle, SERCA2a is preferentially expressed in cardiac and skeletal muscles. SERCA2b differs from SERCA2a by an extension of 46 amino acids.  Diversity of SERCA isoforms in the same cell suggests that each of them could be responsible for controlling unique cell functions.

  • RyR are structurally and functionally analogous to IP3R, although they are approximately twice as large and have twice the conductance of IP3R [27]; RyR channels are sensitive to store loading and IP3R channels are sensitized by the agonist-dependent formation of IP3.

SERCA2’s activity depends on its interaction with phospholamban and is inhibitory in its de-phosphorylated form. PKA phosphorylation of phospholamban results in its dissociation from SERCA2, thus activating the Ca2+ pumps. Cyclic ADP-ribose was also reported to stimulate SERCA pump activity.

As previously mentioned, SR Ca2+ content controls the sensitivity of SR Ca2+ channels, RyR and IP3R, as well as functioning of SOC-mediated Ca2+ entry, thereby determining the type of intracellular calcium transient. Since SOCs opening depends on Ca2+ content of the store, one may suggest that SERCA participates to its regulation. Consistent with this, SOCs open up when the leak of Ca2+ from intracellular stores is not compensated with SERCA activity; SERCA inhibitors such as thapsigargin which prevent Ca2+ uptake are commonly used to chemically induce SOC currents; several works have established that SERCA can cluster with STIM1 and Orai1 in various cellular types.

Mechanisms of cytosolic Ca2+ oscillations in VSMC

Ca2+ oscillations are one of the ways that VSMCs respond to agonists. These Ca2+ oscillations are maintained during receptor occupancy and are driven by an endogenous pacemaker mechanism, called the cellular Ca2+ oscillator. Ca2+ oscillators were classified into two main types, the membrane oscillators and the cytosolic oscillators.

Membrane oscillators are those which generate oscillations at the cell membrane by successive membrane depolarization. In most small resistance arteries, inhibitors of plasma membrane voltage-dependent channels reduce or even abolish the membrane potential oscillations which precede rhythmical contractions. This suggests that rhythmic extracellular Ca2+ influx can be required for calcium oscillatory transient. Besides, membrane oscillators greatly depend on Ca2+ entry in order to provide enough Ca2+ to charge up the intracellular stores for each oscillatory cycle.

Cytosolic oscillators do not depend on the cell membrane to generate oscillations. Instead, they arise from intracellular store membrane instability. The pacemaker mechanism of cytosolic Ca2+ oscillator is based on the velocity of luminal Ca2+ loading and luminal Ca2+ content. The mechanism responsible for initiating Ca2+ release depends either on RyRs or IP3R activation. As soon as stores are sufficiently charged with Ca2+, the SR Ca2+ channels become sensitive to cytosolic Ca2+ and can participate to the process of Ca2+-induced Ca2+-release, which is responsible for orchestrating the regenerative release of Ca2+ from the SR/ER. Importantly, extracellular Ca2+ influx is not required for cytosolic oscillator function. Indeed, the Ca2+ oscillations can be observed in the absence of extracellular Ca2+.

In mature vessels, VSMCs mainly exhibit a tonic or phasic contractile phenotype. In contractile VSMCs extracellular calcium influx predominantly takes place through the voltage-dependent L-type calcium channel, LTCC9 (Figure 3). Extracellular Ca2* influx causes a small increase of cytosolic Ca2* generated by the opening of IP3R clusters, called puff and/or RyR2 clusters, called spark. These local rises of cytosolic Ca2* generate a larger SR Ca2* release through the Ca2*-induced Ca2* release phenomenon. Elevation of free cytosolic calcium triggers VSMC contraction.

  • In contractile VSMCs, NFAT can be activated by sustained Ca2* influx (persistent Ca2* sparklets) mediated by clusters of L-type Ca2* channels operating in a high open probability mode

Steady state increase in cytosolic Ca2* triggers tonic contraction; oscillatory type of Ca2* transient triggers phasic contraction. It is worth mentioning that accumulating evidence indicate that SR Ca2*ATPase functioning/location within the cell (which greatly influences the velocity of calcium upload) determines the mode of Ca2* transient in VSMCs. Consistent with this, i) “phasic” VSMCs display a greater number of peripherally located SR than “tonic” VSMCs; indeed “tonic” VSMCs exhibit centrally located SR; (rev in [61, 77]); ii) drugs which interfere with the IP3 pathway or intracellular stores abolish spontaneous vaso-motion; iii) blocking SERCA strongly inhibits the Ca2* oscillations, demonstrating that they are induced by SR Ca2* release; this latter argument is further supported by the fact that oscillations are present even in the absence of extracellular Ca2*

SERCA2a has a higher catalytic turnover when compared to SERCA2b due to a higher rate of de-phosphorylation and a lower affinity for Ca2+; ii) SER-CA2a is absent in synthetic VSMCs, which only exhibit tonic contraction, iii) transferring the SERCA2a gene to synthetic cultured VSMCs modifies the agonist-induced calcium transient from steady-state to oscillatory mode. Therefore, one might suggest that the physiological role of SERCA2a in VSMCs consists of controlling the “cytosolic oscillator”, thereby determining phasic vs tonic type of smooth muscle contraction.

SERCA2a as a potential target for treating vascular proliferative diseases

Abundant proliferation of VSMCs is an important component of the chronic inflammatory response associated to atherosclerosis and related vascular occlusive diseases (intra-stent restenosis, transplant vasculopathy, and vessel bypass graft failure). Great efforts have been made to prevent/reduce trans-differentiation and proliferation of synthetic VSMCs. Anti-proliferative therapies including the use of pharmacological agents and gene therapy approaches are, until now, considered as a suitable approach in the treatment of these disorders. Indeed, coronary stenting is the only procedure that has been proven to reduce the incidence of late restenosis after percutaneous transluminal coronary angioplasty. Nevertheless, post-interventional intra-stent restenosis, characterized by the re-narrowing of the arteries caused by VSMC proliferation, occurs in 10 to 20 % of patients. These disorders remain the major limitation of revascularization by percutaneous transluminal angioplasty and artery bypass surgery. The use of drug-eluting stents (stent eluting anti-proliferative drug) significantly reduces restenosis but impairs the re-endothelialization process and subsequently often induces late thrombosis. In human, trans-differentiation of contractile VSMCs towards a synthetic/proliferating inflammatory/migratory phenotype after percutaneous transluminal angioplasty appears to be a fundamental process of vascularproliferative disease.

Concluding remarks

Over the last decade, great progress has been made in the understanding of the various intracellular molecular mechanisms in VSMCs which control calcium cycling and excitation/contraction or excitation/transcription coupling. VSMCs employ a great variety of Ca2+ signaling systems that are adapted to control their different contractile functions. Alterations in the expressions of Ca2+ handling molecules are closely associated with VSMC phenotype modulation. Furthermore, these changes in expression are inter-connected and each acquired or lost Ca2+ signaling molecule represents a component of signaling module functioning as a single unit.

In non-excitable synthetic VSMCs, calcium cycling results from the protein module ROC/IP3R/STIM1/ORAI1 which controls SOC influx. Agonist stimulation of synthetic VSMCs translates into a sustained increase in cytosolic Ca2+. This increase is required for the activation of NFAT downstream cellular signaling pathways inducing proliferation, migration and possibly an inflammatory response. Calcium cycling in excitable contractile VSMCs is governed by the protein module composed of ROC/LTCC/RyR2/SERCA2a and controls the contractile response.

Author details
Larissa Lipskaia
Mount Sinai School of Medicine, Department of Cardiology, New York, NY, USA

Isabelle Limon
Univ Paris 6, UR4 stress inflammation and aging, Paris, France


BK – large-conductance Ca2+ sensitive K+ channel; cADPR – cyclic Adenosine Diphosphate Ribose; CICR – Ca2+- Induced Ca2+ Release; CRAC – Ca2+- Release Activated Ca2+ Channels; DAG – Diacyl Glycerol; IP3R – sarco/endoplasmic reticulum Ca2+ channel Inositol tri-Phosphate Receptor; LTCC – voltage-dependent L-type Ca2+ channels; NCX – Na+/Ca2+ exchanger; PKA – Protein Kinase A (activated by cAMP, cyclic adenosine monophosphate); PLC – Phospholipase C; PMCA – Plasmic Membrane Ca2+ ATPase; RyR – sarco/endoplasmic reticulum Ca2+ channel Ryanodin Receptor

B.  cardiomyocyte or smooth muscle.  Let’s look a little further.

CaM kinase  and disordering of intracellular calcium homeostasis , molecular link to arrhythmias

Mark E. Anderson, MD, PhD, Professor of Medicine and Pharmacology, University of Iowa, Iowa City, IADr. Anderson has presented a large body of work done at Vanderbilt University and University of Iowa Medical Schools for over a decade.  The major hypothesis is that in the aftermath of a heart attack, the structural and electrical remodeling renders the heart prone to arrhythmias .  The signaling molecule called calmodulin (CaM) kinase is a key and the work suggests that drugs that block CaM kinase activity might make good anti-arrhythmic medications.  CaM kinase is a molecule that is intricately involved in calcium signaling and regulation.  CaM kinase regulates calcium entry into the cell and calcium storage and release inside the cell.

Calcium enters heart cells through proteins called L-type calcium channels, donut-like pores in the cell membrane that open and close. If these channels stay open and let too much calcium into the cell, the risk of arrhythmia increases. Studies have shown that CaM kinase activity is increased in animal models and human heart disease.  Dr. Anderson poses the question – does CaM kinase — which we know is elevated in heart disease — drive arrhythmias?  The question is driven by their findings that the addition of activated CaM kinase allowed more calcium than normal to flow into isolated heart cells. The investigators measured the opening and closing of single calcium channels using a technique called patch-clamp electrophysiology. Then they added an already-activated form of CaM kinase to the preparation.  When we added the activated CaM kinase, the calcium channels opened like crazy,” Anderson said. “In fact, they were more likely to open and stay open for long periods of time.

They also showed that cardiac cells with added CaM kinase had electrical changes called early afterdepolarizations (EADs). EADs are believed to be the triggering cause of arrhythmias in cardiomyopathy, hypertrophy, and long QT syndrome.  The investigators implanted tiny telemeters into the mice and recorded electrocardiograms (ECGs) , which revealed not only the electrical changes expected in diseased hearts, Anderson said, but also an increased tendency for arrhythmias.  Next, they treated the mice  with a drug that blocks CaM kinase activity significantly suppressed the arrhythmias.  They also found that cardiac cells isolated from the mice and found spontaneous EADs, which disappeared when the cells were treated with the CaM kinase-blocking drug. The evidence all points to CaM kinase driving arrhythmias.

They have demonstrated that CaM kinase is also important for calcium-activated gene expression and that it may be involved in the changes that occur in association with cardiac hypertrophy and heart failure.  Anderson suggests that CaM kinase could be the link to explain why calcium channels open more frequently in heart failure, why people in heart failure have arrhythmias.  He postulates that it would good to have a target that addresses both phenotypic disorders — the arrhythmia phenotype and the heart failure phenotype — and CaM kinase may be that target.  Further, he observes that with the exception of so-called beta blockers, none of the current anti-arrhythmic drugs have been shown to reduce the mortality rate.  More recent work in Iowa has identified a new link – a link between the inflammation in heart muscle following a heart attack and the enzyme calcium/calmodulin-dependent protein kinase II or CaM kinase II.

CaM kinase II, a pivotal enzyme that registers changes in calcium levels and oxidative stress and translates these signals into cellular effects, including changes in heart rate, cell proliferation and cell death. CaM kinase II also regulates gene expression — which genes are turned on or off at any given time. We have seen how Inhibition of CaM kinase II in mice protects the animals’ hearts against some of the damaging effects of a heart attack.  A study compared a large number of genes that were expressed in the protected mice compared to the non-protected control mice. A particularly interesting finding was that a cluster of inflammatory genes was differently expressed depending on whether CaM kinase II was active or inhibited. Specifically, the research showed that heart attack triggered increased expression of a set of pro-inflammatory genes, and inhibition of CaM kinase II substantially reduced this effect.

The main research themes pursued by the Anderson laboratory are

  1. Oxidative activation of CaMKII;
  2. CaMKII signaling to ion channels;
  3. The role of CaMKII in inflammation;
  4. The role of CaMKII in cardiac pacemaker cells;
  5. The role of CaMKII in cell survival.

Keywords:  Calcium-Calmodulin-Dependent Protein Kinase Type 2, Calcium, Calcium-Calmodulin-Dependent Protein Kinases, Calcium Channels, L-Type, Calmodulin, Arrhythmia, Ion channel, Hypertrophy, Cell Signaling, Signal Transduction

Regulation of cardiac ATP-sensitive potassium channel surface expression by calcium/calmodulin-dependent protein kinase II.
Ana Sierra; Asipu Sivaprasadarao; Peter M Snyder; Ekaterina Subbotina; Michel Vivaudou; Zhiyong Zhu; Leonid V Zingman; et al.

Differential regulated interactions of calcium/calmodulin-dependent protein kinase II with isoforms of voltage-gated calcium channel beta subunits.
Grueter, CE, Abiria, SA, Wu, Y, Anderson, ME, Colbran, RJ.
Biochemistry, 47(6), 1760-7, 2008.

Differential effects of phospholamban and Ca2+/calmodulin-dependent kinase II on [Ca2+]i transients in cardiac myocytes at physiological stimulation frequencies.
Werdich, AA, Lima, EA, Dzhura, I, Singh, MV, Li, J, Anderson, ME, Baudenbacher, FJ.
Am J Physiol Heart Circ Physiol, 294(5), H2352-62, 2008.

Conserved Regulation of Cardiac Calcium Uptake by Peptides Encoded in Small Open Reading Frames

Emile G. Magny1, Jose Ignacio Pueyo1, Frances M.G. Pearl1,2, MA Cespedes1, et al.
1 School of Life Sciences, University of Sussex, Falmer, Brighton, East Sussex, UK.
2 Institute of Cancer Research, Sutton, Surrey SM2 5NG, UK Science

Small Open Reading Frames (smORFs) are short DNA sequences able to encode small peptides of less than 100 amino acids. Study of these elements has been neglected despite thousands existing in our genomes. We and others showed previously that peptides as short as 11 amino acids are translated and provide essential functions during insect development. Here, we describe two peptides of less than 30 amino acids regulating calcium transport in the Drosophila heart influencing regular muscle contraction. These peptides seem conserved for more than 550 million years in a range of species from flies to humans, where they have been implicated in cardiac pathologies. Such conservation suggests that the mechanisms for heart regulation are ancient and that smORFs may be a fundamental genome component that should be studied systematically.

Excitation-contraction coupling in the heart: the state of the question.

MD Stern, EG Lakatta
Laboratory of Cardiovascular Science, National Institute on Aging, NIH, Baltimore, Md.
The FASEB Journal (impact factor: 5.71). 10/1992; 6(12):3092-100.
Source: PubMed

Recent developments have led to great progress toward determining the mechanism by which calcium is released from the sarcoplasmic reticulum in the heart. The data support the notion of calcium-induced calcium release via a calcium-sensitive release channel. Calcium release channels have been isolated and cloned. This situation creates a paradox, as it has also been found that calcium release is smoothly graded and closely responsive to sarcolemmal membrane potential, properties that would not be expected of calcium-induced calcium release, which has intrinsic positive feedback. There is, therefore, no quantitative understanding of how the properties of the calcium release channel can lead to the macroscopic physiology of the whole cell. This problem could, in principle, be solved by various schemes involving heterogeneity at the ultrastructural level. The simplest of these require only that the sarcolemmal calcium channel be located in close proximity to one or more sarcoplasmic reticulum release channels. Theoretical modeling shows that such arrangements can, in fact, resolve the positive feedback paradox. An agenda is proposed for future studies required in order to reach a specific, quantitative understanding of the functioning of calcium-induced calcium release.

The role of protein kinases and protein phosphatases in the regulation of cardiac sarcoplasmic reticulum function

EG Kranias, RC Gupta, G Jakab, HW Kim, NAE Steenaart, ST Rapundalo
Molecular and Cellular Biochemistry 06/1988; 82(1):37-44. · 2.06 Impact Factor

Canine cardiac sarcoplasmic reticulum is phosphorylated by adenosine 3,5-monophosphate (cAMP)-dependent and by calcium calmodulin-dependent protein kinases on a 27 000 proteolipid, called phospholamban. Both types of phosphorylation are associated with an increase in the initial rates of Ca(2+) transport by SR vesicles which reflects an increased turnover of elementary steps of the calcium ATPase reaction sequence. The stimulatory effects of the protein kinases on the calcium pump may be reversed by an endogenous protein phosphatase, which can dephosphorylate both the CAMP-dependent and the calcium calmodulin-dependent sites on phospholamban. Thus, the calcium pump in cardiac sarcoplasmic reticulum appears to be under reversible regulation mediated by protein kinases and protein phosphatases.

Regulation of the cardiac ryanodine receptor channel by luminal Ca2+ involves luminal Ca2+ sensing sites

I Györke, S Györke
Biophysical Journal 01/1999; 75(6):2801-10. · 3.65 Impact Factor

The mechanism of activation of the cardiac calcium release channel/ryanodine receptor (RyR) by luminal Ca(2+) was investigated in native canine cardiac RyRs incorporated into lipid bilayers in the presence of 0.01 microM to 2 mM Ca(2+) (free) and 3 mM ATP (total) on the cytosolic (cis) side and 20 microM to 20 mM Ca(2+) on the luminal (trans) side of the channel and with Cs+ as the charge carrier. Under conditions of low [trans Ca(2+)] (20 microM), increasing [cis Ca(2+)] from 0.1 to 10 microM caused a gradual increase in channel open probability (Po). Elevating [cis Ca(2+)] above 100 microM resulted in a gradual decrease in Po. Elevating trans [Ca(2+)] enhanced channel activity (EC50 approximately 2.5 mM at 1 microM cis Ca2+) primarily by increasing the frequency of channel openings. The dependency of Po on trans [Ca2+] was similar at negative and positive holding potentials and was not influenced by high cytosolic concentrations of the fast Ca(2+) chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N, N-tetraacetic acid. Elevated luminal Ca(2+) enhanced the sensitivity of the channel to activating cytosolic Ca(2+), and it essentially reversed the inhibition of the channel by high cytosolic Ca(2+). Potentiation of Po by increased luminal Ca(2+) occurred irrespective of whether the electrochemical gradient for Ca(2+) supported a cytosolic-to-luminal or a luminal-to-cytosolic flow of Ca(2+) through the channel. These results rule out the possibility that under our experimental conditions, luminal Ca(2+) acts by interacting with the cytosolic activation site of the channel and suggest that the effects of luminal Ca2+ are mediated by distinct Ca(2+)-sensitive site(s) at the luminal face of the channel or associated protein.

Contemporary Definitions and Classification of the Cardiomyopathies

AHA Scientific Statement: Council on Clin. Cardiol.; HF and Transplant. Committee; Quality of Care and Outcomes Res. and Functional Genomics and Translational Biology Interdisciplinary Working Groups; and Council on Epidemiology and Prevention
BJ Maron, Chair; JA Towbin; G Thiene; C Antzelevitch; D Corrado; D Arnett; AJ Moss; et al.
Circulation. 2006; 113: 1807-1816    http://dx.doi.org/10.1161/CIRCULATIONAHA.106.174287

Classifications of heart muscle diseases have proved to be exceedingly complex and in many respects contradictory. Indeed, the precise language used to describe these diseases is profoundly important. A new contemporary and rigorous classification of cardiomyopathies (with definitions) is proposed here. This reference document affords an important framework and measure of clarity to this heterogeneous group of diseases. Of particular note, the present classification scheme recognizes the rapid evolution of molecular genetics in cardiology, as well as the introduction of several recently described diseases, and is unique in that it incorporates ion channelopathies as a primary cardiomyopathy.

Ryanopathy: causes and manifestations of RyR2 dysfunction in heart failure

Belevych AE, Radwański PB, Carnes CA, Györke S.
College of Medicine, The Ohio State University, Columbus, OH.
Cardiovasc Res. 2013; 98(2):240-7.   http://dx.doi.org/10.1093/cvr/cvt024.
Epub 2013 Feb 12.  PMID: 23408344 PMCID: PMC3633158 [Available on 2014/5/1]

The cardiac ryanodine receptor (RyR2), a Ca(2+) release channel on the membrane of the sarcoplasmic reticulum (SR), plays a key role in determining the strength of the heartbeat by supplying Ca(2+) required for contractile activation. Abnormal RyR2 function is recognized as an important part of the pathophysiology of heart failure (HF). While in the normal heart, the balance between the cytosolic and intra-SR Ca(2+) regulation of RyR2 function maintains the contraction-relaxation cycle, in HF, this behaviour is compromised by excessive post-translational modifications of the RyR2. Such modification of the Ca(2+) release channel impairs the ability of the RyR2 to properly deactivate leading to a spectrum of Ca(2+)-dependent pathologies that include cardiac systolic and diastolic dysfunction, arrhythmias, and structural remodeling. In this article, we present an overview of recent advances in our understanding of the underlying causes and pathological consequences of abnormal RyR2 function in the failing heart. We also discuss the implications of these findings for HF therapy.

Up-regulation of Sarcoplasmic Reticulum Ca(2+) Uptake Leads to Cardiac Hypertrophy, Contractile Dysfunction and Early Mortality in mice deficient in CASQ2

Kalyanasundaram A, Lacombe VA, Belevych AE, Brunello L, Carnes CA, Janssen PM, … Gyørke S.
Department of Physiology and Cell Biology, College of Medicine, Ohio State University, Columbus, OH.
Cardiovasc Res. May 2013; 98(2):297-306.   http://dx.doi.org/10.1093/cvr/cvs334.  Epub 2012 Nov 6.

Aberrant Ca(2+) release (i.e. Ca(2+) ‘leak’) from the sarcoplasmic reticulum (SR) through cardiac ryanodine receptors (RyR2) is linked to heart failure (HF). Does SR-derived Ca(2+) can actually cause HF? We ask whether and by what mechanism combining dysregulated RyR2 function with facilitated Ca(2+) uptake into SR exacerbates abnormal SR Ca(2+) release and induces HF.

We crossbred mice deficient in expression of cardiac calsequestrin (CASQ2) with mice overexpressing the skeletal muscle isoform of SR Ca(2+)ATPase (SERCA1a). The new double-mutant strains displayed early mortality, congestive HF with left ventricular dilated hypertrophy, and decreased ejection fraction. Intact right ventricular muscle preparations from double-mutant mice preserved normal systolic contractile force but were susceptible to spontaneous contractions. Double-mutant cardiomyocytes while preserving normal amplitude of systolic Ca(2+) transients displayed marked disturbances in diastolic Ca(2+) handling in the form of multiple, periodic Ca(2+) waves and wavelets. Dysregulated myocyte Ca(2+) handling and structural and functional cardiac pathology in double-mutant mice were associated with increased rate of apoptotic cell death. Qualitatively similar results were obtained in a hybrid strain created by crossing CASQ2 knockout mice with mice deficient in phospholamban.

We demonstrate that enhanced SR Ca(2+) uptake combined with dysregulated RyR2s results in sustained diastolic Ca(2+) release causing apoptosis, dilated cardiomyopathy, and early mortality. Further, up-regulation of SERCA activity must be advocated with caution as a therapy for HF in the context of abnormal RyR2 function.

Comment in

Mind the store: modulating Ca(2+) reuptake with a leaky sarcoplasmic reticulum. [Cardiovasc Res. 2013] PMID: 23135969 [PubMed – in process] PMCID: PMC3633154 [Available on 2014/5/1]

Myocardial Delivery of Stromal Cell-Derived Factor 1 in Patients With Ischemic Heart Disease: Safe and Promising    Circ. Res.. 2013;112:746-747

Circulation Research Thematic Synopsis: Cardiovascular Genetics Circ. Res.2013;112:e34-e50,

Ryanodine Receptor Phosphorylation and Heart Failure: Phasing Out S2808 and ³Criminalizing² S2814 ,

Héctor H Valdivia
Center for Arrhythmia Research, University of Michigan, Ann Arbor, MI.
Circ. Res.. 2012;110:1398-1402   http://dx.doi.org/10.1161/CIRCRESAHA.112.270876 (IF: 9.49).

By the time the heart reaches the pathological state clinically recognized as heart failure (HF), it has undergone profound and often irreversible alterations in structure and function at the molecular, cellular and organ level. Although the etiologies of HF are diverse:

  • hypertension,
  • myocardial infarction,
  • atherosclerosis,
  • valvular insufficiency,
  • mutations in genes encoding sarcomeric proteins

Some alterations are commonly found in most forms of HF, and they may account for the maladaptive structural remodeling and systolic dysfunction that characterize this syndrome.

At the cellular level, there are well documented changes in

  • ionic channel density and function (electrical remodeling),
  • increased ROS production,
  • mitochondrial dysfunction,
  • imbalanced energy intake and consumption,
  • genetic reprogramming,
  • altered excitation-contraction coupling,

and in general, dysregulation of a multitude of other processes and pathways that are essential for proper cardiac function. Combined, this myriad of alterations leads to

  1. loss in contractility and
  2. loss ejection fraction,
  3. ventricular wall remodeling,
  4. increased vascular resistance, and
  5. dysregulated fluid homeostasis.

In this issue of Circulation Research, Respress et al.2 report that preventing phosphorylation of cardiac ryanodine receptors (RyR2) at a single residue, S2814, is sufficient to avert many of these alterations and improve cardiac function in HF. The results presented here follow a string of papers that touch on the delicate and controversial subject of ryanodine receptor phosphorylation and HF. They offer a new twist to a contentious story and attempt to reconcile many apparently contradicting results, but key issues remain.

Calcium “Leak” in HF

It appears that suppressing the dysfunction of a select group of biological and molecular signaling pathways may substantially improve or even reverse the cardiac deterioration observed in HF. For example, correcting the characteristically depressed sarcoplasmic reticulum (SR) calcium content of failing cardiomyocytes is a target of HF gene therapy. SR calcium “leak”, an operational term that indicates increased and untimely calcium release by RyR2s, also appears common to several models of HF. Therefore, stemming off calcium “leak” may prevent the progression of cardiac malfunction in HF patients. However, a rationalized therapy towards this aim must be founded on the precise knowledge of the mechanisms leading to calcium leak. Marks group, in a landmark publication in 2000 (ref. 6) and later in multiple other high-impact factor papers (many of them co-authored by Wehrens 7-10) postulated that RyR2 “hyperphosphorylation” at S2808 by PKA was the primary mechanism leading to increased calcium “leak” in HF. This idea was initially appealing and fueled intensive research in the subject, but many groups failed to reproduce central tenets of this hypothesis. (11 and 12)  The controversies surrounding the Marks-Wehrens hypothesis of increased calcium leak by hyperphosphorylation of RyR2-S2808 have been recently and comprehensibly reviewed by Bers.13  Here I will focus on the modifications to this hypothesis as derived from the new findings of Respress et al.2 Emerging points from these new findings will be the demotion of S2808, to intervene not as universal player in HF but only in selective forms of this syndrome, and the role of S2814 as pre-eminent generator of calcium leak that leads to arrhythmias and exacerbates other forms of HF. The “criminalization” of S2814 has begun in earnest.

CaMKII Effect on Calcium Leak and the Role of S2808 and S2814

Many studies have provided evidence that persistent CaMKII activity can lead to cardiac arrhythmias and promote HF.14-16 Animals and patients with congestive HF display increased levels of CaMKII,17,18 and overexpression of AC3-I, a peptide inhibitor of CaMKII, delays the onset of HF in mice.19 There is also good agreement4,20 (although not universal21) that CaMKII, and not PKA, increases calcium leak, and therefore, it is likely that the arrhythmogenic and deleterious activity of CaMKII in HF may be associated with this effect. Obviously, if PKA does not cause calcium leak directly, this by itself imposes insurmountable constraints on the Marks-Wehrens hypothesis that posits that PKA phosphorylation of RyR2-S2808 is responsible for the high calcium leak of HF. With the focus now on CaMKII, the obligated question is then, by what mechanisms CaMKII increases calcium leak from the SR? To increase calcium leak, the cell must either increase SR calcium content, and/or increase the activity of the RyR2 (albeit the latter alone would have only transient effects due to autoregulatory mechanisms22). Since both PKA and CaMKII increase SR calcium load by phosphorylating phospholamban (but at different residues) and relieving the inhibition it exerts on SERCA2a, the differential effect of these kinases must result from the regulation they exert on RyR2s. Wehrens group offers here2 at least a partial explanation of this complex mechanism and, along with previous papers co- authored with Marks, these groups set specific roles for S2808 and S2814 on regulation of RyR2 activity and their protective effect (or lack thereof) in HF. In their view, PKA exclusively phosphorylates S2808 and dissociates FKBP12.6, which destabilizes the closed state of the channel and increases RyR2 activity, whereas CaMKII (almost) exclusively phosphorylates S2814, has no effect on FKBP12.6 binding, and equally activates RyR2s. In this issue, Respress et al.2 report that preventing phosphorylation of S2814 (by genetic substitution of Ser by Ala, S2814A) protects against non-ischemic (pressure overload) HF but has no effect on ischemic HF; conversely, and against other data by the same groups, S2808 phosphorylation was not significantly different in non-ischemic HF, implying that it is relevant only in ischemic HF. This clean targeting of RyR2 phospho-epitopes by PKA and CaMKII and their nice “division of labor” for pathogenicity in distinct forms of HF would really simplify phosphorylation schemes and reconcile apparent contradictions. However, as is generally the case, the proposal appears oversimplified and almost too good to be true. Let’s discuss each of the premises on which the Respress et al.2 results have been interpreted and the problems associated with these premises.

One kinase = one site = one effect. Is it really that simple?

The RyR2 is a huge protein. It is assembled as a tetrameric complex of ~2 million Da, with each subunit composed of ~5,000 amino acids.

Using canonical phosphorylation consensus and high confidence values, the RyR2 may be phosphorylated in silico at more than 100 sites by the combined action of PKA,

  • CaMKII,
  • PKG, and
  • PKC, to name a few.11

Granted, a “potential” phosphorylation site is very different than a demonstrated, physiologically-relevant phosphorylation site and it is possible that many of the predicted residues are not phosphorylated in vivo. Even then, several groups have demonstrated that CaMKII phosphorylates RyR2 with stoichiometry of at least 3 or 4 to 1 with respect to PKA.23-26 This fact is by itself compelling evidence that there are multiple phosphorylation sites in RyR2. Now, let’s make the optimistic assumption that all the PKA sites have already been mapped, and that S2808 and S2030 (ref. 27) are the only PKA sites. Taking into account the CaMKII:PKA phosphorylation ratio (3:1 or 4:1), this would then yield a minimum of ~6 – 8 CaMKII phosphorylation sites (per channel subunit!). In this perspective, it is almost disingenuous to label S2808 as “the” PKA site, and we may purposely deceive ourselves when we label S2814 “the” CaMKII site. Against this sense of pessimism and intractability, let’s not forget that S2808 was actually discovered as a CaMKII site.24 It is possible then that the number of CaMKII sites is smaller if only S2030 remains as a bona fide PKA site. Still, neither scheme supports one CaMKII site per channel subunit.

But let’s go along for a moment with the possibility, however unlikely, that PKA phosphorylates S2808 only, and CaMKII phosphorylates S2814 only. When calling these sites by their distinctive numbers, it is easy to forget that these phospho-sites are only 6 residues apart, that is, a minuscule proportion (~0.000003%) in the context of the whole channel protein. How can the same reaction (phosphorylation) that occurs at sites so close to one another be differentially transmitted to the very distant gating domains of the channel? If these residues were lining the pore of the channel, where critical differences emerge by substituting one residue but not the neighboring one, then it would be easier to understand how S2808 and S2814 could transmit distinct signals. But both are part of a “phosphorylation hot spot”, a cytoplasmic loop that contains additional potential phospho-sites11 and that has been mapped to the external surface of the channel.28 Marks and Wehrens groups have shown that phosphorylation of S2808A by CaMKII or of S2814A by PKA fully activate the channel.7,9 At face value, this means that knocking out one phospho-residue does not cripple this “hot spot” and that phosphorylation of at least one residue in this external loop enables it to transmit conformational changes to the gating domains of the channel. Seen in this structural context in which the “hot spot” works in unison upon phosphorylation of at least one residue, it is very difficult (but not impossible) to accommodate the notion that phosphorylation of S2808 or S2814 alone dictates the differential response of the RyR2 to PKA and CaMKII.

An Alternative Model to explain Differential PKA and CaMKII Effects

An alternative model to explain the differential effect of PKA and CaMKII to elicit calcium leak from RyR2 that takes into account other phospho-sites is needed. Before formulating it, let’s consider some important points. First, it is not difficult to assume that the role of the “phosphorylation hot spot” is to readily pick up signals from different kinases. The multi-valence of this “hot spot” is demonstrated so far by the fact that S2808 may be phosphorylated by CaMKII24,25,26 and by PKA,6,25,26 and its eagerness to undergo phosphorylation by the fact that S2808 is at least ~50% phosphorylated even at basal state25-27,29,30 and phospho-signals from these sites may be readily detected upon β-adrenergic stimulation of the heart.30,31Second, if we accept the Shannon and Bers results that CaMKII, and not PKA, elicits calcium leak from the SR,4,20 this obligatorily means that PKA phosphorylation of S2808 is not responsible for eliciting calcium leak (in direct conflict with the Marks-Wehrens hypothesis). In support of this notion, studies by the Houser and Valdivia groups have provided evidence that preventing S2808 phosphorylation has negligible impact on the β-adrenergic response of the heart and on the progression of non-ischemic and ischemic HF.30-32 Third, another PKA site, S2030, largely ignored in the Marks-Wehrens scheme, has been mapped and shown to activate channel openings27 and although its place in the larger context of RyR2 phosphorylation has not been determined yet, I think it is illogical to assume that its existence is futile and that it contributes nothing to regulation of the channel. Thus, according to the preceding discussion, it is almost unsustainable to postulate that the differential effects of CaMKII and PKA to elicit calcium leak stems from their effects on the RyR2 “phosphorylation hot spot” alone. Instead, I would like to posit an alternative model that integrates findings by many of the above-referenced groups (Fig. 1). In this model, the surface domain of the RyR2 comprising residues 2804-2814 (mouse nomenclature) is an eager target for phosphorylation by PKA, CaMKII and probably other kinases (4 Ser/Thr).11,24-26,29 Phosphorylation of this “hot spot” by either PKA or CaMKII (or both) “primes” the RyR2 for subsequent signals and is probably responsible for the coordinated openings in response to fast calcium stimuli detected in single channel recordings33 and in cellular settings34 (but this has yet to be demonstrated). The differential effect of PKA and CaMKII on RyR2 activity would then depend on the integrated response of the phosphorylated “hot spot” and of additional phosphorylation sites. For example, phosphorylation of S2808 and S2030 by PKA could coordinate channel openings in response to fast calcium stimuli, and phosphorylation of S2814 and other CaMKII site(s) could open RyR2s at diastolic [Ca2+], which would translate in calcium leak. Examples of proteins acting as molecular switchboards in response to various degrees of phosphorylation are not unprecedented.35 In fact, RyR2s are activated by phosphorylation and dephosphorylation as well36,37 and their relative degree of phosphorylation determines a final functional output.38 It is therefore conceivable that the complex response of RyR2s to any type of phosphorylation and the variable results obtained by investigators apparently using the same experimental conditions may be due to the variable degree of phosphorylation in which the RyR2s were found. Of course, until the 3D structure of the RyR2 is solved and we understand the mechanism by which the “phosphorylation hot spot” and other phospho-sites “talk” to the channel’s gating domains this structurally-based model will remain speculative, but it at least takes into consideration compelling evidence on the existence of various phosphorylation sites and departs substantially from the simplified notion of one kinase = one site = one effect.

Fig. 1  Models of RyR2 modulation by phosphorylation

Marks-Wehrens Model and multiphosphorylation  site model

See –  Ryanodine Receptor Phosphorylation and Heart Failure – Phasing Out S2808 and “Criminalizing” S2814.  Héctor H. Valdiviahttp://circres.ahajournals.org/content/110/11/1398.full  www.ncbi.nlm.nih.gov/pmc/articles/PMC3386797

Models of RyR2 modulation by phosphorylation. In the Marks-Wehrens model (A), S2808 is the only site phosphorylated by PKA, and S2814 by CaMKII. PKA phosphorylation of S2808 dissociates FKBP12.6, which destabilizes the closed state of the channel and induces subconductance states, eliciting calcium leak. Calcium leak from the SR then causes deleterious effects such as arrhythmias and worsening of (ischemic) HF. CaMKII phosphorylation of S2814 does not dissociate FKBP12.6 but also causes calcium leak. This leak is also arrhythmogenic but is not relevant in ischemic HF, only in nonischemic HF. In the multiphosphorylation site model (B), S2808 and S2814 are part of a “phosphorylation hot spot” that is located in a protruding part of the channel, is targeted by several kinases, and may contain other phospho-epitopes not yet characterized. Phosphorylation of individual residues within this “hot spot” may be undistinguishable by the channel’s gating domains; instead, the differential regulation of PKA and CaMKII on channel gating may come about by the combined effect of each kinase on phospho-residues of the “hot spot” and other phosphorylation sites.

see- Is ryanodine receptor phosphorylation key to the fight or flight response and heart failure? Thomas Eschenhagen.  JCI 210; 120(12): 4197-4203.   http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2994341/

In situations of stress the heart beats faster and stronger. According to Marks and colleagues, this response is, to a large extent, the consequence of facilitated Ca2+ release from intracellular Ca2+ stores via ryanodine receptor 2 (RyR2), thought to be due to catecholamine-induced increases in RyR2 phosphorylation at serine 2808 (S2808). If catecholamine stimulation is sustained (for example, as occurs in heart failure), RyR2 becomes hyperphosphorylated and “leaky,” leading to arrhythmias and other pathology. This “leaky RyR2 hypothesis” is highly controversial. In this issue of the JCI, Marks and colleagues report on two new mouse lines with mutations in S2808 that provide strong evidence supporting their theory.


JCI45251.f1  classical view of cardiomyocyte excit-contraction coupling and nregulation by beta adrenergic receptors

In the signalling scheme outlined in  ​Figure1 of this commentary, which prevailed until the end of the last century, the two major determinants of intracellular Ca2+  transients and thereby the contractile force of the heart were (a) the size of the Ca2+ current entering via the LTCC (well exemplified by the negative inotropic effects of LTCC blockers) and (b) the activity of SERCA and thus the Ca2+ load of the SR. The critical role of the latter was convincingly demonstrated by the fact that Plb–/– mice, which have maximal SERCA activity, exhibit higher basal force and reduced inotropic response to isoprenaline (1).

See also Table 1


T1  hyperphosphorylation of RyR2 in heart failure and effect of beta adrenergic stimulation of FKBP12.6 binding

In the Marks-Wehrens model, S2808 is the only site phosphorylated by PKA, and S2814 by CaMKII. PKA phosphorylation of S2808 dissociates FKBP12.6, which destabilizes the closed state of the channel and induces subconductance states, eliciting calcium leak. Calcium leak from the SR then causes deleterious effects such as arrhythmias and worsening of (ischemic) HF. CaMKII phosphorylation of S2814 does not dissociate FKBP12.6 but also causes calcium leak. This leak is also arrhythmogenic, but is not relevant in ischemic HF, only in non-ischemic HF. In the multi-phosphorylation site model, S2808 and S2814 are part of a “phosphorylation hot spot” that is located in a protruding part of the channel, is targeted by several kinases, and may contain other phospho-epitopes not yet characterized. Phosphorylation of individual residues within this “hot spot” may be undistinguishable by the channel’s gating domains; instead, the differential regulation of PKA and CaMKII on channel gating may come about by the combined effect of each kinase on phospho-residues of the “hot spot” and other phosphorylation sites.

F1.large  calcium movement and RyR2 receptor F1.large   RyR unzipping ncpcardio0419-f4   calcium leak

Appealing as Marks’ theory is, the concept has been challenged and remains controversial  ​(Tables1 and ​2). On the one hand, some theoretical considerations argue against it. For example, it seems counterintuitive that phosphorylation at a single residue in a protein of more than 5,000 amino acids could profoundly affect channel open probability. Second, S2808, the proposed site of phosphorylation by PKA, is located in an area distant from the FKBP12.6/RyR2 interaction site (3), making it somewhat unlikely that phosphorylation affects FKPB12.6 binding. Third, it seems unlikely and to contradict experimental results (4) that an isolated increase in RyR2 open probability has more than a transient consequence on Ca2+ handling, because an isolated increase in Ca2+release from the RyR2 will automatically lead to reduced Ca2+ load in the SR and therefore fast normalization of Ca2+ transients (autoregulation).

More concerning than theoretical considerations are numerous reports that failed to reproduce important aspects of the data that support the leaky RyR2 hypothesis and the critical importance of S2808 (Tables ​(Tables11and ​and2).2). (a) Phosphorylation of RyR2 at S2808 has been found by others to be either not altered in heart failure at all or to be only moderately increased (58). Others have reported that 75% of the available RyR2 S2808 sites are phosphorylated under normal conditions, making a 9-fold change in chronic heart failure somewhat unlikely (9). (b) Whereas general consensus exists that β-adrenergic stimulation increases spontaneous Ca2+ release (the “Ca2+ leak”) from the SR, the role of RyR2 phosphorylation and FKBP12.6 dissociation remains controversial. Importantly, PKA had no effect on Ca2+release in permeabilized Plb–/– mouse myocytes, i.e., cells in which the SR is maximally loaded with Ca2+ and one would have expected a particularly strong effect of increasing RyR2 open probability.


Now, let’s go back to the results of Respress et al.2 and consider them in this light. They found that preventing phosphorylation of S2814 alone mitigates non-ischemic HF induced by transverse aortic constriction (TAC) in mice. This implies that other CaMKII sites are not necessary to mitigate the CaMKII-induced calcium leak that they propose is responsible for the deleterious effect in WT mice subjected to TAC. If phosphorylation of the “hot spot” is compulsory to prime the RyR2 to process and discriminate other phosphorylation signals, then other residues in that “hot spot” must have been phosphorylated to fulfill this need. Surprisingly, S2808 was not significantly phosphorylated in this setting. This leaves a very difficult conundrum: if S2808 was not phosphorylated significantly and the other CaMKII sites are not necessary to stop calcium leak, how then can we explain the results of Respress et al.2? Of course there are always alternatives, and we would be inconsistent if we rigidly adhere to one model and fell into the dogmatism we are criticizing. The conclusions of Respress et al.2 are in line with their findings, but at this point the numbers do not add up and it’s obvious that the great complexity of this process (RyR2 phosphorylation) precludes simplified and neatly organized schemes. As a clear example of this, in the landmark paper by Marks group,6 S2808 was found substantially hyperphosphorylated in tachypacing-induced failing dogs, also a non-ischemic model of HF. This does not fit well in the current scheme of Wehrens where S2808A protects against ischemic HF, but has no prominent role in non-ischemic HF.

Marks-Wehrens Model and multiphosphorylation  site model

In summary, CaMKII and PKA may have specific roles in calcium leak and, since they both increase SR calcium load, their differential effect likely resides on their effect on RyR2s. However, the effect of PKA- or CaMKII-phosphorylation of RyR2s does not appear solved yet. Starting in 2000 and up to the present day, Marks and Wehrens have provided high-quality data in prominent journals aggressively pursuing the notion that PKA phosphorylates S2808 only, that CaMKII phosphorylates S2814 only, and that these sites alone integrate multiple signals to open RyR2s. Many key aspects of their general hypothesis including dissociation of FKBP12.6 by PKA phosphorylation of S2808, subconductance states as hallmarks of phosphorylation, and the prominent role of S2808 as promoter of arrhythmias and HF have not been confirmed by several groups. The present paper by the Wehrens group modifies slightly the original claim that S2808 was involved in ischemic and non-ischemic forms of HF and continues to shift the lion’s share of pathogenicity to S2814. However, as discussed above, the Marks-Wehrens model largely ignores compelling data on the presence of multiple phosphorylation sites and the complexity they add to the finely graded response of RyR2s to phosphorylation.

2. Respress JL, van Oort RJ, Li N, Rolim N, Dixit S, Dealmeida A, Voigt N, Lawrence WS, Skapura DG, Skårdal K, Wisloff U, Wieland T, Ai X, Pogwizd SM, Dobrev D, Wehrens XH. Role of RyR2 Phosphorylation at S2814 During Heart Failure Progression. Circ Res. 2012;xx:xx–xx. [in the issue; printer, please update] [PMC free article] [PubMed]

6. Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N, Marks AR. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell. 2000;101(4):365–376. [PubMed]

7. Wehrens XH, Lehnart SE, Reiken S, Vest JA, Wronska A, Marks AR. Ryanodine receptor/calcium release channel PKA phosphorylation: a critical mediator of heart failure progression. Proc Natl Acad Sci U S A. 2006;103:511–518. [PMC free article] [PubMed]

36. Lokuta AJ, Rogers TB, Lederer WJ, Valdivia HH. Modulation of cardiac ryanodine receptors of swine and rabbit by a phosphorylation-dephosphorylation mechanism. J Physiol. 1995;487:609–622. [PMC free article] [PubMed]

37. Terentyev D, Viatchenko-Karpinski S, Gyorke I, Terentyeva R, Gyorke S. Protein phosphatases decrease sarcoplasmic reticulum calcium content by stimulating calcium release in cardiac myocytes. J Physiol. 2003;552(Pt 1):109–118. [PMC free article] [PubMed]

38. Carter S, Colyer J, Sitsapesan R. Maximum phosphorylation of the cardiac ryanodine receptor at Ser-2809 by protein kinase A produces unique modifications to channel gating and conductance not observed at lower levels of phosphorylation. Circ Res. 2006; 98:1506–1513. [PubMed]

The Cardiac Ryanodine Receptor (calcium release channel) – Emerging role in Heart Failure and Arrhythmia Pathogenesis

Cardiovasc Res (2002) 56 (3): 359-372.    http://dx.doi.org/10.1016/S0008-6363(02)00574-6

The cardiac sarcoplasmic reticulum calcium release channel, commonly referred to as the ryanodine receptor, is a key component in cardiac excitation–contraction coupling, where it is responsible for the release of calcium from the sarcoplasmic reticulum. As our knowledge of the ryanodine receptor has advanced an appreciation that this key E–C coupling component may have a role in the pathogenesis of human cardiac disease has emerged. Heart failure and arrhythmia generation are both pathophysiological states that can result from deranged excitation–contraction coupling. Evidence is now emerging that hyperphosphorylation of the cardiac ryanodine receptor is an important event in chronic heart failure, contributing to impaired contraction and the generation of triggered ventricular arrhythmias.

Furthermore the therapeutic benefits of β blockers in heart failure appear to be partly explained through a reversal of this phenomenon. Two rare inherited arrhythmogenic conditions, which can cause sudden death in children, have also been shown to result from mutations in the cardiac ryanodine receptor. These conditions,

  1. catecholaminergic polymorphic ventricular tachycardia and
  2. arrhythmogenic right ventricular cardiomyopathy (subtype 2),

further implicate the ryanodine receptor as a potentially arrhythmogenic substrate and suggest this channel may offer a new therapeutic target in the treatment of both cardiac arrhythmias and heart failure.

Protein phosphatases decrease sarcoplasmic reticulum calcium content by stimulating calcium release in cardiac myocytes

D Terentyev, S Viatchenko-Karpinski, I Gyorke, R Terentyeva and S Gyorke
Texas Tech University Health Sciences Center, Lubbock, TX
J Physiol 2003; 552(1), pp. 109–118.    http:/dx.doi.org/10.1113/jphysiol.2003.046367

Phosphorylation/dephosphorylation of Ca2+ transport proteins by cellular kinases and phosphatases plays an important role in regulation of cardiac excitation–contraction coupling; furthermore, abnormal protein kinase and phosphatase activities have been implicated in heart failure. However, the precise mechanisms of action of these enzymes on intracellular Ca2+ handling in normal and diseased hearts remains poorly understood. We have investigated the effects of protein phosphatases PP1 and PP2A on spontaneous Ca(2+) sparks and SR Ca(2+) load in myocytes permeabilized with saponin. Exposure of myocytes to PP1 or PP2A caused a dramatic increase in frequency of Ca(2+) sparks followed by a nearly complete disappearance of events. These effects were accompanied by depletion of the SR Ca(2+) stores, as determined by application of caffeine. These changes in Ca(2+) release and SR Ca(2+) load could be prevented by the inhibitors of PP1 and PP2A phosphatase activities okadaic acid and calyculin A. At the single channel level, PP1 increased the open probability of RyRs incorporated into lipid bilayers. PP1-medited RyR dephosphorylation in our permeabilized myocytes preparations was confirmed biochemically by quantitative immunoblotting using a phosphospecific anti-RyR antibody. Our results suggest that increased intracellular phosphatase activity stimulates RyR mediated SR Ca(2+) release leading to depleted SR Ca(2+) stores in cardiac myocytes.

In heart muscle cells, the process of excitation–contraction (EC) coupling is mediated by Ca(2+) influx through sarcolemmal L-type Ca(2+) channels activating Ca(2+) release channels (ryanodine receptors, RyRs) in the sarcoplasmicreticulum (SR). Once activated, the RyR channels allow Ca(2+) to be released from the SR into the cytosol to induce contraction. This mechanism is known as Ca(2+)-induced calcium release (CICR) (Fabiato, 1985; Bers, 2002).

During relaxation, most of the Ca(2+) is resequestered into the SR by the Ca(2+)-ATPase. The amount of Ca(2+) released and the force of contraction depend on the magnitude of the Ca(2+) trigger signal, the functional state of the RyRs and the amount of Ca(2+) stored in the SR. Reversible phosphorylation of proteins composing the EC coupling machinery plays an important role in regulation of cardiac contractility (Bers, 2002). Thus, during stimulation of the b-adrenergic pathway, phosphorylation of several target proteins, including the L-type Ca(2+) channels, RyRs and phospholamban, by protein kinase A (PKA) leads to an overall increase in SR Ca2+ release and contractile force in heart cells (Callewaert et al. 1988, Spurgeon et al. 1990; Hussain & Orchard, 1997; Zhou et al. 1999; Song et al. 2001; Viatchenko-Karpinski & Gyorke, 2001). PKA-dependent phosphorylation of the L-type Ca(2+) channels increases the Ca2+ current (ICa), increasing both the Ca2+ trigger for SR Ca2+ release and the SR Ca(2+) content (Callewaert et al. 1988; Hussain & Orchard, 1997; Del Principe et al. 2001). Phosphorylation of phospholamban (PLB) relieves the tonic inhibition dephosphorylated PLB exerts on the SR Ca(2+)-ATPase (SERCA) resulting in enhanced SR Ca(2+) accumulation and enlarged Ca(2+) release (Kranias et al. 1985; Simmermann & Jones, 1998). With regard to the RyR, despite clear demonstration of phosphorylation of the channel in biochemical studies (Takasago et al. 1989; Yoshida et al. 1992), the consequences of this reaction to channel function have not been clearly defined. RyR phosphorylation by PKA and Ca(2+)–calmodulin dependent protein kinase (CaMKII) has been reported to increase RyR activity in lipid bilayers (Hain et al. 1995; Marx et al. 2000; Uehara et al. 2002). Moreover, it has been reported that in heart failure (HF), hyperphosphorylation of RyR causes the release of FK-506 binding protein (FKBP12.6) from the RyR, rendering the channel excessively leaky for Ca(2+) (Marx et al. 2000). However, other studies have reported no functional effects (Li et al. 2002) or even found phosphorylation to reduce RyR channel steady-state open probability (Valdivia et al. 1995; Lokuta et al. 1995).

The Action of Protein Kinases is Opposed by Dephosphorylating Phosphatases.

Three types of protein: phosphatases (PPs), referred to as

  1. PP1,
  2. PP2A and
  3. PP2B (calcineurin),

have been shown to influence cardiac performance (Neumann et al. 1993; Rusnak & Mertz, 2000). Overall, according to most studies phosphatases appear to downregulate SR Ca(2+) release and contractile performance (Neumann et al. 1993; duBell et al. 1996, 2002; Carr et al. 2002; Santana et al. 2002). Furthermore, PP1 and PP2A activities appear to be increased in heart failure (Neumann, 2002; Carr et al. 2002). However, again the precise mode of action of these enzymes on intracellular Ca(2+) handling in normal and diseased hearts remains poorly understood. In the present study, we have investigated the effects of protein phosphatases PP1 and PP2A on local Ca(2+) release events, Ca(2+) sparks, in cardiac cells. Our results show that phosphatases activate RyR mediated SR Ca(2+) release leading to depletion of SR Ca(2+) stores. These results provide novel insights into the mechanisms and potential role of protein phosphorylation/dephosphorylation in regulation of Ca(2+) signaling in normal and diseased hearts.


Effects of PP1 and PP2A on Ca2+ Sparks and SR Ca(2+) Content.

  • PP1 caused an early transient potentiation of Ca2+ spark frequency followed by a delayed inhibition of event occurrence.
  • PP1 produced similar biphasic effects on the magnitude and spatio-temporal characteristics of Ca(2+) sparks

Specifically, during the potentiatory phase (1 min after addition of the enzyme), PP1 significantly increased the amplitude, rise-time, duration and width of Ca(2+) sparks; during the inhibitory phase (5 min after addition of the enzyme), all these parameters were significantly suppressed by PP1.

  • The SR Ca(2+) content decreased by 35 % or 69 % following the exposure of myocytes to either 0.5 or 2Uml_1 PP1, respectively (Fig. 1C).

Qualitatively similar results were obtained with phosphatase PP2A. Similar to the effects of PP1, PP2A (5Uml_1) produced a transient increase in Ca(2+) spark frequency (~4-fold) followed by a depression of event occurrence and decreased SR Ca(2+) content (by 82 % and 65 %, respectively). Also similar to the action of PP1, PP2A increased the amplitude and spatio-temporal spread (i.e. rise-time, duration and width) of Ca(2+) sparks at 1 min and suppressed the same parameters at 5 min of exposure to the enzyme (Table 1).  Together, these results suggest that phosphatases enhance spark-mediated SR Ca2+ release, leading to decreased SR Ca(2+) content.

  • Preventive effects of calyculin A and okadaic acid
  • Preventive effects of ryanodine

PP1-mediated RyR dephosphorylation

The cardiac RyR is phosphorylated at Ser-2809 (in the rabbit sequence) by both PKA and CAMKII (Witcher et al. 1991; Marx et al. 2000). Although additional phosphorylation sites may exist on the RyR (Rodriguez et al. 2003), Ser-2809 is believed to be the only site that is phosphorylated by PKA, and RyR hyperphosphorylation at this site has been reported in heart failure (Marx et al. 2000). To test whether indeed phosphatases dephosphorylated the RyR in our permeabilized myocyte experiments we performed quantitative immunoblotting using an antibody that specifically recognizes the phosphorylated form of the RyR at Ser-2809 (Rodriguez et al. 2003). Myocytes exhibited a significant level of phosphorylation under baseline conditions. Maximal phosphorylation was 201 % of control. When exposed to 2Uml_1 PP1, RyR phosphorylation was 58 % of the control basal condition. Exposing to a higher PP1 concentration (10Uml_1) further reduced RyR phosphorylation to 22% of control. Thus, consistent with the results of our functional measurements, PP1 decreased RyR phosphorylation in cardiac myocytes.

Figure 1. Effects of PP1 on properties of Ca(2+) sparks and SR Ca(2+) content in rat permeabilized myocytes

A, spontaneous Ca(2+) spark images recorded under reference conditions, and 1 or 5 min after exposure of the cell to 2Uml_1 PP1. Traces below the images are Ca(2+) transients induced by application of 10 mM caffeine immediately following the acquisition of sparks before (3 min) and after (5 min) application of PP1 in the same cell. The Ca(2+) transients were elicited by a whole bath application of 10 mM caffeine. B, averaged spark frequency at early (1 min) and late (5 min) times following the addition of either 0.5 or 2Uml_1 of PP1 to the bathing solution. C, averaged SR Ca(2+) content for 0.5 or 2Uml_1 of PP1 measured before and 5 min after exposure to the enzyme. Data are presented as means ± S.E.M. of 6 experiments in different cells.

Figure 2. Effects of PP2A on properties of Ca2+ sparks and SR Ca2+ content in rat permeabilized myocytes

A, spontaneous Ca(2+) spark images recorded under reference conditions, and 1 or 5 min after exposure of the cell to 5Uml_1 PP2A. Traces below the images are Ca(2+) transients induced by application of 10 mM caffeine immediately following the acquisition of sparks before (3 min) and after (5 min) application of PP2A in the same cell. B and C, averaged spark frequency (B) and SR Ca(2+) content (C) for the same conditions as in A. Data are presented as means ± S.E.M. of 6 experiments in different cells.

coupled receptors


In the present study, we have investigated the impact of physiologically relevant exogenous protein phosphatases PP1 and PP2A on RyR-mediated SR Ca(2+) release (measured as Ca(2+) sparks) in permeabilized heart cells. Our principal finding is that phosphatases stimulated RyR channels leading to depleted SR Ca(2+) stores. These results have important ramifications for understanding the mechanisms and role of protein phosphorylation/dephosphorylation in modulation of Ca(2+) handling in normal and diseased heart.

Ca(2+) and contraction


Modulation of SR Ca2+ release by Protein Phosphorylation/Dephophorylation

Since protein dephosphorylation clearly resulted in increased functional activity of the Ca(+)release channel, our results imply that a reverse, phosphorylation reaction should reduce RyR activity. If indeed such effects take place, why do they not manifest in inhibition of Ca(+)sparks? One possibility is that enhanced Ca(+) uptake by SERCA masks or overcomes the effects phosphorylation may have on RyRs. In

addition, the potential inhibitory influence of protein phosphorylation on RyR activity in myocytes could be countered by feedback mechanisms involving changes in luminal Ca(+)(Trafford et al. 2002; Gyorke et al. 2002). In particular, reduced open probability of RyRs would be expected to lead to increased Ca2+ accumulation in the SR; increased intra-SR [Ca(2+)] in turn would increase activity of RyRs at their luminal Ca(2+) regulatory sites as demonstrated for the RyR channel inhibitor tetracaine (Gyorke et al. 1997; Overend et al. 1997). Thus potentiation of SERCA combined with the intrinsic capacity of the release mechanism to self-regulate could explain at least in part why PKA-mediated protein phoshorylation results in maintained potentiation of Ca(2+) sparks despite a potential initial decrease in RyR activity.

F2.large   RyR and calcium

Role of altered RyR Phosphorylation in Heart Failure

Marx et al. (2000) have proposed that enhanced levels of circulating catecholamines lead to increased phosphorylation of RyR in heart failure. Based on biochemical observations as well as on studying properties of single RyRs incorporated into artificial lipid bilayers, these investigators have hypothesized that hyperphosphorylation of RyRs contributes to pathogenesis of heart failure by making the channel excessively leaky due to dissociation of FKBP12.6 from the channel. We show that the mode of modulation of RyRs by phosphatases does not support this hypothesis as dephosphorylation caused activation instead of inhibition of activity of RyR channels in a relatively intact setting. Interestingly, our results provide the basis for a different possibility in which dephophosphorylation of RyR rather than its phosphorylation causes depletion of SR Ca(2+) stores by stimulating RyRs in failing hearts. It has been reported that PP1 and PP2 activities are increased in heart failure (Huang et al. 1999; Neumann et al. 1997; Neuman, 2002). Furthermore, overexpression of PP1 or ablation of the endogenous PP1 inhibitor, l-1, results in depressed contractile performance and heart failure (Carr et al. 2002). Our finding that PP1 causes depletion of SR Ca(2+) stores by activating RyRs could account for, or contribute to, these results.

DelPrincipe F, Egger M, Pignier C & Niggli E (2001). Enhanced E-C coupling efficiency after beta-stimulation of cardiac myocytes. Biophys J 80, 64a.

Gyorke I & Gyorke S (1998). Regulation of the cardiac ryanodine receptor channel by luminal Ca2+ involves luminal Ca2+ sensing sites. Biophys J 75, 2801–2810.

Gyorke S, Gyorke I, Lukyanenko V, Terentyev D, Viatchenko-Karpinski S & Wiesner TF (2002). Regulation of sarcoplasmic reticulum calcium release by luminal calcium in cardiac muscle. Front Biosci 7, d1454–d1463.

Gyorke I, Lukyanenko V & Gyorke S (1997). Dual effects of tetracaine on spontaneous calcium release in rat ventricular myocytes. J Physiol 500, 297–309.

MacDougall LK, Jones LR & Cohen P (1991). Identification of the major protein phosphatases in mammalian cardiac muscle which dephosphorylate phospholamban. Eur J Biochem 196, 725–734.

Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N & Marks AR (2000). PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell 101, 365–376.

Rodriguez P, Bhogal MS & Colyer J (2003). Stoichiometric phosphorylation of cardiac ryanodine receptor on serine-2809 by calmodulin-dependent kinase II and protein kinase A. J Biol Chem (in press).

Proc Natl Acad Sci U S A. 2010 August 3; 107(31): E124.
Published online 2010 July 21. doi:  10.1073/pnas.1009086107
PMCID: PMC2922260

Reply to Eisner et al.: CaMKII phosphorylation of RyR2 increases cardiac contractility

The ryanodine receptor/calcium-release channel (RyR2) on the sarcoplasmic reticulum (SR) is the source of Ca2+ required for myocardial excitation–contraction (EC) coupling. During stress (i.e., exercise), contractility of the cardiac muscle is increased largely because of phosphorylation and activation of key proteins that regulate SR Ca2+ release. These include the voltage-gated calcium channel (Cav1.2) on the plasma membrane through which Ca2+ enters the cardiomyocyte, the sarco/endoplasmic reticulum calcium ATPase (SERCA2a)/phospholamban complex that pumps Ca2+ into the SR, and the RyR2 channel that releases Ca2+ from the SR, all of which are activated by phosphorylation.

For the past 10 y, Eisner et al. (1) have advanced the idea that activation of the RyR2 channel (e.g., by phosphorylation) cannot play a role in regulating systolic Ca2+ release and cardiac contractility. They base their position on an experiment in which they used caffeine to activate the RyR2 channel and showed that Ca2+ release was increased but after a few beats, returned to baseline (1). However, their experiment is not a good model for the physiological response to stress in which the three key regulators of EC coupling are all activated by the same signal (i.e., phosphorylation) such that there is increased Ca2+ influx, increased SR Ca2+ uptake, and increased SR Ca2+ release.

In the Eisner caffeine experiment, RyR2 was activated, but the Cav1.2 and SERCA2a were not. Selective activation of RyR2 is not physiological, and the outcome of their experiment was predictable. Caffeine-induced activation of RyR2 resulted in a transient increase in SR Ca2+ release, but because there was no concomitant increase in Ca2+ influx or SR Ca2+ uptake, the increase in SR Ca2+ release could not be sustained. However, on the basis of this experiment, Eisner et al. (1) concluded that activation of RyR2 plays no role in stress-induced increased cardiac contractility.

We have shown that, during stress, the increased heart rate results in a rate-dependent activation of CaMKII that phosphorylates and activates RyR2. We showed the essential role of this rate-dependent activation of RyR2 by CaMKII by showing that genetically engineered mice, lacking the CaMKII phosphorylation site on RyR2 (RyR2-S2814A), exhibit blunted increases in systolic Ca2+-transient amplitudes and contractile responses as heart rate increases (2). We also showed that a reduction in the amount of CaMKII in the RyR2 complex in failing hearts results in defective regulation of the channel, which could explain the loss of the rate-dependent increase in contractility in heart failure.

Eisner et al. (3) challenge all of our findings based on their caffeine experiment. However, our experiments have been conducted under physiological conditions in which all three components involved in Ca2+signaling during muscle contraction are activated, not just one. The only perturbation that we have introduced is to ablate the CaMKII phosphorylation site on RyR2 using a single amino acid substitution. This results in a blunted contractile response, leading us to conclude that CaMKII phosphorylation of RyR2 does indeed play a key role in enhancing contractility as the heart rate increases.

Cardiac Ryanodine Receptor Function and Regulation in Heart Disease

Annals NY Acad Sci JAN 2006    http://dx.doi.org/10.1196/annals.1302.012

Cardiac Engineering: From Genes and Cells to Structure and Function  2004; 1015(1), pp 144–159

The cardiac ryanodine receptor (RyR2) located on the sarcoplasmic reticulum (SR) controls intracellular Ca2+ release and muscle contraction in the heart. Ca2+ release via RyR2 is regulated by several physiological mediators. Protein kinase (PKA) phosphorylation dissociates the stabilizing FKBP12.6 subunit (calstabin2) from the RyR2 complex, resulting in increased contractility and cardiac output. Congestive heart failure is associated with

  • elevated plasma catecholamine levels, and
  • chronic stimulation of β-adrenergic receptors
  • leads to PKA hyperphosphorylation of RyR2 in failing hearts.
  • PKA hyperphosphorylation results in calstabin2-depleted RyR2 that displays altered channel gating and
    • may cause aberrant SR Ca2+ release,
    • depletion of SR Ca2+ stores, and
    • reduced myocardial contractility in heart failure.

Calstabin2-depleted RyR2 may also trigger cardiac arrhythmias that cause sudden cardiac death. In patients with catecholaminergic polymorphic ventricular tachycardia (CPVT), RyR2 missense mutations cause reduced calstabin2 binding to RyR2. Increased RyR2 phosphorylation and pathologically increased calstabin2 dissociation during exercise results in aberrant diastolic calcium release, which may trigger ventricular arrhythmias and sudden cardiac death. In conclusion, heart failure and exercise-induced sudden cardiac death have been linked to defects in RyR2-calstabin2 regulation, and this may represent a novel target for the prevention and treatment of these forms of heart disease

F3.large  cardiomyocyte SR

The δC Isoform of CaMKII Is Activated in Cardiac Hypertrophy and Induces Dilated Cardiomyopathy and Heart Failure

T Zhang, LS Maier, ND Dalton, S Miyamoto, J Ross, DM Bers, JH Brown
University of California, San Diego, La Jolla, Calif; and Loyola University, Chicago, Ill.
Circ Res. 2003;92:912-919.      http://dx.doi.org/10.1161/01.RES.0000069686.31472.C5

Recent studies have demonstrated that transgenic (TG) expression of either Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) or CaMKIIδB, both of which localize to the nucleus, induces cardiac hypertrophy. However, CaMKIV is not present in heart, and cardiomyocytes express not only the nuclear CaMKIIδB but also a cytoplasmic isoform, CaMKII δC. In the present study, we demonstrate that expression of the δC isoform of CaMKII is selectively increased and its phosphorylation elevated as early as 2 days and continuously for up to 7 days after pressure overload. To determine whether enhanced activity of this cytoplasmic δC isoform of CaMKII can lead to phosphorylation of Ca(2+) regulatory proteins and induce hypertrophy, we generated TG mice that expressed the δC isoform of CaMKII.  Immunocytochemical staining demonstrated that the expressed transgene is confined to the cytoplasm of cardiomyocytes isolated from these mice. These mice develop a dilated cardiomyopathy with up to a 65% decrease in fractional shortening and die prematurely. Isolated myocytes are enlarged and exhibit reduced contractility and altered Ca2(2+) handling. Phosphorylation of the ryanodine receptor (RyR) at a CaMKII site is increased even before development of heart failure, and CaMKII is found associated with the RyR in immunoprecipitates from the CaMKII TG mice. Phosphorylation of phospholamban is also increased specifically at the CaMKII but not at the PKA phosphorylation site. These findings are the first to demonstrate that CaMKIIδC can mediate phosphorylation of Ca(2+) regulatory proteins in vivo and provide evidence for the involvement of CaMKIIδC activation in the pathogenesis of dilated cardiomyopathy and heart failure.

Multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaM kinases or CaMKs) are transducers of Ca2+ signals that phosphorylate a wide range of substrates and thereby affect Ca(2+)-mediated cellular responses.1 The family includes CaMKI and CaMKIV, monomeric enzymes activated by CaM kinase kinase,2,3 and CaMKII, a multimer of 6 to 12 subunits activated by autophosphorylation.1 The CaMKII subunits α, β, γ, and δ show different tissue distributions,1 with the δisoform predominating in the heart.4–7 Splice variants of the δisoform, characterized by the presence of a second variable domain,4,7 include δB, which contains a nuclear localization signal (NLS), and δC, which does not. CaMKII composed of δB subunits localizes to the nucleus, whereas CaMKIIδC localizes to the cytoplasm.4,8,9

CaMKII has been implicated in several key aspects of acute cellular Ca(2+) regulation related to cardiac excitation-contraction (E-C) coupling. CaMKII phosphorylates sarcoplasmic reticulum (SR) proteins including the ryanodine receptors (RyR2) and phospholamban (PLB).10–14 Phosphorylation of RyR has been suggested to alter the channel open probability,14,15 whereas phosphorylation of PLB has been suggested to regulate SR Ca(2+) uptake.14 It is also likely that CaMKII phosphorylates the L-type Ca2 channel complex or an associated regulatory protein and thus mediates Ca2 current (ICa) facilitation.16-18 and the development of early after-depolarizations and arrhythmias.19 Thus, CaMKII has significant effects on E-C coupling and cellular Ca2 regulation. Nothing is known about the CaMKII isoforms regulating these responses.

Contractile dysfunction develops with hypertrophy, characterizes heart failure, and is associated with changes in cardiomyocyte Ca2homeostasis.20 CaMKII expression and activity are altered in the myocardium of rat models of hypertensive cardiac hypertrophy21,22 and heart failure,23 and in cardiac tissue from patients with dilated cardiomyopathy.24,25

Several transgenic mouse models have confirmed a role for CaMK in the development of cardiac hypertrophy, as originally suggested by studies in isolated neonatal rat ventricular myocytes.9,26–28 Hypertrophy develops in transgenic mice that overexpress CaMKIV,27 but this isoform is not detectable in the heart,4,29 and CaMKIV knockout mice still develop hypertrophy after transverse aortic constriction (TAC).29

Transgenic mice overexpressing calmodulin developed severe cardiac hypertrophy,30 later shown to be associated with an increase in activated CaMKII31; the isoform of CaMKII involved in hypertrophy could not be determined from these studies. We recently reported that transgenic mice that overexpress CaMKIIδB, which is highly concentrated in cardiomyocyte nuclei, develop hypertrophy and dilated cardiomyopathy.32 To determine whether in vivo expression of the cytoplasmic CaMKIIδC can phosphorylate cytoplasmic Ca2regulatory proteins and induce hypertrophy or heart failure, we generated transgenic (TG) mice that expressed the δC isoform of CaMKII under the control of the cardiac specific α-myosin heavy chain (MHC) promoter. Our findings implicate CaMKIIδC in the pathogenesis of dilated cardiomyopathy and heart failure and suggest that this occurs at least in part via alterations in Ca2handling proteins.33


Expression and Activation of CaMKIIδC Isoform After TAC

To determine whether CaMKII was regulated in pressureoverload–induced hypertrophy, CaMKIIδ expression and phosphorylation were examined by Western blot analysis using left ventricular samples obtained at various times after TAC. A selective increase (1.6-fold) in the lower band of CaMKIIδwas observed as early as 1 day and continuously for 4 days (2.3-fold) and 7 days (2-fold) after TAC (Figure 1A).  To confirm that CaMKIIδC was increased and determine whether this occurred at the transcriptional level, we performed semiquantitative RT-PCR using primers specific for the CaMKIIδC isoform. These experiments revealed that mRNA levels for CaMKIIδC were increased 1 to 7 days after TAC (Figure 1B). In addition to examining CaMKII expression, the activation state of CaMKII was monitored by its autophosphorylation, which confers Ca2-independent activity.

Figure 1. Expression and activation of CaMKII δC isoform after TAC.
see http://dx.doi.org/10.1161/01.RES.0000069686.31472.C5

A, Western blot analysis of total CaMKII in left ventricular (LV) homogenates obtained at indicated times after TAC. Cardiomyocytes transfected with CaMKIIδB and δC (right) served as positive controls and molecular markers. Top band (58 kDa) represents CaMKIIδB plus δ9, and the bottom band (56 kDa) corresponds to CaMKIIδC. *P0.05 vs control. B, Semiquantitative RT-PCR using primers specific for CaMKIIδC isoform (24 cycles) and GAPDH (19 cycles) using total RNA isolated from the same LV samples. C, Western blot analysis of phospho-CaMKII in LV homogenates obtained at various times after TAC. Three bands seen for each sample represent CaMKIIγ subunit (uppermost), CaMKIIδB plus δ9 (58 kDa), and CaMKIIδC (56 kDa). Quantitation is based on the sum of all of the bands. *P0.05 vs control.

Figure 2. Expression and activation of CaMKII in CaMKIIδC transgenic mice.
see  http://dx.doi.org/10.1161/01.RES.0000069686.31472.C5

A, Transgene copy number based on Southern blots using genomic DNA isolated from mouse tails (digested with EcoRI). Probe (a 32P-labeled 1.7-kb EcoRI-SalI -MHC fragment) was hybridized to a 2.3-kb endogenous fragment (En) and a 3.9-kb transgenic fragment (TG). Transgene copy number was determined from the ratio of the 3.9-kb/2.3-kb multiplied by 2. B, Immunocytochemical staining of ventricular myocytes isolated from WT and CaMKIIδTG mice. Myocytes were cultured on laminin-coated slides overnight. Transgene was detected by indirect immunofluorescence staining using rabbit anti-HA antibody (1:100 dilution) followed by FITC-conjugated goat antirabbit IgG antibody (1:100 dilution). CaMKIIδB localization to the nucleus in CaMKIIδB TG mice (see Reference 32) is shown here for comparative purpose. C, Quantitation of the fold increase in CaMKIIδprotein expression in TGL and TGM lines. Different amounts of ventricular protein (numbers) from WT control, TG () and their littermates () were immunoblotted with an anti-CaMKIIδ antibody. Standard curve from the WT control was used to calculate fold increases in protein expression in TGL and TGM lines. D, Phosphorylated CaMKII in ventricular homogenates was measured by Western blot analysis (n5 for each group). **P0.01 vs WT.

Generation and Identification of CaMKIIδC Transgenic Mice

TG mice expressing HA-tagged rat wild-type CaMKIIδC under the control of the cardiac-specific α-MHC promoter were generated as described in Materials and Methods. By Southern blot analysis, 3 independent TG founder lines carrying 3, 5, and 15 copies of the transgene were identified. They were designated as TGL (low copy number), TGM (medium copy number), and TGH (high copy number),

The founder mice from the TGH line died at 5 weeks of age with marked cardiac enlargement.  The other two lines showed germline transmission of the transgene. The transgene was expressed only in the heart.

Although CaMKII protein levels in TGL and TGM hearts were increased 12- and 17-fold over wild-type (WT) controls

(Figure 2C), the amount of activated CaMKII was only increased 1.7- and 3-fold in TGL and TGM hearts (Figure 2D). The relatively small increase in CaMKII activity in the TG lines probably reflects the fact that the enzyme is not constitutively activated and that the availability of Ca2/CaM, necessary for activation of the overexpressed CaMKII, is limited. Importantly, the extent of increase in active CaMKII in the TG lines was similar to that elicited by TAC.

Cardiac Overexpression of CaMKIIδC Induces Cardiac Hypertrophy and Dilated Cardiomyopathy

There was significant enlargement of hearts from CaMKIIδC TGM mice by 8 to 10 weeks (Figure 3A) and from TGL mice by 12 to 16 weeks. Histological analysis showed ventricular dilation (Figure 3B), cardiomyocyte enlargement (Figure 3C), and mild fibrosis (Figure 3D) in CaMKIIδC TG mice. Quantitative analysis of cardiomyocyte cell volume from 12-week-old TGM mice gave values of 54.7 +­ 0.1 pL for TGM (n = 96) versus 28.6 + 0.1 pL for WT littermates (n=94; P0.001).

Ventricular dilation and cardiac dysfunction developed over time in proportion to the extent of transgene expression. Left ventricular end diastolic diameter (LVEDD) was increased by 35% to 45%, left ventricular posterior wall thickness (LVPW) decreased by 26% to 29% and fractional shortening decreased by 50% to 60% at 8 weeks for TGM and at 16 weeks for TGL. None of these parameters were significantly altered at 4 weeks in TGM or up to 11 weeks in TGL mice, indicating that heart failure had not yet developed.  Contractile function was significantly decreased.

Figure 6. Dilated cardiomyopathy and dysfunction in CaMKIIδC TG mice at both whole heart and single cell levels.
see Fig 6  http://dx.doi.org/10.1161/01.RES.0000069686.31472.C5

C, Decreased contractile function in ventricular myocytes isolated from 12-week old TGM and WT controls presented as percent change of resting cell length (RCL) stimulated at 0.5 Hz. Representative trace and mean values are shown. *P0.05 vs WT.

Figure 7. Phosphorylation of PLB in CaMKIIδC TG mice.

see Fig 7: http://dx.doi.org/10.1161/01.RES.0000069686.31472.C5

(Figures 7A and 7B). (see  http://dx.doi.org/10.1161/01.RES.0000069686.31472.C6

(Figure 8C).  (http://dx.doi.org/10.1161/01.RES.0000069686.31472.C5

Thr17 and Ser16 phosphorylated PLB was measured by Western blots using specific anti-phospho antibodies. Ventricular homogenates were from 12- to 14-week-old WT and TGM mice (A) or 4 to 5-week-old WT and TGM mice (B). Data were normalized to total PLB examined by Western blots (data not shown here). n = 6 to 8 mice per group; *P0.05 vs WT.

Cardiac Overexpression of CaMKIIδC Results in Changes in the Phosphorylation of Ca2 Handling Proteins

To assess the possible involvement of phosphorylation of Ca2cycling proteins in the phenotypic changes observed in the CaMKIIC TG mice, we first compared PLB phosphorylation state in homogenates from 12- to 14-week-old TGM and WT littermates. Western blots using antibodies specific for phosphorylated PLB showed a 2.3-fold increase in phosphorylation of Thr17 (the CaMKII site) in hearts from TGM versus WT (Figure 7A). Phosphorylation of PLB at the CaMKII site was also increased 2-fold in 4- to 5-week-old TGM mice (Figure 7B). Significantly, phosphorylation of the PKA site (Ser16) was unchanged in either the older or the younger TGM mice (Figures 7A and 7B).

To demonstrate that the RyR2 phosphorylation changes observed in the CaMKII transgenic mice are not secondary to development of heart failure, we performed biochemical studies examining RyR2 phosphorylation in 4- to 5-week-old TGM mice. At this age, most mice showed no signs of hypertrophy or heart failure (see Figure 6B) and there was no significant increase in myocyte size (21.3 + 1.3 versus 27.7 + 4.6 pL; P0.14). Also, twitch Ca2 transient amplitude was not yet significantly depressed, and mean δ   [Ca2+]i (1 Hz) was only 20% lower (192 + 36 versus 156 + 13 nmol/L; P0.47) versus 50% lower in TGM at 13 weeks.33

The in vivo phosphorylation of RyR2, determined by back phosphorylation, was significantly (2.10.3-fold; P0.05) increased in these 4- to 5-week-old TGM animals (Figure 8C), an increase equivalent to that seen in 12- to 14-week-old mice. We also performed the RyR2 back-phosphorylation assay using purified CaMKII rather than PKA. RyR2 phosphorylation at the CaMKII site was also significantly increased (2.2 + 0.3-fold; P0.05) in 4- to 5-week-old TGM mice (Figure 8C).

The association of CaMKII with the RyR2 is consistent with a physical interaction between this protein kinase and its substrate. The catalytic subunit of PKA and the phosphatases PP1 and PP2A were also present in the RyR2 immunoprecipitates, but not different in WT versus TG mouse hearts (Figure 8D). These data provide further evidence that the increase in RyR2 phosphorylation, which precedes development of failure in the 4- to 5-week-old CaMKIIδC TG hearts, can be attributed to the increased activity of CaMKII.


CaMKII is involved in the dynamic modulation of cellular Ca2 regulation and has been implicated in the development of cardiac hypertrophy and heart failure.14 Published data from CaMK-expressing TG mice demonstrate that forced expression of CaMK can induce cardiac hypertrophy and lead to heart failure.27,32 However, the CaMK genes expressed in these mice are neither the endogenous isoforms of the enzyme nor the isoforms likely to regulate cytoplasmic Ca(2+) handling, because they localize to the nucleus.

First, we demonstrate that the cytoplasmic cardiac isoform of CaMKII is upregulated at the expression level and is in the active state (based on autophosphorylation) after pressure overload induced by TAC. Second, we demonstrate that two cytoplasmic CaMKII substrates (PLB and RyR) are phosphorylated in vivo when CaMKII is overexpressed and its activity increased to an extent seen under pathophysiological conditions. Moreover, CaMKIIδis found to associate physically with the RyR in the heart. Finally, our data indicate that heart failure can result from activation of the cytoplasmic form of CaMKII and this may be due to altered Ca(2+) handling.

Differential Regulation of CaMKIIδ Isoforms in Cardiac Hypertrophy

The isoform of CaMKII that predominates in the heart is the δ isoform.4–7 Neither the α nor the β isoforms are expressed and there is only a low level of expression of the γ isoforms.39 Both δB and δC splice variants of CaMKIIδ are present in the adult mammalian myocardium36,40 and expressed in distinct cellular compartments.4,8,9

We suggest that the CaMKIIδisoforms are differentially regulated in pressure-overload–induced hypertrophy, because the expression of CaMKIIδC is selectively increased as early as 1 day after TAC. Studies using RT-PCR confirm that CaMKIIδC is regulated at the transcriptional level in response to

TAC. In addition, activation of both CaMKIIδB and CaMKIIδC, as indexed by autophosphorylation, increases as early as 2 days after TAC. Activation of CaMKIIδB by TAC is relevant to our previous work indicating its role in hypertrophy.9,32 The increased expression, as well as activation of the CaMKIIδC isoform, suggests that it could also play a critical role in both the acute and longer responses to pressure overload.

In conclusion, we demonstrate here that CaMKIIδC can phosphorylate RyR2 and PLB when expressed in vivo at levels leading to 2- to 3-fold increases in its activity. Similar increases in CaMKII activity occur with TAC or in heart failure. Data presented in this study and in the accompanying article33 suggest that altered phosphorylation of Ca(2+) cycling proteins is a major component of the observed decrease in contractile function in CaMKIIδC TG mice. The early occurrence of increased CaMKII activity after TAC, and of RyR and PLB phosphorylation in the CaMKIIδC TG mice suggest that CaMKIIδC plays an important role in the pathogenesis of dilated cardiomyopathy and heart failure. These results have major implications for considering CaMKII and its isoforms in exploring new treatment strategies for heart failure.

Unique phosphorylation site on the cardiac ryanodine receptor regulates calcium channel activity.

DR Witcher, RJ Kovacs, H Schulman, DC Cefali, LR Jones
Krannert Institute of Cardiology and the Indiana University School of Medicine, Indianapolis,
Stanford University School of Medicine, Stanford.
Journal of Biological Chemistry 07/1991; 266(17):11144-52. · 4.77 Impact

Ryanodine receptors have recently been shown to be the Ca2+ release channels of sarcoplasmic reticulum in both cardiac muscle and skeletal muscle. Several regulatory sites are postulated to exist on these receptors, but to date, none have been definitively identified. In the work described here, we localize one of these sites by showing that the cardiac isoform of the ryanodine receptor is a preferred substrate for multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase). Phosphorylation by CaM kinase occurs at a single site encompassing serine 2809. Antibodies generated to this site react only with the cardiac isoform of the ryanodine receptor, and immunoprecipitate only cardiac [3H]ryanodine-binding sites. When cardiac junctional sarcoplasmic reticulum vesicles or partially purified ryanodine receptors are fused with planar bilayers, phosphorylation at this site activates the Ca2+ channel. In tissues expressing the cardiac isoform of the ryanodine receptor, such as heart and brain, phosphorylation of the Ca(2+) release channel by CaM kinase may provide a unique mechanism for regulating intracellular (Ca2+) release.

The Ca(2+) release from the SR causes an increase in Ca(2+) concentration which leads to muscle contraction (1). Recently, the sites of Ca(2+) release have been identified and purified from both cardiac (2-4) and skeletal muscle SR (5- 7) and shown to be the same as the ryanodine receptors or high molecular weight proteins. The structures attach the transverse tubules to the junctional SR both in intact tissues and isolated membrane fractions (1, 8-10). Although the Ca(2+) release channels from cardiac and skeletal muscle show many similarities such as nearly identical

  • myoplasmic 3- EGTA,
  • Ca2+ conductances (2-7),
  • protease sensitivities (11, E ) ,
  • calmodulin-binding capabilities (ll), and
  • modulation by allosteric regulators such as Ca2+, Mg2+, ATP, and calmodulin (13-15),

they also exhibit several differences in protein structure and function. Quantitative differences have been noted on the effects of modulators on ryanodine binding to the two proteins (16-18), as well as on Ca(2+) channel kinetics. In addition, the cardiac ryanodine smaller apparent molecular weight than the skeletal muscle receptor on SDS-PAGE (ll), and monoclonal antibodies can be made which react with the cardiac receptor but not the skeletal receptor (16).

Recent work on characterization receptors has culminated in elucidation of structures of the proteins by sequencing of their cDNAs (19-21). Consistent with the differences between the two protein iso- forms noted above, the cardiac and skeletal muscle receptors have been found to be the products of different genes, with overall amino acid identities of 66% (21). Both protein isoforms are very large, containing approximately 5,000 amino acids and exhibiting predicted molecular weights of 564,711 for the cardiac protein (21) and 565,223 (19) or 563,584 (20) for the skeletal muscle protein. In the native state, ryanodine receptors are arranged as tetramers (1-7). In an earlier study (22), we demonstrated that the canine cardiac high molecular weight protein (or ryanodine receptor; Ref. 3) was an excellent substrate CaM kinase (23,24) endogenous to junctional SR membranes. In the work described here, we show that phosphorylation of the cardiac receptor by CaM kinase occurs at a single site, which is not substantially phosphorylated in the skeletal muscle receptor, and that phosphorylation ryanodine receptor at this site activates the Ca2+ channel.

Our data are the first to support the hypothesis (21), that the modulator-binding sites of the cardiac ryanodine receptor are contained within residues 2619-3016. (13, 14). The ryanodine receptor is compared with the primary structure for the multifunctional of the cardiac model of Otsu et al. (21).

Experimental Procedures.

See Figs 1-6    http://www.jbc.org/content/266/17/11144.full.pdf


Preferential Phosphorylation Receptor-(Fig. 1, arrowheads) is phosphorylated in junctional vesicles by an endogenous calmodulin-requiring proteinase and this phosphorylation is stimulated several fold when exogenous CaM kinase is added. In contrast, the ryanodine receptor in canine fast and vesicles, which migrates with weight on SDS-PAGE (2, 11, 16), is not significantly phosphorylated by either endogenous or exogenous protein kinase (Fig. 1, small arrows).

Similar results were obtained with rabbit skeletal muscle SR vesicles. The identity of the skeletal muscle ryanodine receptor in these studies (Fig. 1, small arrow) was confirmed by immunoblotting with a skeletal muscle isoform-specific antibody (supplied by K. Campbell, University of Iowa). We did detect a low level of phosphorylation of a protein in slow skeletal muscle samples migrating slightly faster than the cardiac receptor, but this protein did not cross-react with skeletal muscle (or cardiac, see below) antibodies, suggesting that it is unrelated to the ryanodine receptor.  CaM kinase-catalyzed phosphorylation of the cardiac ryanodine receptor was always at least 10-fold greater than skeletal receptor phosphorylation. These results demonstrate that the skeletal muscle ryanodine receptor phosphorylation is insignificant compared to cardiac protein phosphorylation. Consistent with our results, Otsu et al. (21) have recently shown that, the cardiac isoform receptor is absent from fast and slow skeletal muscle. Phosphorylation of the cardiac ryanodine receptor by cAMP kinase also occurs, but phosphorylation by added cAMP kinase is no greater than that achieved with endogenous CaM kinase. (Fig. 2). In contrast, the amount of exogenous CaM kinase increases receptor phosphorylation 4-fold, to a maximal level of 26 pmol of P/mg of SR protein (Fig. 2). We observed no significant phosphorylation of canine fast and slow or rabbit skeletal muscle ryanodine. Maximal ryanodine binding (3) in these preparations ranged between 5 and 6 pmol/mg of protein, a value nearly identical to the level of receptor phosphorylation achieved with exogenous cAMP kinase (see CaM kinase), but one-fourth the value achieved with added CaM kinase. Since the functional unit release channel contains only one high affinity ryanodine- binding site/tetramer (4), our results suggest that the endogenous CaM kinase is capable of phosphorylating only one-fourth of the available sites, whereas the exogenous kinase can fully phosphorylate the receptor (below) of the Cardiac Ryanodine. The canine Slow skeletal muscle SR receptor of the ryanodine it was recently reported is phosphorylated 1/20th by the of the CaM kinase.


Immunoprecipitation of Ryanodine receptors from CHAPS-solubilized canine SR membranes. Values are expressed for aliquots of the following fractions: S, solubilized receptors after treatment of membranes with 2% CHAPS; B, bound fraction, containing ryanodine receptors immunoprecipitated from CHAPS superna- tant; F, free fraction, containing ryanodine receptors not immunoprecipitated. Total binding was measured using 20 nM [3H]ryanodine. For nonspecific binding, 10 PM cold ryanodine was added. FIG. 7.

Effect of ATP and calmodulin on the cardiac Ca(2+) release channel. Holding potential was 0 mV, with upward current deflections representing movement of Ba(2+) from the trans to the cis chamber. Gaussian distributions were fit to the peaks of activity in the histograms. Signals were filtered at 300 Hz (low pass Bessel) and digitized at 1 KHz (Axotape, Axon Instruments) for * off-line analysis. In the control (A), p(open) was 0.26. Addition of 1 mM ATP (B) produced prolonged openings of the channel, increasing p(0pen) to 0.81. Subsequent addition of calmodulin (C) decreased p(open) to 0.12, producing long closures and brief aborted openings.

Sequencing of the Cardiac Phosphorylation Site.  In order to sequence the phosphorylation site of the cardiac ryanodine receptor, we phosphorylated junctional SR membranes on large scale with added CaM kinase and purified the phosphorylated denatured ryanodine receptor to homogeneity in one step using SDS-gel filtration chromatography (Fig. 3). The purified cardiac ryanodine receptor was digested with trypsin, and the radioactive peptides recovered using Fe(3+) affinity chromatography (30,37). 90% of the loaded radioactivity was recovered in the pH 8.6 and 10 eluates from the Fe column (Fig. 4). These fractions were then combined and subjected to reverse-phase chromatography, yielding a single major radioactive peptide peak eluting at approximately 24% acetonitrile (Fig. 4, inset).


Gas-phase sequencing of the radioactive tryptic peptide gave a single sequence of 18 consecutive residues, which corresponded exactly to residues 2807-2824 reported for the rabbit cardiac ryanodine receptor from cDNA cloning (Fig. 5) (21). When CNBr and endoproteinase Lys-C were used to cleave the receptor, another “P-labeled peptide was isolated and sequenced, which matched with residues 2800-2811 of the rabbit cardiac ryanodine receptor (Fig. 5).

Serine 2809 within the phosphorylated tryptic peptide is situated on the carboxyl-terminal side of 2 arginine residues. The fact that R-R-X-S and R-X-X-S/T are minimal consensus phosphorylation sequences (38,39) for CAMP kinase and CaM kinase, respectively, makes this residue the likely phosphorylation site. Consistent with this, the ratio threitol-serine to phenylthiohydantoin-serine recovered dur- ing cycle 3 of sequencing of this peptide was 10 times greater than that recovered during cycles 6 and 9. It is known that dithiothreitol-serine is the predominant breakdown product of phosphoserine (40, 41). Phosphoamino acid analysis revealed that this peptide contained only phosphoserine; more- over, >90% of the 3’Pi was released from the peptide by cycle 10 (40, 42), demonstrating that no serine residue downstream of this region was significantly labeled.

Based on these results, we conclude that serine 2809 is the amino acid phosphorylated by CaM kinase. When only endogenous CaM kinase was used to phosphorylate the cardiac ryanodine receptor, the same labeled tryptic peptide was recovered and sequenced in four separate runs. Thus, although exogenously added kinase gives a 4-fold stimulation of receptor phosphorylation (Fig. 2), no new sites are phosphorylated. The reason for the low level of phosphorylation obtained with endogenous CaM kinase remains undefined.

Cardiac Electrophysiological Dynamics From the Cellular Level to the Organ Level

Daisuke Sato and Colleen E. Clancy
Department of Pharmacology, University of California – Davis, Davis, CA.
Biomedical Engineering and Computational Biology 2013:5: 69–75

http://www.la-press.com.   http://dx.doi.org/10.4137/BECB.S10960

Figure 1. (Top): APD and DI. (Bottom): The physiological mechanism of APD alternans involves recovery from inactivation of ICaL.  [see  http://dx.doi.org/10.4137/BECB.S10960]

Figure 2. APD restitution and dynamical mechanism of APD alternans.   [see  http://dx.doi.org/10.4137/BECB.S10960]
Review Series.  Genetic Causes of Human Heart Failure

Hiroyuki Morita, Jonathan Seidman and Christine E. Seidman
Harvard Medical School, Brigham and Women’s Hospital, Howard Hughes Medical Institute, Boston, MA
J Clin Invest. 2005;115(3):518–526.    http://dx.doi.org/10.1172/JCI24351.

Correspondence to: Christine E. Seidman, Department of Genetics, Harvard Medical School, Boston, MA. Ph: (617) 432-7871; E-mail: cseidman@genetics.med.harvard.edu

Factors that render patients with cardiovascular disease at high risk for heart failure remain incompletely defined. Recent insights into molecular genetic causes of myocardial diseases have highlighted the importance of single-gene defects in the pathogenesis of heart failure. Through analyses of the mechanisms by which a mutation selectively perturbs one component of cardiac physiology and triggers cell and molecular responses, studies of human gene mutations provide a window into the complex processes of cardiac remodeling and heart failure. Knowledge gleaned from these studies shows promise for defining novel therapeutic targets for genetic and acquired causes of heart failure.


Heart failure currently affects 4.8 million Americans, and each year over 500,000 new cases are diagnosed. In 2003 heart failure contributed to over 280,000 deaths and accounted for 17.8 billion health care dollars (1).

Heart failure almost universally arises in the context of antecedent cardiovascular disease:

  • atherosclerosis,
  • cardiomyopathy,
  • myocarditis,
  • congenital malformations, or
  • valvular disease.

The study of single-gene mutations that trigger heart failure provides an opportunity for defining important molecules involved in these processes. Although these monogenic disorders account for only a small subset of overall heart failure cases, insights into the responses triggered by gene mutations are likely to also be relevant to more common etiologies of heart failure.

Early Manifestation – Heart Failure – Ventricular Remodeling.

One of 2 distinct morphologies occurs: left ventricular hypertrophy (increased wall thickness without chamber expansion) or dilation (normal or thinned walls with enlarged chamber volumes).

Each is associated with specific hemodynamic changes. Systolic function is normal, but diastolic relaxation is impaired in hypertrophic remodeling; diminished systolic function characterizes dilated remodeling. Clinical recognition of these cardiac findings usually prompts diagnosis of hypertrophic cardiomyopathy (HCM) or dilated cardiomyopathy (DCM). There is now considerable evidence that many different gene mutations can cause these pathologies (Figure 1), and with these discoveries has come recognition of distinct histopathologic features that further delineate several subtypes of remodeling. The current compendia of genes that remodel the heart already suggest a multiplicity of pathways by which the human heart can fail.

To facilitate a discussion, we have grouped known cardiomyopathy genes according to the probable functional consequences of mutations on

  • force generation and transmission,
  • metabolism,
  • calcium homeostasis, or
  • transcriptional control.

Gene mutations in one functional category inevitably have an impact on multiple myocyte processes, and, the eventual delineation of signals between functional groups may be critical to understanding cardiac decompensation and heart failure development.

Figure 1.  see  http:/dx.doi.org/10.1172/JCI24351

Human gene mutations can cause cardiac hypertrophy (blue), dilation (yellow), or both (green). In addition to these two patterns of remodeling, particular gene defects produce hypertrophic remodeling with glycogen accumulation (pink) or dilated remodeling with fibrofatty degeneration of the myocardium (orange). Sarcomere proteins denote β-myosin heavy chain, cardiac troponin T, cardiac troponin I, α-tropomyosin, cardiac actin, and titin. Metabolic/storage proteins denote AMP-activated protein kinase γ subunit, LAMP2, lysosomal acid α 1,4–glucosidase, and lysosomal hydrolase α-galactosidase A. Z-disc proteins denote MLP and telethonin. Dystrophin-complex proteins denote δ-sarcoglycan, β-sarcoglycan, and dystrophin. Ca2+ cycling proteins denote PLN and RyR2. Desmosome proteins denote plakoglobin, desmoplakin, and plakophilin-2.

Force generation and propagation. Generation of contractile force by the sarcomere and its transmission to the extracellular matrix are the fundamental functions of heart cells. Inadequate performance in either component prompts cardiac remodeling (hypertrophy or dilation), produces symptoms, and leads to heart failure. Given the importance of these processes for normal heart function and overt clinical manifestations of deficits in either force generation or transmission, it is not surprising that more single-gene mutations have been identified in molecules involved in these critical processes than in those of other functional classes.

Figure 2  see http:/dx.doi.org/10.1172/JCI24351

Human mutations affecting contractile and Z-disc proteins. The schematic depicts one sarcomere,

the fundamental unit of contraction encompassing the protein segment between flanking Z discs. Sarcomere thin filament proteins are composed of actin and troponins C, T, and I. Sarcomere thick filament proteins include myosin heavy chain, myosin essential and regulatory light chains, myosin-binding protein-C and titin. The sarcomere is anchored through titin and actin interactions with Z disc proteins α-actinin, calsarcin-1, MLP, telethonin (T-cap), and ZASP. Human mutations (orange text) in contractile proteins and Z-disc proteins can cause HCM or DCM.

Sarcomere protein mutations. Human mutations in the genes encoding protein components of the sarcomere cause either HCM or DCM. While progression to heart failure occurs with both patterns of remodeling, the histopathology, hemodynamic profiles, and biophysical consequences of HCM or DCM mutations suggest that distinct molecular processes are involved.

Over 300 dominant mutations in genes encoding β-cardiac myosin heavy chain (MYH7), cardiac myosin-binding protein-C (MYBPC3), cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), essential myosin light chain (MYL3), regulatory myosin light chain (MYL2), α-tropomyosin (TPM1), cardiac actin (ACTC), and titin (TTN) have been reported to cause HCM (Figure 2) (2, 3). Recent reports of comprehensive sequencing of sarcomere protein genes in diverse patient populations indicate that MYBPC3 and MYH7 mutations are most frequent (4, 5). Sarcomere gene mutations that cause HCM produce a shared histopathology with enlarged myocytes that are disorganized and die prematurely, which results in increased cardiac fibrosis.

The severity and pattern of ventricular hypertrophy,

  • age at onset of clinical manifestations, and
  • progression to heart failure

are, in part, dependent on the precise sarcomere protein gene mutation. For example, TNNT2 mutations are generally associated with a high incidence of sudden death despite only mild left ventricular hypertrophy (6, 7). While only a small subset (10–15%) of HCM patients develop heart failure, this end-stage phenotype has a markedly poor prognosis and often necessitates cardiac transplantation. Accelerated clinical deterioration has been observed with MYH7 Arg719Trp, TNNT2 Lys273Glu, TNNI3 Lys183del, and TPM1 Glu180Val mutations (8–11).

Most HCM mutations encode defective polypeptides containing missense residues or small deletions; these are likely to be stably incorporated into cardiac myofilaments and to produce hypertrophy because normal sarcomere function is disturbed. Many HCM mutations in MYBPC3 fall within carboxyl domains that interact with titin and myosin; however, the exact biophysical properties altered by these defects remain unknown (Figure 2). HCM mutations in myosin are found in virtually every functional domain, which suggests that the biophysical consequences of these defects may vary. Genetic engineering of some human myosin mutations into mice has indicated more consistent sequelae. Isolated single-mutant myosin molecules containing different HCM mutations

  • had increased actin-activated ATPase activity and
  • showed greater force production and
  • faster actin-filament sliding,

biophysical properties that may account for hyperdynamic contractile performance observed in HCM hearts and that suggest a mechanism for premature myocyte death in HCM (12–14). Uncoordinated contraction due to

  • heterogeneity of mutant and normal sarcomere proteins,
  • increased energy consumption, and
  • changes in Ca2+ homeostasis

could diminish myocyte survival and trigger replacement fibrosis. With insidious myocyte loss and increased fibrosis, the HCM heart transitions from hypertrophy to failure.

Mice that are engineered to carry a sarcomere mutation replicate the genetics of human disease; heterozygous mutations cause HCM. One exception is a deletion of proximal myosin-binding protein-C sequences; heterozygous mutant mice exhibited normal heart structure while homozygous mutant mice developed hypertrophy (15). Remarkably, while most heterozygous mouse models with a mutation in myosin heavy chain, myosin-binding protein-C, or troponin T developed HCM (16–18), homozygous mutant mice (19, 20) developed DCM with fulminant heart failure and, in some cases, premature death. These mouse studies might indicate that HCM, DCM, and heart failure reflect gradations of a single molecular pathway. Alternatively, significant myocyte death caused by homozygous sarcomere mutations may result in heart failure. Human data suggest a more complicated scenario. The clinical phenotype of rare individuals who carry homozygous sarcomere mutations in either MYH7 (21) or in TNNT2 (22) is severe hypertrophy, not DCM. Furthermore, individuals with compound heterozygous sarcomere mutations exhibit HCM, not DCM. The absence of ventricular dilation in human hearts with 2 copies of mutant sarcomere proteins is consistent with distinct cellular signaling programs that remodel the heart into hypertrophic or dilated morphologies.

DCM sarcomere protein gene mutations affect distinct amino acids from HCM-causing mutations, although the proximity of altered residues is remarkable. The histopathology of sarcomere DCM mutations is quite different from those causing HCM, and is remarkably nonspecific. Degenerating myocytes with increased interstitial fibrosis are present, but myocyte disarray is notably absent. There are 2 mechanisms by which sarcomere mutations may cause DCM and heart failure: deficits of force production and deficits of force transmission. Diminished force may occur in myosin mutations (e.g., MYH7 Ser532Pro) that alter actin-binding residues involved in initiating the power stroke of contraction. Impaired contractile force may also occur in DCM troponin mutations (TNNT2 ΔLys210, ref. 23; and TNNI3 Ala2Val, ref. 24) that alter residues implicated in tight binary troponin interactions. Because troponin molecules modulate calcium-stimulated actomyosin ATPase activity, these defects may cause inefficient ATP hydrolysis and therein decrease contractile power.

Other DCM sarcomere mutations are more likely to impair force transmission (Figure 2). For example, a myosin mutation (at residue 764) located within the flexible fulcrum that transmits movement from the head of myosin to the thick filament is likely to render ineffectual the force generated by actomyosin interactions (23). DCM TPM1 mutations (25) are predicted to destabilize actin interactions and compromise force transmission to neighboring sarcomere. Likewise, ACTC mutations (26) that impair binding of actin to Z-disc may compromise force propagation. TTN mutations provide quintessential evidence that deficits in force transmission cause DCM and heart failure. By spanning the sarcomere from Z-disc to M-line, this giant muscle protein assembles contractile filaments and provides elasticity through serial spring elements. Titin interacts with α-actinin and telethonin (T-cap) at the Z-disc, with calpain3 and obscurin at the I-band (the extensible thin filament regions flanking Z-discs), and with myosin-binding protein-C, calmodulin, and calpain3 at the M-line region. Human mutations identified in

  • the Z-disc–I-band transition zone (27),
  • in the telethonin and α-actinin–binding domain, and
  • in the cardiac-specific N2B domain (an I-band subregion; ref. 28) each cause DCM and heart failure.

Intermediate filaments and dystrophin-associated glycoprotein mutations. Intermediate filaments function as cytoskeletal proteins linking the Z-disc to the sarcolemma. Desmin is a type III intermediate filament protein, which, when mutated, causes skeletal and cardiac muscle disease (Figure 3). The hearts of mice deficient in desmin (29) are more susceptible to mechanical stress, which is consistent with the function of intermediate proteins in force transmission.

Figure 3

Human mutations (orange text) in components of myocyte cytoarchitecture cause DCM and heart failure. Force produced by sarcomeric actin-myosin interactions is propagated through the actin cytoskeleton and dystrophin to the dystrophin-associated glycoprotein complex (composed of α- and β-dystroglycans, α-, β-, γ- and δ-sarcoglycans, caveolin-3, syntrophin, and dystrobrevin). Desmosome proteins plakoglobin, desmoplakin, and plakophilin-2, provide functional and structural contacts between adjacent cells and are linked through intermediate filament proteins, including desmin, to the nuclear membrane, where lamin A/C is localized. (Adapted from ref. 96.)

Through dystrophin and actin interactions, the dystrophin-associated glycoprotein complex (composed of α- and β-dystroglycans, α-, β-, γ- and δ-sarcoglycans, caveolin-3, syntrophin, and dystrobrevin) provides stability to the sarcomere and transmits force to the extracellular matrix. Human mutations in these proteins cause muscular dystrophy with associated DCM and heart failure (Figure 3). Skeletal muscle manifestations can be minimal in female carriers of X-linked dystrophin defects, and some individuals present primarily with heart failure (30). In the mouse experiment, coxsackievirus B3–encoded protease2A, which can cleave dystrophin, was shown to produce sarcolemmal disruption and cause DCM, which suggests that dystrophin is also involved in the pathologic mechanism of DCM and heart failure that follow viral myocarditis (31).

While deficiencies of proteins that link the sarcomere to the extracellular matrix are likely to impair force transmission, recent studies of mice engineered to carry mutations in these molecules indicate other mechanisms for heart failure. A model of desmin-related cardiomyopathies (32) uncovered striking intracellular aggresomes, electron dense accumulations of heat shock and chaperone protein, α-B-crystalline, desmin, and amyloid in association with sarcomeres. While particularly abundant in the amyloid heart, aggresomes were also found in some DCM and HCM specimens, which suggests that excessive degenerative processing induced by myocyte stress or gene mutation may be toxic to sarcomere function.

Analyses of δ-sarcoglycan null mice (33) also yielded unexpected disease mechanisms, primary coronary vasospasm and myocardial ischemia. Selective restoration of δ-sarcoglycan to the cardiac myocytes extinguished this pathology, thereby implicating chronic ischemia as a contributing factor to heart failure development in patients with sarcoglycan mutations.

Mutations in intercalated and Z-disc proteins. To generate contraction, one end of each actin thin filament must be immobilized. The Z-disc defines the lateral boundary of the sarcomere, where actin filaments, titin, and nebulette filaments are anchored. Metavinculin provides attachment of thin filaments to the plasma membrane and plays a key role in productive force transmission. Two metavinculin gene mutations cause DCM by disruption of disc structure and actin-filament organization (34).

Other Z-disc protein constituents may also function as mechano-stretch receptors (35). Critical components include α-actinin, which aligns actin and titin from neighboring sarcomeres and interacts with muscle LIM protein (MLP encoded by CSRP3), telethonin (encoded by TCAP), which interacts with titin and MLP to subserve overall sarcomere function, and Cypher/Z-band alternatively spliced PDZ-motif protein (Cypher/ZASP), a striated muscle-restricted protein that interacts with α-actinin–2 through a PDZ domain and couples to PKC-mediated signaling via its LIM domains (Figure 2). Mutations in these molecules cause either DCM (35, 36) or HCM (37, 38) and predispose the affected individuals to heart failure. Genetically engineered mice with MLP deficiency (39) help to model the mechanism by which mutations in distinct proteins cause disease. Without MLP, telethonin is destabilized and gradually lost from the Z-disc; as a consequence, MLP-deficient cardiac papillary muscle shows an impairment in tension generation following the delivery of a 10% increase in passive stretch of the muscle and a loss of stretch-dependent induction of molecular markers (e.g., brain natriuretic peptide), which suggests that an MLP-telethonin–titin complex is an essential component of the cardiac muscle mechanical stretch sensor machinery. An important question is how signaling proteins (e.g., Cyper/ZASP) within the Z-disc translate mechanosensing into activation of survival or cell death pathways.

Lamin A/C mutations. The inner nuclear-membrane protein complex contains emerin and lamin A/C. Defects in emerin cause X-linked Emery-Dreifuss muscular dystrophy, joint contractures, conduction system disease, and DCM. Dominant lamin A/C mutations exhibit a more cardiac-restricted phenotype with fibrofatty degeneration of the myocardium and conducting cells, although subclinical involvement of skeletal muscles and contractures are sometimes apparent. The remarkable electrophysiologic deficits (progressive atrioventricular block and atrial arrhythmias) observed in mutations of lamin A/C and emerin indicate the particular importance of these proteins in electrophysiologic cells. A recent study of lamin A/C mutant mice showed evidence of marked nuclear deformation, fragmentation of heterochromatin, and defects in mechanotransduction (40, 41), all of which likely contribute to reduced myocyte viability. The similarities of cardiac histopathology (fibrofatty degeneration) observed in mutations of the nuclear envelope and desmosomes raise the possibility that these structures may both function as important mechanosensors in myocytes (Figure 3).

Desmosome protein mutations. Arrhythmogenic right ventricular cardiomyopathy (ARVD) identifies an unusual group of cardiomyopathies characterized by progressive fibrofatty degeneration of the myocardium, electrical instability, and sudden death (42). While right ventricular dysplasia predominates, involvement of the left ventricle also occurs. Progressive myocardial dysfunction is seen late in the course of disease, often with right-sided heart failure. ARVD occurs in isolation or in the context of Naxos syndrome, an inherited syndrome characterized by prominent skin (palmar-plantar keratosis), hair, and cardiac manifestations. Mutations in protein components of the desmosomes (Figure 3) (plakoglobin, ref. 43; desmoplakin, refs. 44, 45; and plakophilin-2, ref. 46) and in the cardiac ryanodine receptor (RyR2) (ref. 47; discussed below) cause syndromic and nonsydromic ARVD. Desmosomes are organized cell membrane structures that provide functional and structural contacts between adjacent cells and that may be involved in signaling processes. Whether mutations in the desmosomal proteins render cells of the heart (and skin) inappropriately sensitive to normal mechanical stress or cause dysplasia via another mechanism is unknown.

Energy production and regulation

Mitochondrial mutations. Five critical multiprotein complexes, located within the mitochondria, synthesize ATP by oxidative phosphorylation. While many of the protein components of these complexes are encoded by the nuclear genome, 13 are encoded by the mitochondrial genome. Unlike nuclear gene mutations, mitochondrial gene mutations exhibit matrilineal inheritance. In addition, the mitochondrial genome is present in multiple copies, and mutations are often heteroplasmic, affecting some but not all copies. These complexities, coupled with the dependence of virtually all tissues on mitochondrial-derived energy supplies, account for the considerable clinical diversity of mitochondrial gene mutations (Figure 4). While most defects cause either dilated or hypertrophic cardiac remodeling in the context of mitochondrial syndromes such as Kearns-Sayre syndrome, ocular myopathy, mitochondrial encephalomyopathy with lactic-acidosis and stroke-like episodes (MELAS), and myoclonus epilepsy with ragged-red fibers (MERFF) (48), there is some evidence that particular mitochondrial mutations can produce predominant or exclusive cardiac disease (49, 50). An association between heteroplasmic mitochondrial mutations and DCM has been recognized (51).

Figure 4

Human gene mutations affecting cardiac energetics and metabolism. Energy substrate utilization is directed by critical metabolic sensors in myocytes, including AMP-activated protein kinase (AMPK), which, in response to increased AMP/ATP levels, phosphorylates target proteins and thereby regulates glycogen and fatty acid metabolism, critical energy sources for the heart. Glycogen metabolism involves a large number of proteins including α-galactosidase A (mutated in Fabry disease) and LAMP2 (mutated in Danon disease). Glycogen and fatty acids are substrates for multiprotein complexes located within the mitochondria for the synthesis of ATP. KATP channels composed of an enzyme complex and a potassium pore participate in decoding metabolic signals to maximize cellular functions during stress adaptation. Human mutations (orange text) that cause cardiomyopathies have been identified in the regulatory SUR2A subunit of KATP, the γ2 subunit of AMPK, mitochondrial proteins, α-galactosidase A, and LAMP2.

Nuclear-encoded metabolic mutations. Nuclear gene mutations affecting key regulators of cardiac metabolism are emerging as recognized causes of hypertrophic cardiac remodeling and heart failure (Figure 4). Mutations in genes encoding the γ2 subunit of AMP-activated protein kinase (PRKAG2), α-galactosidase A (GLA), and lysosome-associated membrane protein-2 (LAMP2) can cause profound myocardial hypertrophy in association with electrophysiologic defects (52). AMP-activated protein kinase functions as a metabolic-stress sensor in all cells. This heterotrimeric enzyme complex becomes activated during energy-deficiency states (low ATP, high ADP) and modulates (by phosphorylation) a large number of proteins involved in cell metabolism and energy (53). Most GLA mutations can cause multisystem classic Fabry disease (angiokeratoma, corneal dystrophy, renal insufficiency, acroparesthesia, and cardiac hypertrophy), but some defects produce primarily cardiomyopathy. LAMP2 mutations can also produce either multisystem Danon disease (with skeletal muscle, neurologic, and hepatic manifestations) or a more restricted cardiac phenotype.

Cardiac histopathology reveals that, unlike sarcomere gene mutations, which cause hypertrophic remodeling, the mutations in PRKAG2, LAMP2, and GLA accumulate glycogen in complexes with protein and/or lipids, thereby defining these pathologies as storage cardiomyopathies. Progression from hypertrophy to heart failure is particularly common and occurs earlier with LAMP2 mutations than with other gene mutations that cause metabolic cardiomyopathies. Since both GLA and LAMP2 are encoded on chromosome X, disease expression is more severe in men, but heterozygous mutations in women are not entirely benign, perhaps due to X-inactivation that equally extinguishes a normal or mutant allele. The cellular and molecular pathways that produce either profound hypertrophy or progression to heart failure from PRKAG2, GLA, or LAMP2 mutations are incompletely understood. While accumulated byproducts are likely to produce toxicity, animal models indicate that mutant proteins cause far more profound consequences by changing cardiac metabolism and altering cell signaling. This is particularly evident in PRKAG2 mutations that increase glucose uptake by stimulating translocation of the glucose transporter GLUT-4 to the plasma membrane, increase hexokinase activity, and alter expression of signaling cascades (54).

The cooccurrence of electrophysiologic defects in metabolic mutations raises the possibility that pathologic cardiac conduction and arrhythmias contribute to cardiac remodeling and heart failure in these gene mutations. One mechanism for electrophysiologic defects appears to be the direct consequence of storage: transgenic mice that express a human PRKAG2 mutation (55) developed ventricular pre-excitation due to pathologic atrioventricular connections by glycogen-filled myocytes that ruptured the annulus fibrosis (the normal anatomic insulator which separates atrial and ventricular myocytes). A second and unknown mechanism may be that these gene defects are particularly deleterious to specialized cells of the conduction system. Little is known about the metabolism of these cells, although historical histopathologic data indicate glycogen to be particularly more abundant in the conduction system than in the working myocardium (56–58).

Ca2+ Cycling

Considerable evidence indicates the presence of abnormalities in myocyte calcium homeostasis to be a prevalent and important mechanism for heart failure. Protein and RNA levels of key calcium modulators are altered in acquired and inherited forms of heart failure, and human mutations in molecules directly involved in calcium cycling have been found in several cardiomyopathies (Figure 5).

Figure 5

Human mutations affecting Ca2+ cycling proteins. Intracellular Ca2+ handling is the central coordinator of cardiac contraction and relaxation. Ca2+ entering through L-type channels (LTCC) triggers Ca2+ release (CICR) from the SR via the RyR2, and sarcomere contraction is initiated. Relaxation occurs with SR Ca2+ reuptake through the SERCA2a. Calstabin2 coordinates excitation and contraction by modulating RyR2 release of Ca2+. PLN, an SR transmembrane inhibitor of SERCA2a modulates Ca2+ reuptake. Dynamic regulation of these molecules is effected by PKA-mediated phosphorylation. Ca2+ may further function as a universal signaling molecule, stimulating Ca2+-calmodulin and other molecular cascades. Human mutations (orange text) in molecules involved in calcium cycling cause cardiac remodeling and heart failure. NCX, sodium/calcium exchanger.

Calcium enters the myocyte through voltage-gated L-type Ca2+ channels; this triggers release of calcium from the sarcoplasmic reticulum (SR) via the RyR2. Emerging data define FK506-binding protein (FKBP12.6; calstabin2) as a critical stabilizer of RyR2 function (59), preventing aberrant calcium release during the relaxation phase of the cardiac cycle (Figure 5). Stimuli that phosphorylate RyR2 (such as exercise) by protein kinase A (PKA) dissociate calstabin2 from the receptor, thereby increasing calcium release and enhancing contractility. At low concentrations of intracellular calcium, troponin I and actin interactions block actomyosin ATPase activity; increasing levels foster calcium binding to troponin C, which releases troponin I inhibition and stimulates contraction. Cardiac relaxation occurs when calcium dissociates from troponin C, and intracellular concentrations decline as calcium reuptake into the SR occurs through the cardiac sarcoplasmic reticulum Ca2+-ATPase pump (SERCA2a). Calcium reuptake into SR is regulated by phospholamban (PLN), an inhibitor of SERCA2a activity that when phosphorylated dissociates from SERCA2a and accelerates ventricular relaxation.

RyR2 mutations. While some mutations in the RyR2 are reported to cause ARVD (47) (see discussion of desmosome mutations), defects in this calcium channel are more often associated with catecholaminergic polymorphic ventricular tachycardia (60, 61), a rare inherited arrhythmic disorder characterized by normal heart structure and sudden cardiac death during physical or emotional stress. Mutations in calsequestrin2, an SR calcium-binding protein that interacts with RyR2, also cause catecholaminergic polymorphic ventricular tachycardia (62, 63). Whether the effect of calsequestrin2 mutations directly or indirectly alters RyR2 function is unknown (Figure 5).

While RyR2 mutations affect residues in multiple functional domains of the calcium channel, those affecting residues involved in calstabin2-binding provide mechanistic insights into the substantial arrhythmias found in affected individuals. Mutations that impair calstabin2-binding may foster calcium leak from the SR and trigger depolarization. Diastolic calcium leak can also affect excitation-contraction coupling and impair systolic contractility.

Studies of mice deficient in FKBP12.6 (64) confirmed the relevance of SR calcium leak from RyR2 to clinically important arrhythmias. RyR2 channel activity in FKBP12.6-null mice was significantly increased compared with that of wild-type mice, consistent with a diastolic Ca2+ leak. Mutant myocytes demonstrated delayed after-depolarizations, and exercise-induced syncope, ventricular arrhythmias, and sudden death were observed in FKBP12.6-null mice.

Calcium dysregulation is also a component of hypertrophic remodeling that occurs in sarcomere gene mutations. Calcium cycling is abnormal early in the pathogenesis of murine HCM (65, 66): SR calcium stores are decreased and calcium-binding proteins and RyR2 levels are diminished. Whether calcium changes contribute to ventricular arrhythmias in mouse and human HCM remains an intriguing question.

Related mechanisms may contribute to ventricular dysfunction and arrhythmias in acquired forms of heart failure, in which chronic phosphorylation of RyR2 reduces calstabin2 levels in the channel macromolecular complex and increases calcium loss from SR stores. These data indicate the potential benefit of therapeutics that improve calstabin2-mediated stabilization of RyR2 (67, 68); such agents may both improve ventricular contractility and suppress arrhythmias in heart failure.

PLN mutations. Rare human PLN mutations cause familial DCM and heart failure (69, 70). The pathogenetic mechanism of one mutation (PLN Arg9Cys) was elucidated through biochemical studies, which indicated unusual PKA interactions that inhibited phosphorylation of mutant and wild-type PLN. The functional consequence of the mutation was predicted to be constitutive inhibition of SERCA2a, a result confirmed in transgenic mice expressing mutant, but not wild-type, PLN protein. In mutant transgenic mice, calcium transients were markedly prolonged, myocyte relaxation was delayed, and these abnormalities were unresponsive to β-adrenergic stimulation. Profound biventricular cardiac dilation and heart failure developed in mutant mice, providing clear evidence of the detrimental effects of protracted SERCA2a inhibition due to excess PLN activity.

The biophysical consequences accounting for DCM in humans who are homozygous for a PLN null mutation (Leu39stop; ref. 70) are less clear. PLN-deficient mice show increased calcium reuptake into the SR and enhanced basal contractility (71). Indeed, these effects on calcium cycling appear to account for the mechanism by which PLN ablation rescues DCM in MLP-null mice (72). However, normal responsiveness to β-adrenergic stimulation is blunted in PLN-deficient myocytes, and cells are less able to recover from acidosis that accompanies vigorous contraction or pathologic states, such as ischemia (73). The collective lesson from human PLN mutations appears to be that too little or too much PLN activity is bad for long-term heart function.

Acquired causes of heart failure are also characterized by a relative decrease in SERCA2a function due to excessive PLN inhibition. Downregulation of β-adrenergic responsiveness attenuates PLN phosphorylation, which compromises calcium reuptake and depletes SR calcium levels, which may impair contractile force and enhance arrhythmias. Heterozygote SERCA2 null mice are a good model of this phenotype and exhibit impaired restoration of SR calcium with deficits in systolic and diastolic function (74).

Cardiac ATP-sensitive potassium channel mutations. In response to stress such as hypoxia and ischemia, myocardial cells undergo considerable changes in metabolism and membrane excitability. Cardiac ATP-sensitive potassium channels (KATP channels) contain a potassium pore and an enzyme complex that participate in decoding metabolic signals to maximize cellular functions during stress adaptation (Figure 4) (75). KATP channels are multimeric proteins containing the inwardly rectifying potassium channel pore (Kir6.2) and the regulatory SUR2A subunit, an ATPase-harboring, ATP-binding cassette protein. Recently, human mutations in the regulatory SUR2A subunit (encoded by ABCC9) were identified as a cause of DCM and heart failure (76). These mutations reduced ATP hydrolytic activities, rendered the channels insensitive to ADP-induced conformations, and affected channel opening and closure. Since KATP-null mouse hearts have impaired response to stress and are susceptible to calcium overload (75), some of the pathophysiology of human KATP mutations (DCM and arrhythmias) may reflect calcium increases triggered by myocyte stress.

Transcriptional Regulators

Investigation of the molecular controls of cardiac gene transcription has led to the identification of many key molecules that orchestrate physiologic expression of proteins involved in force production and transmission, metabolism, and calcium cycling. Given that mutation in the structural proteins involved in these complex processes is sufficient to cause cardiac remodeling, it is surprising that defects in transcriptional regulation of these same proteins have not also been identified as primary causes of heart failure. Several possible explanations may account for this. Transcription factor gene mutations may be lethal or may at least substantially impair reproductive fitness so as to be rapidly lost. The consequences of transcription factor gene mutations may be so pleiotropic that these cause systemic rather than single-organ disease. Changes in protein function (produced by a structural protein mutation) may be more potent for remodeling than changes in levels of structural protein (produced by transcription factor mutation). While many other explanations may be relevant, the few human defects discovered in transcriptional regulators that cause heart failure provide an important opportunity to understand molecular mechanisms for heart failure.

Nkx2.5 mutations. The homeodomain-containing transcription factor Nkx2.5, a vertebrate homolog of the Drosophila homeobox gene tinman, is one of the earliest markers of mesoderm. When Nkx2.5 is deleted in the fly, cardiac development is lost (77). Targeted disruption of Nkx2.5 in mice (Nkx2.5–/–) causes embryonic lethality due to the arrested looping morphogenesis of the heart tube and growth retardation (78, 79). Multiple human dominant Nkx2.5 mutations have been identified as causing primarily structural malformations (atrial and ventricular septation defects) accompanied by atrioventricular conduction delay, although cardiac hypertrophic remodeling has also been observed (80). Although the mechanism for ventricular hypertrophy in humans with Nkx2.5 mutations is not fully understood, the pathology is unlike that found in HCM, which perhaps indicates that cardiac hypertrophy is a compensatory event. Several human Nkx2.5 mutations have been shown to abrogate DNA binding (81), which suggests that the level of functional transcription factor is the principle determinant of structural phenotypes. Heterozygous Nkx2.5+/– mice exhibit only congenital malformations with atrioventricular conduction delay (82, 83). Remarkably, however, transgenic mice expressing Nkx2.5 mutations develop profound cardiac conduction disease and heart failure (84) and exhibit increased sensitivity to doxorubicin-induced apoptosis (85), which suggests that this transcription factor plays an important role in postnatal heart function and stress response.

Insights into transcriptional regulation from mouse genetics. Dissection of the combinatorial mechanisms that activate or repress cardiac gene transcription has led to the identification of several key molecules that directly or indirectly lead to cardiac remodeling. While human mutations in these genes have not been identified, these molecules are excellent candidates for triggering cell responses to structural protein gene mutations.

Hypertrophic remodeling is associated with reexpression of cardiac fetal genes. Molecules that activate this program may also regulate genes that directly cause hypertrophy. Activation of calcineurin (Ca2+/calmodulin-dependent serine/threonine phosphatase) results in dephosphorylation and nuclear translocation of nuclear factor of activated T cells 3 (NFAT3), which, in association with the zinc finger transcription factor GATA4, induces cardiac fetal gene expression. Transgenic mice that express activated calcineurin or NFAT3 in the heart develop profound hypertrophy and progressive decompensation to heart failure (86), responses that were prevented by pharmacologic inhibition of calcineurin. Although these data implicated NFAT signaling in hypertrophic heart failure, pharmacologic inhibition of this pathway fails to prevent hypertrophy caused by sarcomere gene mutations in mice and even accelerates disease progression to heart failure (65). Mice lacking calsarcin-1, which is localized with calcineurin to the Z-disc, showed an increase in Z-disc width, marked activation of the fetal gene program, and exaggerated hypertrophy in response to calcineurin activation or mechanical stress, which suggests that calsarcin-1 plays a critical role in linking mechanical stretch sensor machinery to the calcineurin-dependent hypertrophic pathway (87).

Histone deacetylases (HDACs) are emerging as important regulators of cardiac gene transcription. Class II HDACs (4/5/7/9) bind to the cardiac gene transcription factor MEF2 and inhibit MEF2-target gene expression. Stress-responsive HDAC kinases continue to be identified but may include an important calcium-responsive cardiac protein, calmodulin kinase. Kinase-induced phosphorylation of class II HDACs causes nuclear exit, thereby releasing MEF2 for association with histone acetyltransferase proteins (p300/CBP) and activation of hypertrophic genes. Mice deficient in HDAC9 are sensitized to hypertrophic signals and exhibit stress-dependent cardiac hypertrophy. The discovery that HDAC kinase is stimulated by calcineurin (88) implicates crosstalk between these hypertrophic signaling pathways.

Recent attention has also been focused on Hop, an atypical homeodomain-only protein that lacks DNA-binding activity. Hop is expressed in the developing heart, downstream of Nkx2-5. While its functions are not fully elucidated, Hop can repress serum response factor–mediated (SRF-mediated) transcription. Mice with Hop gene ablation have complex phenotypes. Approximately half of Hop-null embryos succumb during mid-gestation with poorly developed myocardium; some have myocardial rupture and pericardial effusion. Other Hop-null embryos survive to adulthood with apparently normal heart structure and function. Cardiac transgenic overexpression of epitope-tagged Hop causes hypertrophy, possibly by recruitment of class I HDACs that may inhibit anti-hypertrophic gene expression (89–92).

PPARα plays important roles in transcriptional control of metabolic genes, particularly those involved in cardiac fatty acid uptake and oxidation. Mice with cardiac-restricted overexpression of PPARα replicate the phenotype of diabetic cardiomyopathy: hypertrophy, fetal gene activation, and systolic ventricular dysfunction (93). Heterozygous PPARγ-deficient mice, when subjected to pressure overload, developed greater hypertrophic remodeling than wild-type controls, implicating the PPARγ-pathway as a protective mechanism for hypertrophy and heart failure (94).

Retinoid X receptor α (RXRα) is a retinoid-dependent transcriptional regulator that binds DNA as an RXR/retinoic acid receptor (RXR/RAR) heterodimer. RXRα-null mice die during embryogenesis with hypoplasia of the ventricular myocardium. In contrast, overexpression of RXRα in the heart does not rescue myocardial hypoplasia but causes DCM (95).

Integrating Functional and Molecular Signals

Study of human gene mutations that cause HCM and DCM provides information about functional triggers of cardiac remodeling. In parallel with evolving information about molecular-signaling cascades that influence cardiac gene expression, there is considerable opportunity to define precise pathways that cause the heart to fail. To understand the integration of functional triggers with molecular responses, a comprehensive data set of the transcriptional and proteomic profiles associated with precise gene mutations is needed. Despite the plethora of information associated with such studies, bioinformatic assembly of data and deduction of pathways should be feasible and productive for defining shared or distinct responses to signals that cause cardiac remodeling and heart failure. Accrual of this data set in humans is a desirable goal, although confounding clinical variables and tissue acquisition pose considerable difficulties that can be more readily addressed by study of animal models with heart disease. With more knowledge about the pathways involved in HCM and DCM, strategies may emerge to attenuate hypertrophy, reduce myocyte death, and diminish myocardial fibrosis, processes that ultimately cause the heart to fail.

CardioGenomics. Genomics of Cardiovascular Development, Adaptation, and Remodeling.

NHLBI program for genomic applications. Harvard Medical School. http://cardiogenomics.med.harvard.edu

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Cardiovascular Autonomic Dysfunction and  Predicting Outcomes in Diabetes

Marlene Busko  Aug 27, 2013   http://www.medscape.com/viewarticle/810063?src=wnl_edit_tpal&uac=62859DN

Autonomic Dysfunction and Risk of a CV Event   In patients with CAD and type 2 diabetes, autonomic dysfunction is common, but its prognostic value is unknown.

data  a substudy of patients enrolled in the ARTEMIS trial

,530 patients with CAD and diabetes matched with 530 patients with CAD without diabetes. The patients had a mean age of 67, and 69% were males

patients performed a test on an exercise bicycle, which allowed the researchers to determine their heart-rate recovery, defined as the drop in heart rate from the rate at maximal exercise to the rate one minute after stopping the exercise  In univariate analysis, among patients with CAD and type 2 diabetes, those who had a blunted heart-rate recovery after exercise–defined as a drop in heart rate of less than 21 beats per minute–had a 1.69-fold greater risk having a cardiovascular event than their peers.  Similarly, those with blunted heart-rate turbulence (<3.4 ms/R-R interval) had a 2.08-fold increased risk of an event, and those with low heart-rate variability (<110 ms) had a 1.96-fold greater risk of having a cardiovascular event.  After multivariate analysis, C-reactive protein (CRP), but none of the three measures of autonomic function, still predicted an increased risk of having a cardiovascular event during this short follow-up.

During a two-year follow-up, 127 patients (13%) reached the composite end point of a cardiovascular event, which included

  • cardiovascular death (2%),
  • acute coronary event (8%),
  • stroke (3%), or
  • hospitalization for heart failure (2%).

B. Autonomic Dysfunction and Risk of Severe Hypoglycemia

Dr Seung-Hyun Ko (Catholic University of Korea, Gyeonggi-do, South Korea

data  894 consecutive patients with type 2 diabetes, aged 25 to 75

heart-rate variability measured at three times: during a Valsalva maneuver, deep breathing, and going from lying down to standing.   During close to 10 years of  follow-up, 77 episodes of severe hypoglycemia occurred among 62 patients (9.9%). About 16% of patients were diagnosed with early autonomic dysfunction and another 15% were diagnosed with definite autonomic dysfunction.  Patients with type 2 diabetes and definite autonomic dysfunction were more than twice as likely to have an episode of severe hypoglycemia as those with normal autonomic function (HR 2.43).

patient education concerning hypoglycemia is essential for patients with definite [cardiovascular autonomic neuropathy] to prevent [severe hypoglycemia] and related mortality

Measurement of heart-rate turbulence (HRT), an ECG phenomenon that reflects hemodynamic responses to premature ventricular contractions (PVCs), can risk-stratify patients in the post-MI setting and may be similarly useful in heart failure or other heart disease, according to a state-of-the-art review in the October 21, 2008 issue of the Journal of the American College of Cardiology [1]. “Several large-scale retrospective and prospective studies have established beyond any doubt that HRT is one of the strongest independent risk predictors after MI. It thus appears that the stage has now been reached when HRT might be used in large prospective intervention studies,” according to the authors, led by Dr Axel Bauer (Deutsches Herzzentrum, Munich, Germany). The group had been asked to write the review by the International Society for Holter and Noninvasive Electrophysiology (ISHNE), it states. HRT, first published as a potential CV risk stratifier in 1999 [2], and other measures of autonomic function aren’t as well established or even studied as much as some other prognostic markers based on electrocardiography, such as T-wave alternans. As the authors note, it’s usually measured from an average of multiple PVCs on 24-hour Holter monitoring.

The strongest support for the parameter’s risk-stratification role comes from “six large-scale studies and from two prospective studies, both of which have been specifically designed to validate the prognostic value of HRT in post-MI patients receiving state-of-the-art treatment,” the report states.

Other evidence suggests a role for HRT evaluation after PCI to assess the strength of perfusion from the treated coronary artery. “Persistent impairment of HRT after PCI in patients with incomplete reperfusion implies prolonged baroreflex impairment and is consistent with poor prognosis,” write Bauer et al. “Thus, early assessment of HRT may be detecting pathological loss of reflex autonomic response due to incomplete reperfusion or severe microvascular dysfunction after PCI.  In heart failure, according to the authors, patients “are known to have significantly impaired baroreflex sensitivity as well as reduced heart-rate variability. . . . This may suggest the possibility of guiding pharmacological therapy [according to HRT responses] in heart-failure patients.” They also note that the prognostic power of HRT in heart failure appears limited to patients with ischemic cardiomyopathy.

Bauer A, Malik M, Schmidt G, et al. Heart rate turbulence: Standards of measurement, physiological interpretation, and clinical use. International Society for Holter and Noninvasive Electrophysiology consensus. J Am Coll Cardiol 2008; 52:1353–1365.

Schmidt G, Malik M, Barthel P, et al. Heart-rate turbulence after ventricular premature beats as a predictor of mortality after acute myocardial infarction. Lancet 1999; 353:1390–1396. Abstract

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Pacemakers, Implantable Cardioverter Defibrillators (ICD) and Cardiac Resynchronization Therapy (CRT)

Curators: Justin D Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN

Updated on 2/16/2015

Mild, non-ischemic heart failure might be more deadly than thought, an Austrian group found, calling for broader ICD use.




The voice of our Series A Content Consultant: Justin D Pearlman, MD, PhD, FACC

Pacemakers place one or more wires into heart muscle to trigger electro-mechanically coupled contraction. A single wire to the right atrium is called an AAI pacemaker (atrial sensing, atrial triggering, inhibit triggering if sensed). A single wire to the right ventricle is called a VVI pacemaker (ventricular sensing, ventricular triggering, inhibit if sensed). With two wires to the heart more combinations are possible, including atrial-ventricular sequential activation, a closer mimic to normal function (DDDR pacemaker: dual sensing, dual triggering, dual functions, and rate-responsive to mimic exercise adjustment of heart rate). Three wires are used for synchronization: one to the right atrium, one to the right ventricle apex, and a third lead into a distal branch of the coronary sinus to activate the far side of the left ventricle. Resynchronization is used to compensate for a dilated ventricle, especially one with conduction delays, where the timing of activation is so unbalanced that the heart contraction approaches a wobbling motion rather than a well coordinated contraction. Adjusting timing of activation of the right ventricle and left ventricle can offset dysynchrony (unbalanced timing) and thereby increase the amount of blood ejected by each heart beat contraction (ejection fraction). Patients with dilated cardiomyopathy and significant conduction delays can improve the ejection fraction by 10 or more percentage points, which offers a significant improvement in exertion tolerance and heart failure symptoms.

Patients with ejection fraction below 35%, among others, have an elevated risk of life-ending arrhythmias such as ventricular tachycardia. Ventricular tachycardia is an extreme example of a wobbling heart in which the electrical activation sequence circles around the heart sequentially activating a portion and blocking its ability to respond until the electric signal comes around again. Whenever a portion of the heart is activated, ions shift location, and further activation of that region is not possible until sufficient time passes so that the compartmentalized ion concentrations can be restored (repolarization). Pacing can interrupt ventricular tachycardia by depolarizing a region that supported the circular activation pattern. Failing that, an electric shock can stop an ineffective rhythm. After all regions stop activation, they will generally reactivate in the normal pulsatile synchronous manner. An implanted cardiac defibrillator is a device designed to apply an internal electric shock to pause all activation and thereby interrupt ventricular tachycardia.
UPDATED on 12/31/2013

Published on Friday, 27 December 2013

S-ICD – Subcutaneous Implantable Cardioverter Defibrillator – Boston Scientific

Boston Scientific Subcutaneous Implantable Cardiodefibrillator Device S-ICD

S-ICD – Subcutaneous Implantable Cardioverter Defibrillator – Boston Scientific

Boston Scientific Subcutaneous Implantable Cardiodefibrillator Device S-ICD

‘Regular’ Pacemaker/ICD with Leads and a ‘Can’
When we think of Pacemakers and ICD’s we naturally think of a ‘Can’ and Leads that track down into the heart. Whilst these devices work fantastically well and will continue to do so. Unfortunately the ‘lead’ part of the device opens the door for a few complications to possibly arise. Those who have a Pacemaker or ICD will probably be familiar with concerns over;
  1. Systemic Infection – Infections travelling down the Leads into the Heart
  2. Lead Displacement – The Lead moving away from the heart tissue and thus becoming pretty useless.
  3. Vascular/Organ Injury – Damage to the blood vessels being used for access or perforation of heart wall.
  4. Pneumothorax (damage to the lining around the Lung), Haemothorax (build up of blood in the chest cavity), and air embolism (air bubble trapped in a blood vessel).
These complications are one of the key motivations behind developing ‘leadless’ devices the first of which the St Jude Nanostim, a small VVI Pacemaker that fits directly into the heart.
Another device to address these issues is the Boston Scientific S-ICD

What is the Boston Scientific S-ICD?

The S-ICD is what is sometimes referred to as a ‘shock box’ it does not have the pacemaker functionality that many other ICD’s do have. It is ONLY there to terminate dangerous Arrhythmias.
*It does not have the pacing functionality of traditional ICD‘s because it DOES NOT HAVE A LEAD THAT ENTERS THE HEART.*
It is not a Pacemaker!
Without the lead(s) ENTERING the heart via a blood vessel there is a reduction in the risks mentioned previously that are associated traditional device. Another of the benefits is that the S-ICD is positioned and implanted using anatomical landmarks (visible parts of your body) and not Fluoroscopy (video X-Ray) which reduces radiation exposure to the patient.

Positioning of the S-ICD.

Boston Scientific Subcutaneous Implantable Cardiodefibrillator Device S-ICD

The ‘Can‘ (metal box that contains all the circuitry and battery), is buried under the skin on the outside of the ribs. Put your arms down by your sides, the device would go where your ribs meet the middle of your bicep. A lead is then run under the skin to the centre of your chest where its is anchored and then north, under the skin again until the tip of the lead is roughly at the top of the sternum.
For you physicians out there the ‘can’ is positioned at the mid-axillary line between the 5th and 6th intercostal spaces, the lead is then tunnelled to a small Xiphoid incision and then tunnelled north to a superior incision.

How is an S-ICD Implanted?

Having spoken to Boston Scientific it is becoming more apparent that the superior incision (cut at the top of the chest) may actually be removed from the procedure guidance as simply tunnelling the lead and ‘wedging’ the tip at that point is satisfactory – THIS IS NOT CONFIRMED AT THE MOMENT AND IS THEREFORE NOT PROCEDURE ADVICE.
Boston Scientific Subcutaneous Implantable Cardiodefibrillator Device S-ICD
Image Courtesy of

How does the S-ICD Work?

A ‘Shock Box’ basically needs to do 2 things. Firstly be able to SENSE if the heart has entered a Dangerous Arrhythmia and Secondly, be able to treat it.
The treatment part of the functionality is the easy bit – it delivers an electric shock across a ‘circuit’ that involves a large amount of the tissue in the heart. The lead has two ‘electrodes’ and the ‘Can’ is a third electrode allowing you different shocking ‘vectors’. By vectors we mean directions and area through which the electricity travels during a shock. This gives us extra options when implanting a device as some vectors will work better than others for the treatment of dangerous arrhythmias.

Shocking Vectors?

This is a concept you are familiar with without even thinking about it… when you are watching ER or another TV program and they Defibrillate the patient using the metal paddles, where do they position them? One either side of the heart? Precisely!! this is creating a ‘vector’ across the heart to involve the cardiac tissue. The paddles would be a lot less effective if you put one on the knee and one on the foot!

Boston Scientific Subcutaneous Implantable Cardiodefibrillator Device S-ICD

Now because the ‘Vectors’ used by the S-ICD are over a larger area than those with a traditional device – more energy has to be delivered to have the same desired affect. The upshot of this is that a larger battery is required to deliver the 80J! Bigger Battery = Bigger Box. This image shows a demo device but this is the exact size compared to a One Pound Coin! Now yes it is big but because of the extra room where they place the device it is pretty discrete and hidden in even slender patients.
The S-ICD System delivers up to 5 shocks per episode at 80 J with up to 128 seconds of ECG storage per episode and storage of up to 45 episodes.
The heart rate that the S-ICD is told to deliver therapy is programable between 170 and 250 bpm. Quite cleverly the device is able to also deliver a small amount of ‘pacing’ after a shock, when the heart can often run slowly. This is external pacing and will be felt!! It can run for 30s.

Sensing in an S-ICD.

The S-ICD uses its electrodes to produce an ECG similar to a surface ECG. 
Now the Sensing functionality is the devices ability to determine what Rhythm the heart is in! Without a lead in the heart to give us really accurate information the device is using a large area of heart, ribs and muscle. This means there is more potential for ‘artefact’. Artefact is the electrical interference and confusion – that could potentially lead to a patient being shocked when they do not require it – or not being shocked when they do…
Boston Scientific have come up with a very clever software/algorithm called ‘Insight’. Insight uses 3 separate methods to determine the nature of a heart rhythm.
  • Normal Sinus Rhythm Template (Do your heart beats look as they should)
  • Dynamic Morphology Analysis (A live comparison of heart beat to previous heart beat, do they all look the same or do they keep changing?)
  • QRS Width analysis (Are the tall ‘peaks’ on your ECG, the QRS’, wider than they normally are?)
These questions (with some very complex maths) and the rate of a rhythm are used to decide whether to ‘shock’ or not.
Insight Algorithm S-ICD

Image Courtesy of  http://www.bostonscientific.com/

How does Insight and the S-ICD compare to other ICD Devices?

The statistics for treatment success and inappropriate shocks (an electrocuted patient that did not need to be) actually compare very similarly if not favourably compared to other devices on the market – these two studies are well worth a read if you have the time 🙂
1. Burke M, et al. Safety and Efficacy of a Subcutaneous Implantable-Debrillator (S-ICD System US IDE Study). Late-Breaking Abstract Session. HRS 2012.
2. Lambiase PD, et al. International Experience with a Subcutaneous ICD; Preliminary Results of the EFFORTLESS S-ICD Registry. Cardiostim 2012.
3. Gold MR, et al. Head-to-head comparison of arrhythmia discrimination performance of subcutaneous and transvenous ICD arrhythmia detection algorithms: the START study. J Cardiovasc Electrophysiol. 2012;23;4:359-366.
Who qualifies?
Template S-ICD Eligibility

Template used to assess eligibility!
Image Courtesy of
Well essentially anyone who qualifies for a normal ‘shock box’ ICD but with one other requirement. The Insight Software requires that a person has certain characteristics on their ECG. This is essentially showing that they have tall enough and narrow enough complexes to allow the algorithm to perform effectively. A simple 12 lead ECG Laying and Standing will be obtained and then a ‘Stencil’ is passed over the Print out – If the complexes fit within the boundaries marked on the ‘stencil’ then you potentially qualify. If your ECG does not meet requirements then it will not be recommended for you to have the S-ICD.

There you have it a quick overview of the Boston Scientific S-ICD.

Thanks for Reading

Cardiac Technician



UPDATED on 10/15/2013

Frequency and Determinants of Implantable Cardioverter Defibrillator Deployment Among Primary Prevention Candidates With Subsequent Sudden Cardiac Arrest in the Community

  1. Kumar Narayanan, MD;
  2. Kyndaron Reinier, PhD;
  3. Audrey Uy-Evanado, MD;
  4. Carmen Teodorescu, MD, PhD;
  5. Harpriya Chugh, BS;
  6. Eloi Marijon, MD;
  7. Karen Gunson, MD;
  8. Jonathan Jui, MD, MPH;
  9. Sumeet S. Chugh, MD

+Author Affiliations

  1. From The Heart Institute, Cedars-Sinai Medical Center, Los Angeles, CA (K.N., K.R., A.U.-E., C.T., H.C., E.M., S.S.C.); and Departments of Pathology (K.G.) and Emergency Medicine (J.J.), Oregon Health and Science University, Portland, OR.
  1. Correspondence to Sumeet S. Chugh, MD, Cedars-Sinai Medical Center, The Heart Institute, AHSP Suite A3100, 127 S. San Vicente Blvd., Los Angeles, CA 90048, Los Angeles, CA 90048. E-mail sumeet.chugh@cshs.org


Background—The prevalence rates and influencing factors for deployment of primary prevention implantable cardioverter defibrillators (ICDs) among subjects who eventually experience sudden cardiac arrest in the general population have not been evaluated.

Methods and Results—Cases of adult sudden cardiac arrest with echocardiographic evaluation before the event were identified from the ongoing Oregon Sudden Unexpected Death Study (population approximately 1 million). Eligibility for primary ICD implantation was determined from medical records based on established guidelines. The frequency of prior primary ICD implantation in eligible subjects was evaluated, and ICD nonrecipients were characterized. Of 2093 cases (2003–2012), 448 had appropriate pre– sudden cardiac arrest left ventricular ejection fraction information available. Of these, 92 (20.5%) were eligible for primary ICD implantation, 304 (67.9%) were ineligible because of left ventricular ejection fraction >35%, and the remainder (52, 11.6%) had left ventricular ejection fraction ≤35% but were ineligible on the basis of clinical guideline criteria. Among eligible subjects, only 12 (13.0%; 95% confidence interval, 6.1%–19.9%) received a primary ICD. Compared with recipients, primary ICD nonrecipients were older (age at ejection fraction assessment, 67.1±13.6 versus 58.5±14.8 years, P=0.05), with 20% aged ≥80 years (versus 0% among recipients, P=0.11). Additionally, a subgroup (26%) had either a clinical history of dementia or were undergoing chronic dialysis.

Conclusions—Only one fifth of the sudden cardiac arrest cases in the community were eligible for a primary prevention ICD before the event, but among these, a small proportion (13%) were actually implanted. Although older age and comorbidity may explain nondeployment in a subgroup of these cases, other determinants such as socioeconomic factors, health insurance, patient preference, and clinical practice patterns warrant further detailed investigation.

Key Words:

  • Received March 11, 2013.
  • Accepted August 21, 2013


UPDATED on 9/15/2013

based on 9/6/2013 Trials and Fibrillations — The Heart.org


Echo-CRT trial: Most important study released at ESC 2013

Cardiac resynchronization therapy (CRT) is a multilead pacing device that can extend lives and improve the quality of life of selected patients who suffer from reduced performance of the heart due to adverse timing of contraction (wobbling motion from conduction delays that cause asynchrony or  delayed activation of one portion of the left ventricle compared to others reducing net blood ejection).

The degree of benefit in CRT responders depends not only on the degree of asynchrony, but also on the delayed activity location in relation to the available locations for lead placement. CRT is an adjustment in the timing of muscle activiation to improve the concerted impact on blood ejection. Only patients likely to improve should be exposed to the risks and costs of CRT.

The Echo-CRT trial, presented September 3, 2013 at the European Society of Cardiology (ESC) 2013 Congressand simultaneously published in the New England Journal of Medicine, helps identify which patients may benefit from CRT devices. (See Steve Stiles’ report on heartwire),

Echo-CRT trial summary

Background is important

Previous CRT studies enrolled patients with QRS duration >120 or >130 ms for synchronizing biventricular pacing. Additional work confirmed the greatest benefit occurred in patients with QRS durations >150 ms and typical left bundle branch block (LBBB). Conflicting observational and small randomized trials were less clear for patients with shorter QRS durations—the majority of heart-failure patients. What’s more, most cardiologists have seen patients with “modest” QRS durations respond to CRT. In theory, wide QRS is only expected if the axis of significant delay projects onto the standard ECG views, whereas significant opportunity for benefit can be missed if the axis of significant delay is not wide in the standard views. CRT implanters have heard of patients with normal-duration QRS where echo shows marked dyssynchrony. This raised the  question: Are there CHF patients with mechanical dyssynchrony (determined by echo) but no electrical delay (as measured by the ECG) benefit from CRT?Unfortunately, echo does not resolve the issue either. Thus there is the residual question of who should be evaluated by a true 3D syncrhony assessment by cardiac MRI.

Echocardiographic techniques held promise to identify mechanical dyssynchrony, but like the standard 12 lead ECG, they also utilize limited orientations of views of the heart and hence the directions in which delays can be detected. Cardiac MRI Research (not limited in view angle) by JDPearlman showed that the axis of maximal delay in patients with asynchrony is within 30 degrees of the ECG and echo views in a majority of patients with asynchrony, but it can be 70-110 degrees away from the views used by echocardiography and by ECG in 20% of cases. Hence some patients who may benefit can be missed by ECG or Echo criteria.


Echo-CRT was an industry-sponsored (Biotronik) investigator-initiated prospective international randomized controlled trial. All patients had mechanical dyssynchrony by echo, QRS <130 ms, and an ICD indication. CRT-D devices were implanted in all patients. Blinded randomization to CRT-on (404 patients) vs CRT-off (405 patients) was performed after implantation. Programming in the CRT-off group was set to minimize RV pacing. The primary outcome was a composite of all-cause mortality or hospitalization.

Six key findings

1. Although entry criteria for the trial was a QRS duration <130 ms, the mean QRS duration of both groups was 105 ms.

2. The data safety monitoring board terminated the trial prematurely because of an increased death rate in the CRT group.

3. No differences were noted in the primary outcome.

4. More patients died in the CRT group (hazard ratio=1.8).

5. The higher death rate in the CRT group was driven by cardiovascular death.

6. More patients in the CRT group were hospitalized, due primarily to device-related issues.

These findings send clear and simple messages to all involved with treating patients with heart failure. My interpretation of Echo-CRT is as follows:

Do not implant CRT devices in patients with “narrow” QRS complexes.

The signal of increased death was strong. A hazard ratio of 1.8 translates to an almost doubling of the risk of death. This finding is unlikely to be a statistical anomaly, as it was driven by CV death. The risks of CRT in nonresponders are well-known and include: increased RV pacing, possible proarrhythmia from LV pacing, and the need for more device-related surgery. Patients who do not respond to CRT get none of the benefits but all the potential harms—an unfavorable ratio indeed.

Echo is not useful for assessing dyssynchrony in patients with narrow QRS complexes.

Dr Samuel Asirvatham explains the concept of electropathy in a review article in the Journal of Cardiovascular Electrophysiology. He teaches us that the later the LV lateral wall is activated relative to the RV, the more the benefit of preexciting the lateral wall with an LV lead. That’s why the benefit from CRT in many cases increases with QRS duration, because—in a majority—a wide QRS means late activation of the lateral LV.

Simple triumphs over complicated—CRT response best estimated with the old-fashioned ECG.

In a right bundle branch block, the left ventricle is activated first; in LBBB, the LV lateral wall is last, and with a nonspecific ICD, there’s delayed conduction in either the His-Purkinje system or in ventricular muscle. What does a normal QRS say? It says the wave front of activation as projected onto the electric views obtained activates the LV and RV simultaneously. If those views capture the worst delay then they can eliminate the  need for resynchrony.

CRT benefit with mild-moderate QRS prolongation still not settled

Dr Robert Myerburg (here and here) teaches us to make a distinction between trial entry criteria and the actual values of the cohort.

Consider how this applies to QRS duration:  COMPANION and CARE-HF are clinical trials that showed definitive CRT benefit. Entry required a QRS duration >120 ms (130 ms in CARE-HF). But the actual mean QRS duration of enrolled patients was 160 ms. A meta-analysis of CRT trials confirmed benefit at longer QRS durations and questioned it below 150 ms. CRT guideline recommendations incorporate study entry criteria, not the mean values of actual patients in the trial. Patients enrolled in Echo-CRT had very narrow QRS complexes (105 ms). What to recommend in the common situation when a patient with a typical LBBB has a QRS duration straddling 130 ms is not entirely clear. The results of Echo-CRT might have been different had the actual QRS duration values been closer to 130 ms.


Echo-CRT study reinforces expectations based on cardiac physiology. In the practice of medicine, it’s quite useful to know when not to do something.

The trial should not dampen enthusiasm for CRT. Rather, it should focus our attention to patient selection—and the value of the 12-lead ECG.


Rethinking QRS Duration as an Indication for CRT


Author Information

  1. Department of Pediatric Cardiology, Cleveland Clinic, Cleveland, Ohio, USA
  2. Division of Cardiovascular Diseases, Department of Medicine, Mayo Clinic, Rochester, Minnesota, USA
  3. Department of Pediatrics and Adolescent Medicine, Mayo Clinic, Rochester, Minnesota, USA

*Samuel J. Asirvatham, M.D., Division of Cardiovascular Diseases, Department of Internal Medicine and Division of Pediatric Cardiology, Department of Pediatric and Adolescent Medicine, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA. E-mail: asirvatham.samuel@mayo.edu

J Cardiovasc Electrophysiol, Vol. 23, pp. 169-171, February 2012.


Indications for Implantable Cardioverter-Defibrillators Based on Evidence and Judgment FREE

Robert J. Myerburg, MD; Vivek Reddy, MD; Agustin Castellanos, MD
J Am Coll Cardiol. 2009;54(9):747-763. doi:10.1016/j.jacc.2009.03.078

Implantable Cardioverter–Defibrillators after Myocardial Infarction

Robert J. Myerburg, M.D.

Division of Cardiology, University of Miami Miller School of Medicine, Miami.

N Engl J Med 2008; 359:2245-2253 November 20, 2008DOI: 10.1056/NEJMra0803409


Electrical conduction of the Human Heart

  • Physiology and
  • Genetics

were explained by us in the following articles:

Genetics of Conduction Disease: Atrioventricular (AV) Conduction Disease (block): Gene Mutations – Transcription, Excitability, and Energy Homeostasis

On Devices and On Algorithms: Prediction of Arrhythmia after Cardiac Surgery and ECG Prediction of an Onset of Paroxysmal Atrial Fibrillation

Dilated Cardiomyopathy: Decisions on implantable cardioverter-defibrillators (ICDs) using left ventricular ejection fraction (LVEF)

Reduction in Inappropriate Therapy and Mortality through ICD Programming

Below, we present the following complementary topics:

Options for Cardiac Resynchronization Therapy (CRT) to Arrhythmias:

  • Implantable Pacemaker
  • Insertable Programmable Cardioverter Defibrillator (ICD)

UPDATED 8/6/2013

Medtronic Pacemaker Recall



Australia’s regulatory authority, the Therapeutic Goods Administration (TGA) has issued a hazard alert pertaining to one of Medtronic’s pacing devices, the Consulta® Cardiac Resynchronization Therapy Pacemaker (CRT-P). The alert coincides somewhat with Medtronic’s own issuance of a field safety notice concerning Consulta and Syncra® CRT-P devices.


Consulta and Syncra CRT-Ps are implantable medical devices used to treat heart failure. The devices provide pacing to help coordinate the heart’s pumping action and improve blood flow.

The two devices are the subject of a global manufacturer recall after Medtronic had identified an issue with a subset of both during production, although as yet there had been no reported or confirmed device failures. However, because of the potential for malfunction, Medtronic is requiring the return of non-implanted devices manufactured between April 1 and May 13, 2013 for re-inspection.

Seemingly this manufacturing issue could compromise the sealing of the device. Should an out-of-spec weld fail this could result in body fluids entering the device, which could cause it to malfunction leading to loss of pacing output. This could potentially see the return of symptoms including

  • fainting or lightheadedness,
  • dyspnoea (shortness of breath),
  • fatigue and
  • oedema.

Medtronic’s recall is thought to relate to 265 devices, 44 of which have been implanted in the US.

The Australian warning letter, issued by the TGA states that only one “at risk” Consulta CRT-P device has been implanted in the country and there have been no reports of device failures or patient injuries relating to this issue.

Neither Medtronic nor the TGA are suggesting any specific patient management measures other than routine follow-up in accordance with labelling instructions.

Pacemaker/Implantable Cardioverter Defibrillator (ICD) Insertion

Procedure Overview

What is a pacemaker/implantable cardioverter defibrillator (ICD) insertion?

A pacemaker/implantable cardioverter defibrillator (ICD) insertion is a procedure in which a pacemaker and/or an ICD is inserted to assist in regulating problems with the heart rate (pacemaker) or heart rhythm (ICD).


When a problem develops with the heart’s rhythm, such as a slow rhythm, a pacemaker may be selected for treatment. A pacemaker is a small electronic device composed of three parts: a generator, one or more leads, and an electrode on each lead. A pacemaker signals the heart to beat when the heartbeat is too slow.

Illustration of a single-chamber pacemaker
Click Image to Enlarge

A generator is the “brain” of the pacemaker device. It is a small metal case that contains electronic circuitry and a battery. The lead (or leads) is an insulated wire that is connected to the generator on one end, with the other end placed inside one of the heart’s chambers.

The electrode on the end of the lead touches the heart wall. In most pacemakers, the lead senses the heart’s electrical activity. This information is relayed to the generator by the lead.

If the heart’s rate is slower than the programmed limit, an electrical impulse is sent through the lead to the electrode and the pacemaker’s electrical impulse causes the heart to beat at a faster rate.

When the heart is beating at a rate faster than the programmed limit, the pacemaker will monitor the heart rate, but will not pace. No electrical impulses will be sent to the heart unless the heart’s natural rate falls below the pacemaker’s low limit.

Pacemaker leads may be positioned in the atrium or ventricle or both, depending on the condition requiring the pacemaker to be inserted. An atrial dysrhythmia/arrhythmia (an abnormal heart rhythm caused by a dysfunction of the sinus node or the development of another atrial pacemaker within the heart tissue that takes over the function of the sinus node) may be treated with an atrial pacemaker.

Illustration of a dual-chamber pacemaker
Click Image to Enlarge

A ventricular dysrhythmia/arrhythmia (an abnormal heart rhythm caused by a dysfunction of the sinus node, an interruption in the conduction pathways, or the development of another pacemaker within the heart tissue that takes over the function of the sinus node) may be treated with a ventricular pacemaker whose lead wire is located in the ventricle.

It is possible to have both atrial and ventricular dysrhythmias, and there are pacemakers that have lead wires positioned in both the atrium and the ventricle. There may be one lead wire for each chamber, or one lead wire may be capable of sensing and pacing both chambers.

A new type of pacemaker, called a biventricular pacemaker, is currently used in the treatment of congestive heart failure. Sometimes in heart failure, the two ventricles (lower heart chambers) do not pump together in a normal manner. When this happens, less blood is pumped by the heart.

A biventricular pacemaker paces both ventricles at the same time, increasing the amount of blood pumped by the heart. This type of treatment is called cardiac resynchronization therapy.

Implantable cardioverter defibrillator (ICD)

An implantable cardioverter defibrillator (ICD) looks very similar to a pacemaker, except that it is slightly larger. It has a generator, one or more leads, and an electrode for each lead. These components work very much like a pacemaker. However, the ICD is designed to deliver an electrical shock to the heart when the heart rate becomes dangerously fast, or €œfibrillates.”

An ICD senses when the heart is beating too fast and delivers an electrical shock to convert the fast rhythm to a normal rhythm. Some devices combine a pacemaker and ICD in one unit for persons who need both functions.

The ICD has another type of treatment for certain fast rhythms called anti-tachycardia pacing (ATP). When ATP is used, a fast pacing impulse is sent to correct the rhythm. After the shock is delivered, a “back-up” pacing mode is used if needed for a short while.

The procedure for inserting a pacemaker or an ICD is the same. The procedure generally is performed in an electrophysiology (EP) lab or a cardiac catheterization lab.

Other related procedures that may be used to assess the heart include resting and exercise electrocardiogram (ECG), Holter monitor, signal-averaged ECG, cardiac catheterization, chest x-ray, computed tomography (CT scan) of the chest, echocardiography, electrophysiology studies, magnetic resonance imaging (MRI) of the heart, myocardial perfusion scans, radionuclide angiography, and ultrafast CT scan.

The heart’s electrical conduction system

Illustration of the anatomy of the heart, view of the electrical system
Click Image to Enlarge

The heart is, in the simplest terms, a pump made up of muscle tissue. Like all pumps, the heart requires a source of energy in order to function. The heart’s pumping energy comes from an indwelling electrical conduction system.

An electrical stimulus is generated by the sinus node (also called the sinoatrial node, or SA node), which is a small mass of specialized tissue located in the right atrium (right upper chamber) of the heart.

The sinus node generates an electrical stimulus regularly at 60 to 100 times per minute under normal conditions. This electrical stimulus travels down through the conduction pathways (similar to the way electricity flows through power lines from the power plant to your house) and causes the heart’s chambers to contract and pump out blood.

The right and left atria (the two upper chambers of the heart) are stimulated first and contract a short period of time before the right and left ventricles (the two lower chambers of the heart).

The electrical impulse travels from the sinus node to the atrioventricular (AV) node, where it stops for a very short period, then continues down the conduction pathways via the “bundle of His” into the ventricles. The bundle of His divides into right and left pathways to provide electrical stimulation to both ventricles.

What is an ECG?

This electrical activity of the heart is measured by an electrocardiogram (ECG or EKG). By placing electrodes at specific locations on the body (chest, arms, and legs), a tracing of the electrical activity can be obtained. Changes in an ECG from the normal tracing can indicate one or more of several heart-related conditions.

Dysrhythmias/arrhythmias (abnormal heart rhythms) are diagnosed by methods such as EKG, Holter monitoring, signal-average EKG, or electrophysiological studies. These symptoms may be treated with medication or procedures such as a cardiac ablation (removal of a location in the heart that is causing a dysrhythmia by freezing or radiofrequency).

Reasons for the Procedure

A pacemaker may be inserted in order to provide stimulation for a faster heart rate when the heart is beating too slowly, and when other treatment methods, such as medication, have not improved the heart rate.

An ICD may be inserted in order to provide fast pacing (ATP), cardioversion (small shock), or defibrillation (larger shock) when the heart beats too fast.

Problems with the heart rhythm may cause difficulties because the heart is unable to pump an adequate amount of blood to the body. If the heart rate is too slow, the blood is pumped too slowly.

If the heart rate is too fast or too irregular, the heart chambers are unable to fill up with enough blood to pump out with each beat. When the body does not receive enough blood, symptoms such as fatigue, dizziness, fainting, and/or chest pain may occur.

Some examples of rhythm problems for which a pacemaker or ICD might be inserted include:

  • atrial fibrillation – occurs when the atria beat irregularly and too fast
  • ventricular fibrillation – occurs when the ventricles beat irregularly and too fast
  • bradycardia – occurs when the heart beats too slow
  • tachycardia – occurs when the heart beats too fast
  • heart block – occurs when the electrical signal is delayed after leaving the SA node; there are several types of heart blocks, and each one has a distinctive ECG tracing

There may be other reasons for your physician to recommend a pacemaker or ICD insertion.

Risks of the Procedure

Possible risks of pacemaker or ICD insertion include, but are not limited to, the following:

  • bleeding from the incision or catheter insertion site
  • damage to the vessel at the catheter insertion site
  • infection of the incision or catheter site
  • pneumothorax – air becomes trapped in the pleural space causing the lung to collapse

If you are pregnant or suspect that you may be pregnant, you should notify your physician. If you are lactating, or breastfeeding, you should notify your physician.

Patients who are allergic to or sensitive to medications or latex should notify their physician.

For some patients, having to lie still on the procedure table for the length of the procedure may cause some discomfort or pain.

There may be other risks depending upon your specific medical condition. Be sure to discuss any concerns with your physician prior to the procedure.

Before the Procedure

  • Your physician will explain the procedure to you and offer you the opportunity to ask any questions that you might have about the procedure.
  • You will be asked to sign a consent form that gives your permission to do the test. Read the form carefully and ask questions if something is not clear.
  • You will need to fast for a certain period of time prior to the procedure. Your physician will notify you how long to fast, usually overnight.
  • If you are pregnant or suspect that you are pregnant, you should notify your physician.
  • Notify your physician if you are sensitive to or are allergic to any medications, iodine, latex, tape, or anesthetic agents (local and general).
  • Notify your physician of all medications (prescription and over-the-counter) and herbal supplements that you are taking.
  • Notify your physician if you have heart valve disease, as you may need to receive an antibiotic prior to the procedure.
  • Notify your physician if you have a history of bleeding disorders or if you are taking any anticoagulant (blood-thinning) medications, aspirin, or other medications that affect blood clotting. It may be necessary for you to stop some of these medications prior to the procedure.
  • Your physician may request a blood test prior to the procedure to determine how long it takes your blood to clot. Other blood tests may be done as well.
  • You may receive a sedative prior to the procedure to help you relax. If a sedative is given, you will need someone to drive you home afterwards.
  • The upper chest may be shaved or clipped prior to the procedure.
  • Based upon your medical condition, your physician may request other specific preparation.

During the Procedure

Picture of a chest X-ray, showing a single-chamber implanted pacemaker
Chest X-ray with Implanted Pacemaker

A pacemaker or implanted cardioverter defibrillator may be performed on an outpatient basis or as part of your stay in a hospital. Procedures may vary depending on your condition and your physician’s practices.

Generally, a pacemaker or ICD insertion follows this process:

  1. You will be asked to remove any jewelry or other objects that may interfere with the procedure.
  2. You will be asked to remove your clothing and will be given a gown to wear.
  3. You will be asked to empty your bladder prior to the procedure.
  4. An intravenous (IV) line will be started in your hand or arm prior to the procedure for injection of medication and to administer IV fluids, if needed.
  5. You will be placed in a supine (on your back) position on the procedure table.
  6. You will be connected to an electrocardiogram (ECG or EKG) monitor that records the electrical activity of the heart and monitors the heart during the procedure using small, adhesive electrodes. Your vital signs (heart rate, blood pressure, breathing rate, and oxygenation level) will be monitored during the procedure.
  7. Large electrode pads will be placed on the front and back of the chest.
  8. You will receive a sedative medication in your IV before the procedure to help you relax. However, you will likely remain awake during the procedure.
  9. The pacemaker or ICD insertion site will be cleansed with antiseptic soap.
  10. Sterile towels and a sheet will be placed around this area.
  11. A local anesthetic will be injected into the skin at the insertion site.
  12. Once the anesthetic has taken effect, the physician will make a small incision at the insertion site.
  13. A sheath, or introducer, is inserted into a blood vessel, usually under the collarbone. The sheath is a plastic tube through which the pacer/ICD lead wire will be inserted into the blood vessel and advanced into the heart.
  14. It will be very important for you to remain still during the procedure so that the catheter placement will not be disturbed and to prevent damage to the insertion site.
  15. The lead wire will be inserted through the introducer into the blood vessel. The physician will advance the lead wire through the blood vessel into the heart.
  16. Once the lead wire is inside the heart, it will be tested to verify proper location and that it works. There may be one, two, or three lead wires inserted, depending on the type of device your physician has chosen for your condition. Fluoroscopy, (a special type of x-ray that will be displayed on a TV monitor), may be used to assist in testing the location of the leads.
  17. Once the lead wire has been tested, an incision will be made close to the location of the catheter insertion (just under the collarbone). You will receive local anesthetic medication before the incision is made.
  18. The pacemaker/ICD generator will be slipped under the skin through the incision after the lead wire is attached to the generator. Generally, the generator will be placed on the non-dominant side. (If you are right-handed, the device will be placed in your upper left chest. If you are left-handed, the device will be placed in your upper right chest).
  19. The ECG will be observed to ensure that the pacer is working correctly.
  20. The skin incision will be closed with sutures, adhesive strips, or a special glue.
  21. A sterile bandage/dressing will be applied.

After the Procedure

In the hospital

After the procedure, you may be taken to the recovery room for observation or returned to your hospital room. A nurse will monitor your vital signs for a specified period of time.

You should immediately inform your nurse if you feel any chest pain or tightness, or any other pain at the incision site.

After the specified period of bed rest has been completed, you may get out of bed. The nurse will assist you the first time you get up, and will check your blood pressure while you are lying in bed, sitting, and standing. You should move slowly when getting up from the bed to avoid any dizziness from the period of bedrest.

You will be able to eat or drink once you are completely awake.

The insertion site may be sore or painful, but pain medication may be administered if needed.

Your physician will visit with you in your room while you are recovering. The physician will give you specific instructions and answer any questions you may have.

Once your blood pressure, pulse, and breathing are stable and you are alert, you will be taken to your hospital room or discharged home.

If the procedure is performed on an outpatient basis, you may be allowed to leave after you have completed the recovery process. However, if there are concerns or problems with your ECG, you may stay in the hospital for an additional day (or longer) for monitoring of the ECG.

You should arrange to have someone drive you home from the hospital following your procedure.

At home

You should be able to return to your daily routine within a few days. Your physician will tell you if you will need to take more time in returning to your normal activities. In addition, you should not do any lifting or pulling on anything for a few weeks. You may be instructed not to lift your arms above your head for a period of time.

You will most likely be able to resume your usual diet, unless your physician instructs you differently.

It will be important to keep the insertion site clean and dry. Your physician will give you specific bathing instructions.

Your physician will give you specific instructions about driving. If you had an ICD, you will not be able to drive until your physician gives you approval. Your physician will explain these limitations to you, if they are applicable to your situation.

You will be given specific instructions about what to do if your ICD discharges a shock. For example, you may be instructed to dial 911 or go to the nearest emergency room in the event of a shock from the ICD.

Ask your physician when you will be able to return to work. The nature of your occupation, your overall health status, and your progress will determine how soon you may return to work.

Notify your physician to report any of the following:

  • fever and/or chills
  • increased pain, redness, swelling, or bleeding or other drainage from the insertion site
  • chest pain/pressure, nausea and/or vomiting, profuse sweating, dizziness and/or fainting
  • palpitations

Your physician may give you additional or alternate instructions after the procedure, depending on your particular situation.

Pacemaker/ICD precautions

The following precautions should always be considered. Discuss the following in detail with your physician, or call the company that made your device:

  • Always carry an ID card that states you are wearing a pacemaker or an ICD. In addition, you should wear a medical identification bracelet that states you have a pacemaker or ICD.
  • Use caution when going through airport security detectors. Check with your physician about the safety of going through such detectors with your type of pacemaker. In particular, you may need to avoid being screened by hand-held detector devices, as these devices may affect your pacemaker.
  • You may not have a magnetic resonance imaging (MRI) procedure. You should also avoid large magnetic fields.
  • Abstain from diathermy (the use of heat in physical therapy to treat muscles).
  • Turn off large motors, such as cars or boats, when working on them (they may temporarily €œconfuse” your device).
  • Avoid certain high-voltage or radar machinery, such as radio or television transmitters, electric arc welders, high-tension wires, radar installations, or smelting furnaces.
  • If you are having a surgical procedure performed by a surgeon or dentist, tell your surgeon or dentist that you have a pacemaker or ICD, so that electrocautery will not be used to control bleeding (the electrocautery device can change the pacemaker settings).
  • You may have to take antibiotic medication before any medically invasive procedure to prevent infections that may affect the pacemaker.
  • Always consult your physician if you have any questions concerning the use of certain equipment near your pacemaker.
  • When involved in a physical, recreational, or sporting activity, you should avoid receiving a blow to the skin over the pacemaker or ICD. A blow to the chest near the pacemaker or ICD can affect its functioning. If you do receive a blow to that area, see your physician.
  • Always consult your physician when you feel ill after an activity, or when you have questions about beginning a new activity.



In Summary: Who Needs a Pacemaker?

Doctors recommend pacemakers for many reasons. The most common reasons are bradycardia and heart block.

Bradycardia is a heartbeat that is slower than normal. Heart block is a disorder that occurs if an electrical signal is slowed or disrupted as it moves through the heart.

Heart block can happen as a result of aging, damage to the heart from a heart attack, or other conditions that disrupt the heart’s electrical activity. Some nerve and muscle disorders also can cause heart block, including muscular dystrophy.

Your doctor also may recommend a pacemaker if:

  • Aging or heart disease damages your sinus node’s ability to set the correct pace for your heartbeat. Such damage can cause slower than normal heartbeats or long pauses between heartbeats. The damage also can cause your heart to switch between slow and fast rhythms. This condition is called sick sinus syndrome.
  • You’ve had a medical procedure to treat an arrhythmia called atrial fibrillation. A pacemaker can help regulate your heartbeat after the procedure.
  • You need to take certain heart medicines, such as beta blockers. These medicines can slow your heartbeat too much.
  • You faint or have other symptoms of a slow heartbeat. For example, this may happen if the main artery in your neck that supplies your brain with blood is sensitive to pressure. Just quickly turning your neck can cause your heart to beat slower than normal. As a result, your brain might not get enough blood flow, causing you to feel faint or collapse.
  • You have heart muscle problems that cause electrical signals to travel too slowly through your heart muscle. Your pacemaker may provide cardiac resynchronization therapy (CRT) for this problem. CRT devices coordinate electrical signaling between the heart’s lower chambers.
  • You have long QT syndrome, which puts you at risk for dangerous arrhythmias.

Doctors also may recommend pacemakers for people who have certain types ofcongenital heart disease or for people who have had heart transplants. Children, teens, and adults can use pacemakers.

Before recommending a pacemaker, your doctor will consider any arrhythmia symptoms you have, such as dizziness, unexplained fainting, or shortness of breath. He or she also will consider whether you have a history of heart disease, what medicines you’re currently taking, and the results of heart tests.

Diagnostic Tests

Many tests are used to detect arrhythmias. You may have one or more of the following tests.

EKG (Electrocardiogram)

An EKG is a simple, painless test that detects and records the heart’s electrical activity. The test shows how fast your heart is beating and its rhythm (steady or irregular).

An EKG also records the strength and timing of electrical signals as they pass through your heart. The test can help diagnose bradycardia and heart block (the most common reasons for needing a pacemaker).

A standard EKG only records the heartbeat for a few seconds. It won’t detect arrhythmias that don’t happen during the test.

To diagnose heart rhythm problems that come and go, your doctor may have you wear a portable EKG monitor. The two most common types of portable EKGs are Holter and event monitors.

Holter and Event Monitors

A Holter monitor records the heart’s electrical activity for a full 24- or 48-hour period. You wear one while you do your normal daily activities. This allows the monitor to record your heart for a longer time than a standard EKG.

An event monitor is similar to a Holter monitor. You wear an event monitor while doing your normal activities. However, an event monitor only records your heart’s electrical activity at certain times while you’re wearing it.

For many event monitors, you push a button to start the monitor when you feel symptoms. Other event monitors start automatically when they sense abnormal heart rhythms.

You can wear an event monitor for weeks or until symptoms occur.


Echocardiography (echo) uses sound waves to create a moving picture of your heart. The test shows the size and shape of your heart and how well your heart chambers and valves are working.

Echo also can show areas of poor blood flow to the heart, areas of heart muscle that aren’t contracting normally, and injury to the heart muscle caused by poor blood flow.

Electrophysiology Study

For this test, a thin, flexible wire is passed through a vein in your groin (upper thigh) or arm to your heart. The wire records the heart’s electrical signals.

Your doctor uses the wire to electrically stimulate your heart. This allows him or her to see how your heart’s electrical system responds. This test helps pinpoint where the heart’s electrical system is damaged.

Stress Test

Some heart problems are easier to diagnose when your heart is working hard and beating fast.

During stress testing, you exercise to make your heart work hard and beat fast while heart tests, such as an EKG or echo, are done. If you can’t exercise, you may be given medicine to raise your heart rate.



What Are the Risks of Pacemaker Surgery?

Pacemaker surgery generally is safe. If problems do occur, they may include:

  • Swelling, bleeding, bruising, or infection in the area where the pacemaker was placed
  • Blood vessel or nerve damage
  • A collapsed lung
  • A bad reaction to the medicine used during the procedure

Talk with your doctor about the benefits and risks of pacemaker surgery.

How Does a Pacemaker Work?

A pacemaker consists of a battery, a computerized generator, and wires with sensors at their tips. (The sensors are called electrodes.) The battery powers the generator, and both are surrounded by a thin metal box. The wires connect the generator to the heart.

A pacemaker helps monitor and control your heartbeat. The electrodes detect your heart’s electrical activity and send data through the wires to the computer in the generator.

If your heart rhythm is abnormal, the computer will direct the generator to send electrical pulses to your heart. The pulses travel through the wires to reach your heart.

Newer pacemakers can monitor your blood temperature, breathing, and other factors. They also can adjust your heart rate to changes in your activity.

The pacemaker’s computer also records your heart’s electrical activity and heart rhythm. Your doctor will use these recordings to adjust your pacemaker so it works better for you.

Your doctor can program the pacemaker’s computer with an external device. He or she doesn’t have to use needles or have direct contact with the pacemaker.

Pacemakers have one to three wires that are each placed in different chambers of the heart.

  • The wires in a single-chamber pacemaker usually carry pulses from the generator to the right ventricle (the lower right chamber of your heart).
  • The wires in a dual-chamber pacemaker carry pulses from the generator to the right atrium (the upper right chamber of your heart) and the right ventricle. The pulses help coordinate the timing of these two chambers’ contractions.
  • The wires in a biventricular pacemaker carry pulses from the generator to an atrium and both ventricles. The pulses help coordinate electrical signaling between the two ventricles. This type of pacemaker also is called a cardiac resynchronization therapy (CRT) device.

Cross-Section of a Chest With a Pacemaker

The image shows a cross-section of a chest with a pacemaker. Figure A shows the location and general size of a double-lead, or dual-chamber, pacemaker in the upper chest. The wires with electrodes are inserted into the heart's right atrium and ventricle through a vein in the upper chest. Figure B shows an electrode electrically stimulating the heart muscle. Figure C shows the location and general size of a single-lead, or single-chamber, pacemaker in the upper chest.

The image shows a cross-section of a chest with a pacemaker. Figure A shows the location and general size of a double-lead, or dual-chamber, pacemaker in the upper chest. The wires with electrodes are inserted into the heart’s right atrium and ventricle through a vein in the upper chest. Figure B shows an electrode electrically stimulating the heart muscle. Figure C shows the location and general size of a single-lead, or single-chamber, pacemaker in the upper chest.

Types of Pacemaker Programming

The two main types of programming for pacemakers are

  • demand pacing and
  • rate-responsive pacing.

A demand pacemaker monitors your heart rhythm. It only sends electrical pulses to your heart if your heart is beating too slow or if it misses a beat.

A rate-responsive pacemaker will speed up or slow down your heart rate depending on how active you are. To do this, the device monitors your

  • sinus node rate,
  • breathing,
  • blood temperature, and
  • other factors to determine your activity level.

Your doctor will work with you to decide which type of pacemaker is best for you.



What To Expect During Pacemaker Surgery

Placing a pacemaker requires minor surgery. The surgery usually is done in a hospital or special heart treatment laboratory.

Before the surgery, an intravenous (IV) line will be inserted into one of your veins. You will receive medicine through the IV line to help you relax. The medicine also might make you sleepy.

Your doctor will numb the area where he or she will put the pacemaker so you don’t feel any pain. Your doctor also may give you antibiotics to prevent infection.

First, your doctor will insert a needle into a large vein, usually near the shoulder opposite your dominant hand. Your doctor will then use the needle to thread the pacemaker wires into the vein and to correctly place them in your heart.

An x-ray “movie” of the wires as they pass through your vein and into your heart will help your doctor place them. Once the wires are in place, your doctor will make a small cut into the skin of your chest or abdomen.

He or she will slip the pacemaker’s small metal box through the cut, place it just under your skin, and connect it to the wires that lead to your heart. The box contains the pacemaker’s battery and generator.

Once the pacemaker is in place, your doctor will test it to make sure it works properly. He or she will then sew up the cut. The entire surgery takes a few hours.



What To Expect After Pacemaker Surgery

Expect to stay in the hospital overnight so your health care team can check your heartbeat and make sure your pacemaker is working well. You’ll likely have to arrange for a ride to and from the hospital because your doctor may not want you to drive yourself.

For a few days to weeks after surgery, you may have pain, swelling, or tenderness in the area where your pacemaker was placed. The pain usually is mild; over-the-counter medicines often can relieve it. Talk to your doctor before taking any pain medicines.

Your doctor may ask you to avoid vigorous activities and heavy lifting for about a month after pacemaker surgery. Most people return to their normal activities within a few days of having the surgery.



How Will a Pacemaker Affect My Lifestyle?

Once you have a pacemaker, you have to avoid close or prolonged contact with electrical devices or devices that have strong magnetic fields. Devices that can interfere with a pacemaker include:

  • Cell phones and MP3 players (for example, iPods)
  • Household appliances, such as microwave ovens
  • High-tension wires
  • Metal detectors
  • Industrial welders
  • Electrical generators

These devices can disrupt the electrical signaling of your pacemaker and stop it from working properly. You may not be able to tell whether your pacemaker has been affected.

How likely a device is to disrupt your pacemaker depends on how long you’re exposed to it and how close it is to your pacemaker.

To be safe, some experts recommend not putting your cell phone or MP3 player in a shirt pocket over your pacemaker (if the devices are turned on).

You may want to hold your cell phone up to the ear that’s opposite the site where your pacemaker is implanted. If you strap your MP3 player to your arm while listening to it, put it on the arm that’s farther from your pacemaker.

You can still use household appliances, but avoid close and prolonged exposure, as it may interfere with your pacemaker.

You can walk through security system metal detectors at your normal pace. Security staff can check you with a metal detector wand as long as it isn’t held for too long over your pacemaker site. You should avoid sitting or standing close to a security system metal detector. Notify security staff if you have a pacemaker.

Also, stay at least 2 feet away from industrial welders and electrical generators.

Some medical procedures can disrupt your pacemaker. These procedures include:

  • Magnetic resonance imaging, or MRI
  • Shock-wave lithotripsy to get rid of kidney stones
  • Electrocauterization to stop bleeding during surgery

Let all of your doctors, dentists, and medical technicians know that you have a pacemaker. Your doctor can give you a card that states what kind of pacemaker you have. Carry this card in your wallet. You may want to wear a medical ID bracelet or necklace that states that you have a pacemaker.

Physical Activity

In most cases, having a pacemaker won’t limit you from doing sports and exercise, including strenuous activities.

You may need to avoid full-contact sports, such as football. Such contact could damage your pacemaker or shake loose the wires in your heart. Ask your doctor how much and what kinds of physical activity are safe for you.

Ongoing Care

Your doctor will want to check your pacemaker regularly (about every 3 months). Over time, a pacemaker can stop working properly because:

  • Its wires get dislodged or broken
  • Its battery gets weak or fails
  • Your heart disease progresses
  • Other devices have disrupted its electrical signaling

To check your pacemaker, your doctor may ask you to come in for an office visit several times a year. Some pacemaker functions can be checked remotely using a phone or the Internet.

Your doctor also may ask you to have an EKG (electrocardiogram) to check for changes in your heart’s electrical activity.

Battery Replacement

Pacemaker batteries last between 5 and 15 years (average 6 to 7 years), depending on how active the pacemaker is. Your doctor will replace the generator along with the battery before the battery starts to run down.

Replacing the generator and battery is less-involved surgery than the original surgery to implant the pacemaker. Your pacemaker wires also may need to be replaced eventually.

Your doctor can tell you whether your pacemaker or its wires need to be replaced when you see him or her for followup visits.



Clinical Trial on Pace Makers

clinical trials related to pacemakers, talk with your doctor. You also can visit the following Web sites to learn more about clinical research and to search for clinical trials:

For more information about clinical trials for children, visit the NHLBI’s Children and Clinical Studies Web page.



RESOUCES on PaceMakers

Links to Other Information About Pacemakers

NHLBI Resources

Non-NHLBI Resources

Clinical Trials



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Reporter: Aviva Lev-Ari, PhD, RN

Relypsa Announces $80 Million Private Financing Transaction

Funds will Support the Advancement of Patiromer (RLY5016) through Phase 3 and NDA Submission

SANTA CLARA, Calif., Aug 15, 2012 (BUSINESS WIRE) — Relypsa, Inc. today announced an $80 million Series C preferred stock financing, including participation from both new and existing investors. Proceeds will be used to fund late-stage development, submission of a new drug application (NDA) and commercial planning for patiromer (RLY5016), the company’s high capacity non-absorbed oral potassium binder being developed for the treatment of hyperkalemia. Relypsa plans to initiate Phase 3 pivotal clinical trials of patiromer this year.

“The strong participation in this financing transaction by our existing investors, as well as the addition of new investors, provides important validation of patiromer’s commercial potential,” stated Gerrit Klaerner, Ph.D., President of Relypsa.

The Series C financing round included existing investors OrbiMed Advisors, 5AM Ventures, New Leaf Venture Partners, Sprout Group, Delphi Ventures and Mediphase Venture Partners. New investor Sibling Capital, LLC also participated in the transaction.

“Based on the data generated to date in nearly 500 patients, we’re optimistic that patiromer can become an important part of improving the standard of care for patients with chronic kidney disease, especially those suffering from diabetic nephropathy,” said Jonathan T. Silverstein, J.D., General Partner of OrbiMed.

Patiromer is currently being evaluated for the treatment of hyperkalemia in an ongoing Phase 2b study designated AMETHYST-DN. Enrollment of 306 subjects at approximately 50 sites was completed in May 2012. In the study, the efficacy of patiromer is being evaluated in an initial 8-week treatment period, followed by the long-term evaluation of safety and tolerability in a subsequent 44-week extended treatment period.

“As a founding investor in Relypsa, we have been delighted to follow the progress of patiromer from an early stage preclinical candidate to a compelling late-stage product opportunity,” commented Scott M. Rocklage, Ph.D., Managing Partner of 5AM Ventures and Chairman of Relypsa.

About Patiromer and Hyperkalemia

Hyperkalemia is a condition frequently prevalent in patients that suffer from renal impairment, hypertension, diabetes and heart failure. It is characterized by elevated serum potassium levels, which can lead to cardiac arrhythmia and sudden death. Patients with chronic kidney disease are at particular risk for developing hyperkalemia, especially those treated with renin-angiotensin-aldosterone-system (RAAS) inhibitors. Although RAAS inhibition has been shown to protect kidney and cardiac function, as well as prolong life, many patients who could benefit from RAAS inhibitors are untreated or undertreated due to the undesirable side effect of increasing serum potassium.

Patiromer (RLY5016) is a high capacity non-absorbed oral potassium binder being developed for the management of elevated serum potassium levels. Relypsa has completed several clinical trials of patiromer that have demonstrated the preliminary efficacy, safety and tolerability of patiromer for the prevention of hyperkalemia.

About Relypsa, Inc.

Relypsa, Inc. is a clinical-stage pharmaceutical company leading the discovery and development of novel non-absorbed polymeric drugs for important applications in cardiovascular and renal diseases. Relypsa’s lead product candidate is patiromer, a non-absorbed potassium binder for the treatment of hyperkalemia. Relypsa is pursuing the discovery of additional product candidates through use of its proprietary polymer platform. More information is available at http://www.relypsa.com .

SOURCE: Relypsa, Inc.

Relypsa, Inc.

Jim Johnson,


Senior VP & CFO


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