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Prefacing the e-Book Epilogue: Metabolic Genomics and Pharmaceutics

Author and Curator: Larry H. Bernstein, MD, FCAP

 

Adieu, adieu, adieu …

Sound of Music

Snoopy - Charlie happiness

Snoopy – Charlie happiness

This work has been a coming to terms with my scientific and medical end of career balancing in a difficult time after retiring, but it has been rewarding.  In the clinical laboratories, radiology, anesthesiology, and in pharmacy, there has been some significant progress in support of surgical, gynecological, developmental, medical practices, and even neuroscience directed disciplines, as well as epidemiology over a period of half a century.  Even then, cancer and neurological diseases have been most difficult because the scientific basic research has either not yet uncovered a framework, or because that framework has proved to be multidimensional.  In the clinical laboratory sciences, there has been enormous progress in instrumental analysis, with the recent opening of molecular methods not yet prepared for routine clinical use, which will be a very great challenge to the profession, which has seen the development of large sample volume, multianalite, high-throughput, low-cost support emerging for decades.  The capabilities now underway will also enrrich the the capabilities of the anatomic pathology suite and the capabilities of 3-dimensional radiological examination.  In both pathology and radiology, we have seen the division of the fields into major subspecialties.  The development of the electronic health record had to take lessons from the first developments in the separate developments of laboratory, radiology, and pharmacy health record systems, to which were added, full cardiology monitoring systems.  These have been unintegrated.  This made it difficult to bring forth a suitable patient health record because the information needed to support decision-making by practitioners was in separate “silos”.  The mathematical methods that are being applied to the -OMICS sciences, can be brought to bear on the simplification and amplification of the clinicians’ ability to make decisions with near “errorless” discrimination, still allowing for an element of “art” in carrying out the history, physical examination, and knowledge unique to every patient.

We are at this time opening a very large, complex, study of biology in relationship to the human condition.  This will require sufficient resources to be invested in the development of these for a better society, which I suspect, will go on beyond the life of my grandchildren.  Hopefully, the long-term dangers of climate change will be controlled in that time.  As a society, or as a group of interdependent societies, we have no long term interest in continuing self-destructive behaviors that have predominated in the history of mankind.  I now top off these discussions with some further elucidation of what lies before us.

Metabolomics and systems pharmacology: why and how to model the human metabolic network for drug discovery

Douglas B. Kell and Royston Goodacre
School of Chemistry and Manchester Institute of Biotechnology, The University of Manchester, Manchester, UK
Drug Discovery Today Feb 2014;19(2)  http://dx.doi.org/10.1016/j.drudis.2013.07.014

Metabolism represents the ‘sharp end’ of systems biology,

  • because changes in metabolite concentrations
  • are necessarily amplified relative to
  • changes in the transcriptome, proteome and enzyme activities,
  • which can be modulated by drugs.

To understand such behaviour, we therefore need
(and increasingly have)

  • reliable consensus (community) models of the human metabolic network
  • that include the important transporters.

Small molecule ‘drug’ transporters are in fact metabolite transporters,

  • because drugs bear structural similarities to metabolites known
  • from the network reconstructions and from measurements of the metabolome.

Recon2 represents the present state-of-the-art human metabolic
network reconstruction; it can predict inter alia:

  1. the effects of inborn errors of metabolism;
  2. which metabolites are exometabolites, and
  3. how metabolism varies between tissues and cellular compartments.

Even these qualitative network models are not yet complete. As our
understanding improves so do we recognize more clearly the need for a systems (poly)pharmacology.

Modelling biochemical networks – why we do so
There are at least four types of reasons as to why one would wish to model a biochemical network:

  1. Assessing whether the model is accurate, in the sense that it
    reflects – or can be made to reflect – known experimental facts.
  2. Establishing what changes in the model would improve the
    consistency of its behaviour with experimental observations
    and improved predictability, such as with respect to metabolite
    concentrations or fluxes.
  3. Analyzing the model, typically by some form of sensitivity
    analysis, to understand which parts of the system contribute
    most to some desired functional properties of interest.
  4. Hypothesis generation and testing, enabling one to analyse
    rapidly the effects of manipulating experimental conditions in
    the model without having to perform complex and costly
    experiments (or to restrict the number that are performed).

In particular, it is normally considerably cheaper to perform
studies of metabolic networks in silico before trying a smaller
number of possibilities experimentally; indeed for combinatorial
reasons it is often the only approach possible. Although
our focus here is on drug discovery, similar principles apply to the
modification of biochemical networks for purposes of ‘industrial’
or ‘white’ biotechnology.
Why we choose to model metabolic networks more than

  • transcriptomic or proteomic networks

comes from the recognition – made particularly clear

  • by workers in the field of metabolic control analysis

– that, although changes in the activities of individual enzymes tend to have

  • rather small effects on metabolic fluxes,
  • they can and do have very large effects on metabolite concentrations (i.e. the metabolome).

Modelling biochemical networks – how we do so

Although one could seek to understand the

  1. time-dependent spatial distribution of signalling and metabolic substances within indivi
    dual cellular compartments and
  2. while spatially discriminating analytical methods such as Raman spectroscopy and
    mass spectrometry do exist for the analysis of drugs in situ,
  • the commonest type of modelling, as in the spread of substances in
    ecosystems,
  • assumes ‘fully mixed’ compartments and thus ‘pools’ of metabolites.

Although an approximation, this ‘bulk’ modelling will be necessary for complex ecosystems such as humans where, in addition to the need for tissue- and cell-specific models, microbial communities inhabit this superorganism and the
gut serves as a source for nutrients courtesy of these symbionts.

Topology and stoichiometry of metabolic networks as major constraints on fluxes
Given their topology, which admits a wide range of parameters for
delivering the same output effects and thereby reflects biological
robustness,

  • metabolic networks have two especially important constraints that assist their accurate modelling:

(i) the conservation of mass and charge, and
(ii) stoichiometric and thermodynamic constraints.

These are tighter constraints than apply to signalling networks.

New developments in modelling the human metabolic network
Since 2007, several groups have been developing improved but nonidentical models of the human metabolic network at a generalised level and in tissue-specific forms. Following a similar community-driven strategy in Saccharomyces cerevisiae, surprisingly similar to humans, and in Salmonella typhimurium,

we focus in particular on a recent consensus paper that provides a highly curated and semantically annotated model of the human metabolic network, termed

In this work, a substantial number of the major groups active in this area came together to provide a carefully and manually constructed/curated network, consisting of some 1789 enzyme-encoding genes, 7440 reactions and 2626 unique metabolites distributed over eight cellular compartments.  A variety of dead-end metabolites and blocked reactions remain (essentially orphans and widows). But Recon2 was able to

  • account for some 235 inborn errors of metabolism,
  • a variety of metabolic ‘tasks’ (defined as a non-zero flux through a reaction or through a pathway leading to the production of a metabolite Q from a metabolite P).
  • filtering based on expression profiling allowed the construction of 65 cell-type-specific models.
  • Excreted or exometabolites are an interesting set of metabolites,
  • and Recon2 could predict successfully a substantial fraction of those

Role of transporters in metabolic fluxes

The uptake and excretion of metabolites between cells and their macrocompartments

  • requires specific transporters and in the order of one third of ‘metabolic’ enzymes,
  • and indeed of membrane proteins, are in fact transporters or equivalent.

What is of particular interest (to drug discovery), based on their structural similarities, is the increasing recognition (Fig. 3) that pharmaceutical drugs also

  • get into and out of cells by ‘hitchhiking’ on such transporters, and not –

to any significant extent –

  • by passing through phospholipid bilayer portions
    of cellular membranes.

This makes drug discovery even more a problem of systems biology than of biophysics.

role of solute carriers and other transporters in cellular drug uptake

role of solute carriers and other transporters in cellular drug uptake

Two views of the role of solute carriers and other transporters in cellular drug uptake. (a) A more traditional view in which all so-called ‘passive’drug uptake occurs through any unperturbed bilayer portion of membrane that might be present.
(b) A view in which the overwhelming fraction of drug is taken up via solute transporters or other carriers that are normally used for the uptake of intermediary metabolites. Noting that the protein:lipid ratio of biomembranes is typically 3:1 to 1:1 and that proteins vary in mass and density (a typical density is 1.37 g/ml) as does their extension, for example, normal to the ca. 4.5 nm lipid bilayer region, the figure attempts to portray a section of a membrane with realistic or typical sizes and amounts of proteins and lipids. Typical protein areas when viewed normal to the membrane are 30%, membranes are rather more ‘mosaic’ than ‘fluid’ and there is some evidence that there might be no genuinely ‘free’ bulk lipids (typical phospholipid masses are 750 Da) in biomembranes that are uninfluenced by proteins. Also shown is a typical drug: atorvastatin (LipitorW) – with a molecular mass of 558.64 Da – for size comparison purposes. If proteins are modelled as
cylinders, a cylinder with a diameter of 3.6 nm and a length of 6 nm has a molecular mass of ca. 50 kDa. Note of course that in a ‘static’ picture we cannot show the dynamics of either phospholipid chains or lipid or protein diffusion.

‘Newly discovered’ metabolites and/or their roles

To illustrate the ‘unfinished’ nature even of Recon2, which concentrates on the metabolites created via enzymes encoded in the human genome, and leaving aside the more exotic metabolites of drugs and foodstuffs and the ‘secondary’ metabolites of microorganisms, there are several examples of interesting ‘new’ (i.e. more or less recently recognised) human metabolites or roles thereof that are worth highlighting, often from studies seeking biomarkers of various diseases – for caveats of biomarker discovery, which is not a topic that we are covering here, and the need for appropriate experimental design. In addition, classes of metabolites not well represented in Recon2 are oxidised molecules such as those caused by nonenzymatic reaction of metabolites with free radicals such as the hydroxyl radical generated by unliganded iron. There is also significant interest in using methods of determining small molecules such as those in the
metabolome (inter alia) for assessing the ‘exposome’, in other words all the potentially polluting agents to which an
individual has been exposed.

Recently discovered effects of metabolites on enzymes 

Another combinatorial problem reflects the fact that in molecular enzymology it is not normally realistic to assess every possible metabolite to determine whether it is an effector (i.e.activator or inhibitor) of the enzyme under study. Typical proteins are highly promiscuous and there is increasing evidence for the comparative promiscuity of metabolites
and pharmaceutical drugs. Certainly the contribution of individual small effects of multiple parameter changes can have substantial effects on the potential flux through an overall pathway, which makes ‘bottom up’ modelling an inexact science. Even merely mimicking the vivo (in Escherichia coli) concentrations of K+, Na+, Mg2+, phosphate, glutamate, sulphate and Cl significantly modulated the activities of several enzymes tested relative to the ‘usual’ assay conditions. Consequently, we need to be alive to the possibility of many (potentially major) interactions of which we are as yet ignorant. One class of example relates to the effects of the very widespread post-translational modification on metabolic
enzyme activities.

A recent and important discovery (Fig. 4) is that a single transcriptome experiment, serving as a surrogate for fluxes through individual steps, provides a huge constraint on possible models, and predicts in a numerically tractable way and
with much improved accuracy the fluxes to exometabolites without the need for such a variable ‘biomass’ term. Other recent and related strategies that exploit modern advances in ‘omics and network biology to limit the search space in constraint-based metabolic modelling.

Fig 4. Workflow for expression-profile-constrained metabolic flux estimation

  1. Genome-scale metabolic model with gene-protein-reaction relationships
  2. Map absolute gene expression levels to reactions
  3. Maximise correlation between absolute gene expression and metabolic flux
  4. Predict fluxes to exometabolites
  5. Compare predicted with experimental fluxes to exometabolites

Drug Discovery Today

The steps in a workflow that uses constraints based on (i) metabolic network stoichiometry and chemical reaction properties (both encoded in the model) plus, and (ii) absolute (RNA-Seq) transcript expression profiles to enable the
accurate modelling of pathway and exometabolite fluxes. .

Concluding remarks – the role of metabolomics in systems pharmacology

What is becoming increasingly clear, as we recognize that to understand living organisms in health and disease we must treat them as systems, is that we must bring together our knowledge of the topologies and kinetics of metabolic networks with our knowledge of the metabolite concentrations (i.e. metabolomes) and fluxes. Because of the huge constraints imposed on metabolism by reaction stoichiometries, mass conservation and thermodynamics, comparatively few well-chosen ‘omics measurements might be needed to do this reliably (Fig. 4). Indeed, a similar approach exploiting constraints has come to the fore in denovo protein folding and interaction studies.

What this leads us to in drug discovery is the need to develop and exploit a ‘systems pharmacology’ where multiple binding targets are chosen purposely and simultaneously. Along with other measures such as phenotypic screening, and the integrating of the full suite of e-science approaches, one can anticipate considerable improvements in the rate of discovery of safe and effective drugs.

Metabolomics: the apogee of the omics trilogy
Gary J.!Patti, Oscar Yanes and Gary Siuzdak

Metabolites, the chemical entities that are transformed during metabolism, provide a functional readout of cellular biochemistry. With emerging technologies in mass spectrometry, thousands of metabolites can now be
quantitatively measured from minimal amounts of biological material, which has thereby enabled systems-level analyses. By performing global metabolite profiling, also known as untargeted metabolomics, new discoveries linking cellular pathways to biological mechanism are being revealed and are shaping our understanding of cell biology, physiology and medicine.

Metabolites are small molecules that are chemically transformed during metabolism and, as such, they provide a functional readout of cellular state. Unlike genes and proteins, the functions of which are subject to epigenetic regulation and posttranslational modifications, respectively, metabolites serve as direct signatures of biochemical activity and are therefore easier to correlate with phenotype. In this context, metabolite profiling, or metabolomics, has become a powerful approach that has been widely adopted for clinical diagnostics.

The field of metabolomics has made remarkable progress within the past decade and has implemented new tools that have offered mechanistic insights by allowing for the correlation of biochemical changes with phenotype.

In this Innovation article, we first define and differentiate between the targeted and untargeted approaches to metabolomics. We then highlight the value of untargeted metabolomics in particular and outline a guide to performing such studies. Finally, we describe selected applications of un targeted metabolomics and discuss their potential in cell biology.

  • metabolites serve as direct signatures of biochemical activity
  1. In some instances, it may be of interest to examine a defined set of metabolites by using a targeted approach.
  2. In other cases, an untargeted or global approach may be taken in which as many metabolites as possible are measured and compared between samples without bias.
  3. Ultimately, the number and chemical composition of metabolites to be studied is a defining attribute of any metabolomic experiment and shapes experimental design with respect to sample preparation and choice of instrumentation.

The targeted and untargeted workflow for LC/MS-based metabolomics.

a | In the triple quadrupole (QqQ)-based targeted metabolomic workflow, standard compounds for the metabolites of interest are first used to set up selected reaction monitoring methods. Here, optimal instrument voltages are determined and response curves are generated for absolute quantification. After the targeted methods have been established
on the basis of standard metabolites, metabolites are extracted from tissues, biofluids or cell cultures and analysed. The data output provides quantification only of those metabolites for which standard methods have been built.

b | In the untargeted metabolomic workflow, metabolites are first isolated from biological samples and subsequently analysed by liquid chromatography followed by mass spectrometry (LC/MS). After data acquisition, the results are processed by using bioinformatic software such as XCMS to perform nonlinear retention time alignment and identify peaks that are changing between the groups of samples measured. The m/z value s for the peaks of interest are searched in metabolite databases to obtain putative identifications. Putative identifications are then confirmed
by comparing tandem mass spectrometry (MS/MS) data and retention time data to that of standard compounds. The untargeted workflow is global in scope and outputs data related to comprehensive cellular metabolism.

Metabolic Biomarker and Kinase Drug Target Discovery in Cancer Using Stable Isotope-Based Dynamic Metabolic Profiling (SIDMAP)

László G. Boros1*, Daniel J. Brackett2 and George G. Harrigan3
1UCLA School of Medicine, Harbor-UCLA Research and Education Institute, Torrance, CA. 2Department of Surgery, University of Oklahoma Health Sciences Center & VA Medical Center, Oklahoma City, OK, 3Global High Throughput
Screening (HTS), Pharmacia Corporation, Chesterfield, MO.
Current Cancer Drug Targets, 2003, 3, 447-455.

Tumor cells respond to growth signals by the activation of protein kinases, altered gene expression and significant modifications in substrate flow and redistribution among biosynthetic pathways. This results in a proliferating phenotype
with altered cellular function. These transformed cells exhibit unique anabolic characteristics, which includes increased and preferential utilization of glucose through the non-oxidative steps of the pentose cycle for nucleic acid synthesis but limited denovo fatty acid synthesis and TCA cycle glucose oxidation. This primarily nonoxidative anabolic profile reflects an undifferentiated highly proliferative aneuploid cell phenotype and serves as a reliable metabolic biomarker to determine cell proliferation rate and the level of cell transformation/differentiation in response to drug treatment. Novel drugs effective in particular cancers exert their anti-proliferative effects by inducing significant reversions of a few specific non-oxidative anabolic pathways. Here we present evidence that cell transformation of various mechanisms is sustained by a unique
disproportional substrate distribution between the two branches of the pentose cycle for nucleic acid synthesis, glycolysis and the TCA cycle for fatty acid synthesis and glucose oxidation. This can be demonstrated by the broad labeling and unique specificity of [1,2-13C2]glucose to trace a large number of metabolites in the metabolome. Stable isotope-based dynamic metabolic profiles (SIDMAP) serve the drug discovery process by providing a powerful new tool that integrates the metabolome into a functional genomics approach to developing new drugs. It can be used in screening kinases and their metabolic targets, which can therefore be more efficiently characterized, speeding up and improving drug testing, approval and labeling processes by saving trial and error type study costs in drug testing.

Navigating the HumanMetabolome for Biomarker Identification and Design of Pharmaceutical Molecules

Irene Kouskoumvekaki and Gianni Panagiotou
Department of Systems Biology, Center for Biological Sequence Analysis, Building 208, Technical University of Denmark, Lyngby, Denmark
Hindawi Publishing Corporation  Journal of Biomedicine and Biotechnology 2011, Article ID 525497, 19 pages
http://dx.doi.org:/10.1155/2011/525497

Metabolomics is a rapidly evolving discipline that involves the systematic study of endogenous small molecules that characterize the metabolic pathways of biological systems. The study of metabolism at a global level has the potential to contribute significantly to biomedical research, clinical medical practice, as well as drug discovery. In this paper, we present the most up-to-date metabolite and metabolic pathway resources, and we summarize the statistical, and machine-learning tools used for the analysis of data from clinical metabolomics.

Through specific applications on cancer, diabetes, neurological and other diseases, we demonstrate how these tools can facilitate diagnosis and identification of potential biomarkers for use within disease diagnosis. Additionally, we
discuss the increasing importance of the integration of metabolomics data in drug discovery. On a case-study based on the Human Metabolome Database (HMDB) and the Chinese Natural Product Database (CNPD), we demonstrate the close relatedness of the two data sets of compounds, and we further illustrate how structural similarity with human metabolites could assist in the design of novel pharmaceuticals and the elucidation of the molecular mechanisms of medicinal plants.

Metabolites are the byproducts of metabolism, which is itself the process of converting food energy to mechanical energy
or heat. Experts believe there are at least 3,000 metabolites that are essential for normal growth and development (primary metabolites) and thousands more unidentified (around 20,000, compared to an estimated 30,000 genes and 100,000 proteins) that are not essential for growth and development (secondary metabolites) but could represent prognostic, diagnostic, and surrogate markers for a disease state and a deeper understanding of mechanisms of disease.

Metabolomics, the study of metabolism at the global level, has the potential to contribute significantly to biomedical
research, and ultimately to clinical medical practice. It is a close counterpart to the genome, the transcriptome and the proteome. Metabolomics, genomics, proteomics, and other “-omics” grew out of the Human Genome Project, a massive research effort that began in the mid-1990s and culminated in 2003 with a complete mapping of all the genes in the human body. When discussing the clinical advantages of metabolomics, scientists point to the “real-world” assessment
of patient physiology that the metabolome provides since it can be regarded as the end-point of the “-omics” cascade. Other functional genomics technologies do not necessarily predict drug effects, toxicological response, or disease states at the phenotype but merely indicate the potential cause for phenotypical response. Metabolomics can bridge this information gap. The identification and measurement of metabolite profile dynamics of host changes provides the closest link to the various phenotypic responses. Thus it is clear that the global mapping of metabolic signatures pre- and postdrug treatment is a promising approach to identify possible functional relationships between medication and medical phenotype.

Human Metabolome Database (HMDB). Focusing on quantitative, analytic, or molecular scale information about
metabolites, the enzymes and transporters associated with them, as well as disease related properties the HMDB represents the most complete bioinformatics and chemoinformatics medical information database. It contains records for
thousands of endogenous metabolites identified by literature surveys (PubMed, OMIM, OMMBID, text books), data
mining (KEGG, Metlin, BioCyc) or experimental analyses performed on urine, blood, and cerebrospinal fluid samples.
The annotation effort is aided by chemical parameter calculators and protein annotation tools originally developed for
DrugBank.

A key feature that distinguishes the HMDB from other metabolic resources is its extensive support for higher level database searching and selecting functions. More than 175 hand-drawn-zoomable, fully hyperlinked human
metabolic pathway maps can be found in HMDB and all these maps are quite specific to human metabolism and
explicitly show the subcellular compartments where specific reactions are known to take place. As an equivalent to
BLAST the HMDB contains a structure similarity search tool for chemical structures and users may sketch or
paste a SMILES string of a query compound into the Chem-Query window. Submitting the query launches a
structure similarity search tool that looks for common substructures from the query compound that match the
HMDB’s metabolite database. The wealth of information and especially the extensive linkage to metabolic diseases
to normal and abnormal metabolite concentration ranges, to mutation/SNP data and to the genes, enzymes, reactions
and pathways associated with many diseases of interest makes the HMDB one the most valuable tool in the hands
of clinical chemists, nutritionists, physicians and medical geneticists.

Metabolomics in Drug Discovery and Polypharmacology Studies

Drug molecules generally act on specific targets at the cellular level, and upon binding to the receptors, they exert
a desirable alteration of the cellular activities, regarded as the pharmaceutical effect. Current drug discovery depends
largely on ransom screening, either high-throughput screening (HTS) in vitro, or virtual screening (VS) in silico. Because
the number of available compounds is huge, several druglikeness filters are proposed to reduce the number of compounds that need to be evaluated. The ability to effectively predict if a chemical compound is “drug-like” or “nondruglike” is, thus, a valuable tool in the design, optimization, and selection of drug candidates for development. Druglikeness is a general descriptor of the potential of a small molecule to become a drug. It is not a unified descriptor
but a global property of a compound processing many specific characteristics such as good solubility, membrane
permeability, half-life, and having a pharmacophore pattern to interact specifically with a target protein. These
characteristics can be reflected as molecular descriptors such as molecular weight, log P, the number of hydrogen bond
donors, the number of hydrogen-bond acceptors, the number of rotatable bonds, the number of rigid bonds, the
number of rings in a molecule, and so forth.

Metabolomics for the Study of Polypharmacology of Natural Compounds

Internationally, there is a growing and sustained interest from both pharmaceutical companies and public in medicine
from natural sources. For the public, natural medicine represent a holistic approach to disease treatment, with
potentially less side effects than conventional medicine. For the pharmaceutical companies, bioactive natural products
constitute attractive drug leads, as they have been optimized in a long-term natural selection process for optimal interaction with biomolecules. To promote the ecological survival of plants, structures of secondary products have evolved to interact with molecular targets affecting the cells, tissues and physiological functions in competing microorganisms,
plants, and animals. In this, respect, some plant secondary products may exert their action by resembling endogenous
metabolites, ligands, hormones, signal transduction molecules, or neurotransmitters and thus have beneficial
effects on humans.

Future Perspectives

Metabolomics, the study of metabolism at the global level, is moving to exciting directions.With the development ofmore
sensitive and advanced instrumentation and computational tools for data interpretation in the physiological context,
metabolomics have the potential to impact our understanding of molecular mechanisms of diseases. A state-of-theart
metabolomics study requires knowledge in many areas and especially at the interface of chemistry, biology, and
computer science. High-quality samples, improvements in automated metabolite identification, complete coverage of
the human metabolome, establishment of spectral databases of metabolites and associated biochemical identities, innovative experimental designs to best address a hypothesis, as well as novel computational tools to handle metabolomics data are critical hurdles that must be overcome to drive the inclusion of metabolomics in all steps of drug discovery and drug development. The examples presented above demonstrated that metabolite profiles reflect both environmental and genetic influences in patients and reveal new links between metabolites and diseases providing needed prognostic,diagnostic, and surrogate biomarkers. The integration of these signatures with other omic technologies is of utmost importance to characterize the entire spectrum of malignant phenotype.

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Larry H Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Intelligence

 

 

Association of heart rate variability and inflammatory response in patients with cardiovascular diseases: current strengths and limitations
V Papaioannou, I Pneumatikos and N Maglaveras
Front Phys 2013.
http://dx.doi.org:/10.3389/fphys.2013.00174

A few clinical studies have assessed the possible inter-relation between neuro-autonomic output, estimated with heart rate variability analysis, which is the variability of R-R in the electrocardiogram, and different inflammatory biomarkers, in patients suffering from stable or unstable coronary artery disease (CAD) and heart failure. Moreover, different indices derived from heart rate signals’ processing, have been proven to correlate strongly with severity of heart disease and predict final outcome. In this review article we will summarize major findings from different investigators, evaluating neuro-immunological interactions through heart rate variability analysis, in different groups of cardiovascular patients. We suggest that markers originating from variability analysis of heart rate signals seem to be related to inflammatory biomarkers.
Atrial Natriuretic Peptide Frameshift Mutation in Familial Atrial Fibrillation  

DM. Hodgson-Zingman, ML. Karst, LV. Zingman, DM. Heublein, et al.
N Engl J Med. 2008 July 10; 359(2): 158–165  http://www.nejm.org/doi/full/10.1056/NEJMoa0706300

We mapped an atrial fibrillation locus to chromosome 1p36-p35 and identified a heterozygous frameshift mutation in the gene encoding atrial natriuretic peptide. Circulating chimeric atrial natriuretic peptide (ANP) was detected in high concentration in subjects with the mutation, and shortened atrial action potentials were seen in an isolated heart model, creating a possible substrate for atrial fibrillation. This report implicates perturbation of the atrial natriuretic peptide–cyclic guanosine monophosphate (cGMP) pathway in cardiac electrical instability.
Impact of anemia on clinical outcome in patients with atrial fibrillation undergoing percutaneous coronary intervention: insights from the AFCAS registry.
M Puurunen, T Kiviniemi, W Nammas, A Schlitt, A Rubboli, K Nyman, et al.
BMJ Open 2014; 4:e004700.
http://dx.doi.org:/10.1136/bmjopen-2013-004700

The study adds to our knowledge on the prevalence and impact of anemia in patients with AF undergoing PCI and thus requiring combination antithrombotic medication. It shows that anemia is a frequent finding and that even mild anemia has an adverse impact on outcome.
Atrial Natriuretic Peptide Single Nucleotide Polymorphisms in Patients with Nonfamilial Structural Atrial Fibrillation.
P Francia, A Ricotta, A Frattari, R Stanzione, A Modestino, et al.
Clinical Medicine Insights: Cardiology 2013:7 153–159
http://dx.doi.org:/10.4137/CMC.S12239

We report lack of association between the rs5065 and −G664C ANP gene SNPs and AF in a Caucasian population of patients with structural AF. Further studies will clarify whether these or other ANP gene variants affect the risk of different subpheno-types of AF driven by distinct pathophysiological mechanisms.
Gene Expression and Genetic Variation in Human Atria.

H Lin, EV. Dolmatova, MP. Morley, KL. Lunetta, et al.
Heart Rhythm HRTHM5533.
http://dx.doi.org/10.1016/j.hrthm.2013.10.051

We studied the gene expression profiles and genetic variations in 53 left atrial and 52 right atrial tissue samples collected from the Myocardial Applied Genomics Network (MAGNet) repository. The tissues were collected from heart failure patients undergoing transplantation and from unused organ donor hearts with normal ventricular function.
A total of 187 and 259 significant cis-associations between transcript levels and genetic variants were identified in left and right atrial tissues, respectively. We also found that a SNP at a known AF locus, rs3740293, was associated with the expression of MYOZ1 in both left and right atrial tissues. Our results implicate MYOZ1 as the causative gene at the chromosome 10q22 locus for AF. 

Global Left Atrial Strain Correlates with CHADS2 Risk Score in Patients with Atrial Fibrillation
SK. Saha, PL. Anderson, G Caracciolo, A Kiotsekoglou, S Wilansky, et al.

J Am Soc Echocardiogr 2011;24:506-12.
http://dx.doi.org:/10.1016/j.echo.2011.02.012

Global longitudinal LA strain was reduced in patients with AF compared with controls (P < .001) and was a predictor of high risk for thromboembolism (CHADS2 score > 2; odds ratio, 0.86; P = .02). LA strain indexes showed good interobserver and intraobserver variability. In sequential Cox models, the prediction of hospitalization and/or death was improved by addition of global LA strain and indexed LA volume to CHADS2 score (P = .003).

Time and Frequency Domain Analysis of Heart Rate Variability and their Correlations in Diabetes Mellitus.
PTA Seyd, VIT Ahamed, J Jacob, P Joseph K.
Int  Biol and Life Sci  2008; 4(1).
http://citeseerx.ist.psu.edu/viewdoc/summary?doi=10.1.1.307.6260

In this paper, changes in ANS activity are quantified by means of frequency and time domain analysis of R-R interval variability. Electrocardiograms (ECG) of 16 patients suffering from DM and of 16 healthy volunteers were recorded. Frequency domain analysis of extracted normal to normal interval (NN interval) data indicates significant difference in very low frequency (VLF) power, low frequency (LF) power and high frequency (HF) power, between the DM patients and control group. Time domain measures, standard deviation of NN interval (SDNN), root mean square of successive NN interval differences (RMSSD), successive NN intervals differing more than 50 ms (NN50 Count), percentage value of NN50 count (pNN50), HRV triangular index and triangular interpolation of NN intervals (TINN) also show significant difference between the DM patients and control group.

Power Spectral Density of the RR interval of a 55 year old healthy volunteer

Power Spectral Density of the RR interval of a 55 year old healthy volunteer

 

 

Power Spectral Density of the RR interval of a 55 year old healthy volunteer

 

Power Spectral Density of the RR interval of a 62 year old woman suffering from diabetes for the last 15 years.

Power Spectral Density of the RR interval of a 62 year old woman suffering from diabetes for the last 15 years.

 

 

Power Spectral Density of the RR interval of a 62 year old woman suffering from diabetes for the last 15 years.

Time domain and frequency domain analysis of the RR interval variability of diabetic and normal subjects shows that there is significant difference in these measures for DM patients with respect to normal subjects. Variation of the HRV parameters indicates changes in ANS activity of DM patients. This can provide valid information regarding autonomic neuropathy in people with diabetes. It may be noted that these methods can detect changes before clinical signs appear.

Quantification of Heart Rate Variability: A Measure based on Unique Heart Rates
VIT Ahamed, P Dhanasekaran, A Naseem, NG Karthick, TKA Jaleel, Paul K

It is established that the instantaneous heart rate (HR) of healthy humans keeps on changing. Analysis of heart rate variability (HRV) has become a popular non invasive tool for assessing the activities of autonomic nervous system. Depressed HRV has been found in several disorders, like diabetes mellitus (DM) and coronary artery disease, characterised by autonomic nervous dysfunction. A new technique, which searches for pattern repeatability in a time series, is proposed specifically for the analysis of heart rate data. These set of indices, which are termed as pattern repeatability measure and pattern repeatability ratio are compared with approximate entropy and sample entropy.

Cardiovascular autonomic neuropathy in patients with diabetes mellitus
International Journal of Pharma and Bio Sciences
http://citeseerx.ist.psu.edu/viewdoc/summary?doi=10.1.1.178.2974

The cardioautonomic reflexes of 82 diabetic subjects and 40 age and sex matched healthy controls were studied using blood pressure and heart rate variation in response to standing, deep breathing, isometric exercise, cold pressor test and determination of QTc interval. Among the 82 patients, 68 patients were found to have cardiac autonomic neuropathy (CAN). Results showed that diabetics had significantly impaired cardioautonomic reflexes compared to non-diabetics, which increases with the duration of diabetes. Out of 68 patients with CAN, QTc prolongation was observed in 64 patients. In conclusion the autonomic nervous system integrity is appeared to be greatly affected by diabetes mellitus and the degree of impairment was dependent on duration of the disease.

Prognostic Value of Heart Rate Variability Analysis in Patients with Depressed Left Ventricular Function Irrespective of Cardiac Rhythm
http://citeseerx.ist.psu.edu/viewdoc/summary?doi=10.1.1.377.9244
 M Sosnowski, Pw Macfarlane, R Parma, J Skrzypek-wanha, M Tendera

A new index of heart rate variability – HRF Fraction – was developed and its value for risk stratification was evaluated in 480 patients with coronary heart disease. The main purpose to introduce the HRVF was to overcome one of the most important constraints – cardiac arrhythmia, especially atrial fibrillation – that limits use of HRV measurement as a routine clinical tool. In 384 patients with sinus rhythm (SR) and 96 with AF HRV measurements from 24h ambulatory ECG were performed. Patients were followed for a median period of 28 months. The HRV indices in those who died were compared to those who survived. Authors found that HRV Fraction and- among standard time-domain indices- only SDANN, possessed properties that allow HRV measurement to be applied for risk stratification studies in unselected population of patients with cardiac arrhythmia.

Short- and long-term reproducibility of heart rate variability in patients with long-standing type I diabetes mellitus.
Burger AJ1, Charlamb M, Weinrauch LA, D’Elia JA
Am J Cardiol. 1997 Nov 1;80(9):1198-202.
http://www.ncbi.nlm.nih.gov/pubmed/9359550

Using Pearson correlation, the time domain indicators of parasympathetic activity demonstrated very strong correlations at 3 and 6 months compared with baseline, with good correlations at 1 year. The average SD of all 5-minute RR intervals maintained a very strong correlation for the entire year (r >0.94). In the frequency domain, the measures of parasympathetic and sympathetic activity maintained a solid correlation for the entire study period. Reproducibility of HRV was also examined using repeated-measures analysis of variance. The time and frequency domain parameters demonstrated very little variation over the study period of 12 months. Thus, our investigation demonstrated that HRV in long-term diabetics using 24-hour ambulatory recordings is abnormal and reproducible over a 12-month interval; very little variation in all HRV parameters, especially in parameters of parasympathetic activity, occurred during the study period.

 

 

 

 

 

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Epilogue: Volume 4 – Translational, Post-Translational and Regenerative Medicine in Cardiology

  • Larry H Bernstein, MD, FCAP, Author and Curator, Volume Four, Co-Editor
  • Justin Pearlman, MD, PhD, FACC, Content Consultant for Series A: Cardiovascular Diseases
  • Aviva Lev-Ari, PhD, RN, Co-Editor of Volume Four and Editor-in-Chief, BioMed e-Series

 

This completes Chapter 4 in two parts on the most dynamic developments in the regulatory pathways guiding cardiovascular dynamics and function in health and disease.  I have covered key features of these in two summaries, so I shall try to look further into important expected future directions and their anticipated implications.

1. Mechanisms of Disease

Signal Transduction: Akt Phosphorylates HK-II at Thr-473 and Increases Mitochondrial HK-II Association to Protect Cardiomyocytes

David J. Roberts, Valerie P. Tan-Sah, Jeffery M. Smith and Shigeki Miyamoto
J. Biol. Chem. 2013, 288:23798-23806.  http://dx.doi.org/ 10.1074/jbc.M113.482026

Backgound: Hexokinase II binds to mitochondria and promotes cell survival.
Results: Akt phosphorylates HK-II but not the threonine 473 mutant. The phosphomimetic T473D mutant decreases its dissociation from mitochondria induced by G-6P and increases cell viability against stress.
Conclusion: Akt phosphorylates HK-II at Thr-473, resulting in increased mitochondrial HK-II and cell protection.
Significance: The Akt-HK-II signaling nexus is important in cell survival.

HK-II Phosphorylation

HK-II Phosphorylation

 

 

 

 

 

 

It has been demonstrated that an increased level of HK-II at mitochondria is protective and is increased by protective interventions but decreased under stress.

It   has not  been fully determined   which  molecular  signals  regulate  the    level    of  HK-II at mitochondria.

Thr-473 in HK-II  is phosphorylated by Akt and this phosphorylation  leads to  increases  in  mitochondrial  HK-II binding  through inhibition  of  G-6P-dependent  dissociation, conferring resistance to oxidative stress  (Fig.     7).

Overexpression of  WTHK-II increases mitochondrial HK-II and confers protection against  hydrogen peroxide,  which  is enhanced significantly  in   HK-II   T473D-expressing  cells, whereas  NHK-II, lacking the ability to bind to mitochondria, does not confer protection.   Conversely,  mitochondrial  HK-II from mitochondria (Fig.6, and B) inhibits  the  IGF-1-mediated increase in mitochondrial HK-II and cellular protection.   Similar   dose-dependent  curves were obtained in mitochondrial   HK-II     against stress    (15–25).

Gene Expression and Genetic Variation in Human Atria

Honghuang Lin PhD, Elena V. Dolmatova MD, Michael P. Morley, PhD, Kathryn L. Lunetta PhD, David D. McManus MD, ScM, et al.
Heart Rhythm  2013   http://dx.doi.org/10.1016/j.hrthm.2013.10.051

Background— The human left and right atria have different susceptibilities to develop atrialfibrillation (AF). However, the molecular events related to structural and functional changes that
enhance AF susceptibility are still poorly understood.
Objective— To characterize gene expression and genetic variation in human atria.
Results— We found that 109 genes were differentially expressed between left and right atrial tissues. A total of 187 and 259 significant cis-associations between transcript levels and genetic
variants were identified in left and right atrial tissues, respectively. We also found that a SNP at a known AF locus, rs3740293, was associated with the expression of MYOZ1 in both left and right
atrial tissues.
Conclusion— We found a distinct transcriptional profile between the right and left atrium, and extensive cis-associations between atrial transcripts and common genetic variants. Our results
implicate MYOZ1 as the causative gene at the chromosome 10q22 locus for AF.

Long-Term Caspase Inhibition Ameliorates Apoptosis, Reduces Myocardial Troponin-I Cleavage, Protects Left Ventricular Function, and Attenuates Remodeling in Rats With Myocardial Infarction

Y. Chandrashekhar,  Soma Sen, Ruth Anway,  Allan Shuros,  Inder Anand,

J Am Col  Cardiol  2004; 43(2)   http://dx.doi.org/10.1016/j.jacc.2003.09.026

This study was designed to evaluate whether in vivo caspase inhibition can prevent myocardial contractile protein degradation, improve myocardial function, and attenuate ventricular remodeling.
Apoptosis is thought to play an important role in the development and progression of heart failure (HF) after a myocardial infarction (MI). However, it is not known whether inhibiting apoptosis can attenuate left ventricular (LV) remodeling and minimize systolic dysfunction.

A 28-day infusion of caspase inhibitor was administeredimmediately after an anterior MI. In addition, five sham-operated rats given the caspase inhibitor were compared with 17 untreated sham-operated animals to study effects in non-MI rats. Left ventricular function, remodeling parameters, and hemodynamics were studied four weeks later. Myocardial caspase 3 activation and troponin-I contractile protein cleavage were studied in the non-infarct, remote LV myocardium using Western blots. Apoptosis was assessed using immunohistochemistry for activated caspase-positive cells as well as the TUNEL method. Collagen volume was estimated using morphometry.

Caspase inhibition reduced myocardial caspase 3 activation. This was accompanied by less cleavage of troponin-I, an important component of the cardiac contractile apparatus, and fewer apoptotic cardiomyocytes. Furthermore, caspase inhibition reduced LV-weight-to- body-weight ratio, decreased myocardial interstitial collagen deposition, attenuated LV remodeling, and better preserved LV systolic function after MI.

Caspase inhibition, started soon after MI and continued for four weeks, preserves myocardial contractile proteins, reduces systolic dysfunction, and attenuates ventricular remodeling.

These findings may have important therapeutic implications in post-MI HF. J Am Col Cardiol 2004;43:295–301)

Precardiac deletion of Numb and Numblike reveals renewal of cardiac progenitors

Lincoln T Shenje,  Peter P Rainer , Gun-sik Cho , Dong-ik Lee , Weimin Zhong , Richard P Harvey , David A Kass , Chulan Kwon *,  et al.
eLife 2014.    http://dx.doi.org/10.7554/eLife.02164.001

Cardiac progenitor cells (CPCs) must control their number and fate to sustain the rapid heart growth during development, yet the intrinsic factors and environment governing these processes remain unclear. Here, we show that deletion of the ancient cell-fate regulator Numb (Nb) and its homologue Numblike (Nbl) depletes CPCs in second pharyngeal arches (PA2s) and is associated with an atrophic heart. With histological, fow cytometric and functional analyses, we fnd that CPCs remain undifferentiated and expansive in the PA2, but differentiate into cardiac cells as they exit the arch. Tracing of Nb- and Nbl-defcient CPCs by lineage-specifc mosaicism reveals that the CPCs normally populate in the PA2, but lose their expansion potential in the PA2. These fndings demonstrate that Nb and Nbl are intrinsic factors crucial for the renewal of CPCs in the PA2 and
that the PA2 serves as a microenvironment for their expansion.

2. Diagnostics and Risk Assessment

Classical and Novel Biomarkers for Cardiovascular Risk Prediction in the United States

Aaron R. Folsom
J Epidemiol 2013;23(3):158-162   http://dx.doi.org/10.2188/jea.JE20120157

Cardiovascular risk prediction models based on classical risk factors identified in epidemiologic cohort studies are useful in primary prevention of cardiovascular disease in individuals. This article briefly reviews aspects of
cardiovascular risk prediction in the United States and efforts to evaluate novel risk factors. Even though many novel risk markers have been found to be associated with cardiovascular disease, few appear to improve risk prediction
beyond the powerful, classical risk factors. A recent US consensus panel concluded that clinical measurement of certain novel markers for risk prediction was reasonable, namely,

  1. hemoglobin A1c (in all adults),
  2. microalbuminuria (in patients with hypertension or diabetes), and
  3. C-reactive protein,
  4. lipoprotein-associated phospholipase,
  5. coronary calcium,
  6. carotid intima-media thickness, and
  7. ankle/brachial index (in patients deemed to be at intermediate cardiovascular risk, based on traditional risk factors).

Diagnostic accuracy of NT-proBNP ratio (BNP-R) for early diagnosis of tachycardia-mediated cardiomyopathy: a pilot study

Amir M. Nia, Natig Gassanov, Kristina M. Dahlem, Evren Caglayan, Martin Hellmich, et al.
Clin Res Cardiol (2011) 100:887–896    http://dx.doi.org/10.1007/s00392-011-0319-y

Tachycardia-mediated cardiomyopathy (TMC) occurs as a consequence of prolonged high heart rate due to ventricular and supraventricular tachycardia. In animal models, rapid pacing induces severe biventricular remodeling with dilation and dysfunction [7]. On a cellular basis, cardiomyocytes exert fundamental morphological and functional roles.

When heart failure and tachycardia occur simultaneously, a useful diagnostic tool for early discrimination of patients with benign tachycardia-mediated  cardiomyopathy (TMC) versus major structural heart disease  (MSHD) is not available. Such a tool is required to prevent unnecessary and wearing diagnostics in patients with reversible TMC. Moreover, it could lead to early additional diagnostics and therapeutic approaches in patients with  MSHD.

A total of 387 consecutive patients with supraventricular arrhythmia underwent assessment.  Of these patients, 40 fulfilled the inclusion criteria
with a resting heart rate C100 bpm and an impaired left ventricular ejection fraction \40%. In all patients, successful electrical cardioversion was performed. At baseline, day 1 and weekly for 4 weeks, levels of NT-proBNP and echocardiographic parameters were evaluated.

NT-proBNP ratio (BNP-R) was calculated as a quotient of baseline NT-proBNP/follow-up NT-proBNP. After 4 weeks, cardiac catheterization was performed to identify patients with a final diagnosis of TMC versus MSHD.

Initial NT-proBNP concentrations were elevated and consecutively decreased after cardioversion in all patients studied. The area under the ROC curve for BNP-R to detect TMC was 0.90 (95% CI 0.79–1.00; p \ 0.001) after 1 week  and 0.995 (95% CI 0.99–1.00; p \ 0.0001) after 4 weeks. One week after cardioversion already, a BNP-R cutoff C2.3 was useful for TMC diagnosis indicated by an accuracy of 90%, sensitivity of 84% and specificity of 95%.

BNP-R was found to be highly accurate for the early diagnosis of TMC.

Omega-3 Index and Cardiovascular Health

Clemens von Schacky
Nutrients 2014; 6: 799-814;  http://dx. doi.org/10.3390/nu602099

Fish, marine oils, and their concentrates all serve as sources of the two marine omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), as do some products from algae.
To demonstrate an effect of EPA + DHA on heart health, a number of randomized, controlled intervention studies with clinical endpoints like overall mortality or a combination of adverse cardiac events were conducted in populations with elevated cardiovascular risk. One early intervention study with oily fish, rich in EPA + DHA, and some early studies with fish oil or fish oil concentrate or even purified EPA at doses ranging between 0.9 and 1.8 g/day indeed demonstrated effects in terms of fewer sudden cardiac deaths, fatal or non-fatal myocardial infarctions, or a combination of adverse cardiac events.

Recent meta-analyses found no significant benefits on total mortality, cardiovascular mortality, and other adverse cardiac or cardiovascular events [13–18]. This is in contrast to findings in epidemiologic studies, where intake of EPA + DHA had been found to correlate generally with an up to 50% lower incidence of adverse cardiac events [18,19], and in even sharper contrast to epidemiologic studies based on levels of EPA + DHA, demonstrating e.g., a 10-fold lower incidence of sudden cardiac death associated with high levels of the
fatty acids, as compared to low levels.

This seemingly contradictory evidence has led the American Heart Association to recommend “omega-3 fatty acids from fish or fish oil capsules (1 g/day) for cardiovascular disease risk reduction” for secondary prevention, whereas the European Society for Cardiology recommends “Fish at least twice a week, one of which to be oily fish”, but no supplements for cardiovascular prevention.

A similar picture emerges for atrial fibrillation: In epidemiologic studies, consumption of EPA + DHA or higher levels of EPA + DHA were associated with lower risk for developing atrial fibrillation, while intervention studies found no effect. Pertinent guidelines do not mention EPA + DHA. A similar picture also emerges for severe ventricular rhythm disturbances.

Why is it that trial results are at odds with results from epidemiology? What needs to be done to better translate the epidemiologic findings into trial results? The current review will try to shed some light on this  issue, with a special consideration of the Omega-3 Index.

Recent large trials with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the cardiovascular field did not demonstrate a beneficial effect in terms of reductions of clinical endpoints like

  • total mortality,
  • sudden cardiac arrest or
  • other major adverse cardiac events.

Pertinent guidelines do not uniformly recommend EPA + DHA for cardiac patients. In contrast,

  • in epidemiologic findings, higher blood levels of EPA + DHA were consistently associated with a lower risk for the endpoints mentioned.

The following points argue for the use of erythrocytes: erythrocyte fatty acid
composition has a low biological variability, erythrocyte fat consists almost exclusively of phospholipids, erythrocyte fatty acid composition reflects tissue fatty acid composition, pre-analytical stability, and other points.  In 2004, EPA + DHA in erythrocyte fatty acids were defined as the Omega-3 Index and suggested as a risk factor for sudden cardiac death [39]. Integral to the definition was a specific and standardized analytical procedure, conforming the quality management routinely implemented in the field of clinical chemistry.

The laboratories adhering to the HS-Omega-3 Index methodology perform regular proficiency testing, as mandated in routine Clinical Chemistry labs. So far, the HS-Omega-3 Index is the only analytical procedure used in several laboratories. A standardized analytical procedure is a prerequisite to generate the data base necessary to transport a laboratory parameter from research into clinical routine. Moreover, standardization of the analytical procedure is the first important criterion for establishing a new biomarker for cardiovascular risk set forth by the American Heart Association and the US Preventive Services Task Force.

Because of low biological and analytical variability, a standardized analytical procedure, a large database and for other reasons,

  • blood levels of EPA + DHA are frequently assessed in erythrocytes, using the HS-Omega-3 Index methodology.

Table 1. Mean HS-Omega-3 Index values in various populations, Mean (±standard deviation (SD)). Please note that in every population studied, a lower value was found to be associated with a worse condition than a higher value. References are given, if not, unpublished, n = number of individuals measured.

All levels of fatty acids are determined by the balance of substance entering the body and those leaving the body. Neither a recent meal, even if rich in EPA + DHA, nor severe cardiac events altered the HS-Omega-3 Index. However, while long-term intake of EPA + DHA, e.g., as assessed with food questionnaires, was the main predictor of the HS-Omega-3 Index, long-term intake explained only 12%–25% of its variability. A hereditary component of 24% exists. A number of other factors correlated positively (+) or negatively (−), like age (+), body mass index (−), socioeconomic status (+), smoking (−), but no other conventional cardiac risk factors. More factors determining the level of the HS-Omega-3 Index, especially regarding efflux remain to be  defined. Therefore, it is impossible to predict the HS-Omega-3 Index in an individual, as it is impossible to predict the increase in the HS-Omega-3 Index in an individual in response to a given dose of EPA + DHA. In Table 2, current evidence is presented on the relation of the HS-Omega-3 Index to CV events.

The HS-Omega-3 Index has made it possible to reclassify individuals from intermediate cardiovascular risk into the respective high risk and low risk strata, the third criterion for establishing a new biomarker for CV  risk.

A low Omega-3 Index fulfills the current criteria for a novel cardiovascular risk factor.

Increasing the HS-Omega-3 Index by increased intake of EPA + DHA in randomized controlled trials improved a number of surrogate parameters for cardiovascular risk:

  1. heart rate was reduced,
  2. heart rate variability was increased,
  3. blood pressure was reduced,
  4. platelet reactivity was reduced,
  5. triglycerides were reduced,
  6. large buoyant low-density lipoprotein (LDL)-particles were increased and
  7. small dense LDL-particles were reduced,
  8. large buoyant high-density lipoproteins (HDL)2 were increased,
  9. very low-density lipoprotein (VLDL1) + 2 was reduced,
  10. pro-inflammatory cytokines (e.g., tumor necrosis factor alpha, interleukin-1β, interleukins-6,8,10 and monocyte chemoattractant protein-1) were reduced,
  11. anti-inflammatory oxylipins were increased.

Importantly, in a two-year randomized double-blind angiographic intervention trial, increased erythrocyte EPA + DHA

  • reduced progression and increased regression of coronary lesions, an intermediate parameter.

Taken together, increasing the HS-Omega-3 Index improved surrogate and intermediate parameters for cardiovascular events. A large intervention trial with clinical endpoints based on the HS-Omega-3 Index remains to be conducted. Therefore, the fourth criterion, proof of therapeutic consequence of determining the HS-Omega- Index, is only partially fulfilled.

 

Neutral results of intervention trials can be explained by issues of bioavailability and trial design that surfaced after the trials were initiated.

In the future, incorporating the Omega-3 Index into trial designs by

  1. recruiting participants with a low Omega-3 Index and
  2. treating them within a pre-specified target range (e.g., 8%–11%),
  3. will make more efficient trials possible and
    • provide clearer answers to the questions asked than previously possible.

 

3. Stem Cells and Regenerative Biology

Adult Stem Cells Reverse Muscle Atrophy In Elderly Mice   http://www.science20.com/profile/news_staff

Bioengineers at the University of California, Berkeley in a new study published in Nature say they have identified two key regulatory pathways that control how well adult stem cells repair and replace damaged tissue. They then tweaked how those stem cells reacted to those biochemical signals to revive the ability of muscle tissue in old mice to repair itself nearly as well as the muscle in the mice’s much younger counterparts. Irina Conboy, an assistant professor of bioengineering and an investigator at the Berkeley Stem Cell Center and at the California Institute for Quantitative Biosciences (QB3), led the research team conducting this study. Because the findings relate to adult stem cells that reside in existing tissue, this approach to rejuvenating degenerating muscle eliminates the ethical and medical complications associated with transplanting tissues grown from embryonic stem cells. The researchers focused on

  • the interplay of two competing molecular pathways that control the stem cells,

which sit next to the mature, differentiated cells that make up our working body parts. When the mature cells are damaged or wear out, the stem cells are called into action to begin the process of rebuilding.

old muscle tissue is left with

old muscle tissue is left with

 

 

 

 

 

 

 

 

 

 

 

 

“We don’t realize it, but as we grow our bodies are constantly being remodeled,” said Conboy. “We are constantly falling apart, but we don’t notice it much when we’re young because we’re always being restored. As we age, our stem cells are prevented, through chemical signals, from doing their jobs.” The good news, the researchers said, is that

  • the stem cells in old tissue are still ready and able to perform their regenerative function
  • if they receive the appropriate chemical signals.

Studies have shown that when old tissue is placed in an environment of young blood, the stem cells behave as if they are young again. “Conversely, we have found in a study published last year that even young stem cells rapidly age when placed among blood and tissue from old mice,” said Carlson, who will stay on at UC Berkeley to expand his work on stem cell engineering.

  • Adult stem cells have a receptor called Notch that, when activated,
  • tells them that it is time to grow and divide
  • stem cells also have a receptor for the protein TGF-beta
  • that sets off a chain reaction activatingthemoleculepSmad3 and
    • ultimately producing cyclin-dependent kinase (CDK) inhibitors, which regulate the cell’s ability to divide.
  • activated Notch competeswithactivatedpSmad3 for
    • binding to the regulatory regions of the same CDK inhibitors in the stem cell

“We found that Notch is capable of physically kicking off pSmad3 from the promoters for the CDK inhibitors within the stem cell’s nucleus, which tells us that a precise manipulation of the balance of these pathways would allow the ability to control stem cell responses.” Notch and TGF-beta are well known in molecular biology, but Conboy’s lab is the first to connect them to the process of aging, and the first to show that they act in opposition to each other within the nucleus of the adult stem cell. Aging and the inevitable march towards death are, in part, due to the progressive decline of Notch and the increased levels of TGF-beta , producing a one-two punch to the stem cell’s capacity to effectively rebuild the body, the researchers said.

The researchers disabled the “aging pathway” that tells stem cells to stop dividing by using an established method of RNA interference that reduced levels of pSmad3. The researchers then examined the muscle of the different groups of mice one to five days after injury to compare how well the tissue repaired itself. As expected,

  •  muscle tissue in the young mice easily replaced damaged cells with new, healthy cells. In contrast,
  • the areas of damaged muscle in the control group of old mice were characterized by fibroblasts and scar tissue. However,
  • muscles in the old mice whose stem cell “aging pathway”had been dampened showed levels of cellular regeneration that were
    • comparable to their much younger peers, and that were 3 to 4 times greater than those of the group of “untreated” old mice.

Adult Stem Cells To Repair Damaged Heart Muscle

http://www.science20.com/profile/news_staff

In the first trial of its kind in the world, 60 patients who have recently suffered a major heart attack will be injected with selected stem cells from their own bone marrow during routine coronary bypass surgery. The Bristol trial will test

  • whether the stem cells will repair heart muscle cells damaged by the heart attack,
  • by preventing late scar formation and hence impaired heart contraction.

“ Cardiac stem cell therapy aims to repair the damaged heart as it has the potential to replace the damaged tissue.” We have elected to use a very promising stem cell type selected from the patient’s own bone marrow. This approach ensures no risk of rejection or infection. It also gets around the ethical issues that would result from use of stem cells from embryonic or foetal tissue.

In this trial (known as TransACT), all patients will have bone marrow harvested before their heart operation. Then either stem cells from their own bone marrow or a placebo will be injected into the patients’ damaged hearts during routine coronary bypass surgery. The feasibility and safety of this technique has already been demonstrated. As a result of the chosen double blind placebo-controlled design, neither the patients nor the surgeon knows whether the patient is going to be injected with stem cells or placebo. This ensures that results are not biased in any way, and is the most powerful way to prove whether or not the new treatment is effective.

Research of Stem Cells Repair Damaged Heart

By Kelvinlew Minhan | March 26th 2008

Under highly specific growth conditions in laboratory culture dishes, stem cells

  • can be coaxed into developing as new cardiomyocytes and vascular endothelial cells (Kirschstein and Skirboll, 2001).

Discoveries that have triggered the interest in the application of adult stem cells to heart muscle repair in animal models have been made by researchers in the past few years (Kirschstein and Skirboll, 2001). One  study demonstrated that cardiac tissue can be regenerated in the mouse heart attack model through the introduction of adult stem cells from mouse bone marrow (Kirschstein and Skirboll, 2001). These cells were transplanted into the marrow of irradiated mice approximately 10 weeks before the recipient mice were subjected to heart attack thru tying off different major heart blood vessel, the left anterior descending (LAD) coronary artery. The survival rate was 26 percent at two to four weeks after the induced cardiac injury (Kirschstein and Skirboll, 2001). Another study of the region surrounding the damaged tissue in surviving mice showed the presence of donor-derived cardiomyocytes and endothelial cells (Kirschstein and Skirboll, 2001).

  • the mouse hematopoietic stem cells transplanted into the bone marrow had migrated to the border part of the damaged area, and differentiated into several types of tissue for cardiac repair.

Regenerating heart tissue through stem cell therapy

http://www.mayo.edu/research/discoverys-edge/regenerating-heart-tissue-stem-cell-therapy

Summary

A groundbreaking study on repairing damaged heart tissue through stem cell therapy has given patients hope that they may again live active lives. An international team of Mayo Clinic researchers and collaborators has done it by discovering a way to regenerate heart tissue.

“It’s a paradigm shift,” says Andre Terzic, M.D., Ph.D., director of Mayo Clinic’s Center for Regenerative Medicine and senior investigator of the stem cell trial. “We are moving from traditional medicine, which addresses the symptoms of disease to cure disease.” Treating patients with cardiac disease has typically involved managing heart damage with medication.  In collaboration with European researchers, Mayo Clinic researchers have discovered a novel way to repair a damaged heart. In Mayo Clinic’s breakthrough process,
  • stem cells are harvested from a patient’s bone marrow.
  •  undergo a laboratory treatment that guides them into becoming cardiac cells,
  • which are then injected into the patient’s heart in an effort to grow healthy heart tissue.
The study is the first successful demonstration in people of the feasibility and safety of transforming adult stem cells into cardiac cells. Beyond heart failure, the Mayo Clinic research also is a milestone in the emerging field of regenerative medicine, which seeks to fully heal damaged tissue and organs.

Creating a heart repair kit

Process of converting bone marrow cells to heart cells
This image shows the process used in the clinical trials to repair damaged hearts. Cardioprogenitor cells is another term for cardiopoietic cells, those that were transformed into cardiac cells.
Stem cells transforming to cardiac tissue
Transformation: The cardiopoietic cells on the left react to the cardiac environment, cluster together with like cells and form tissue.
 Mayo Clinic researchers pursued this research, inspired by an intriguing discovery. In the early 2000s, they analyzed stem cells from 11 patients undergoing heart bypass surgery. The stem cells from two of the patients had an unusually high expression of certain transcription factors — the proteins that control the flow of genetic information between cells. Clinically, the two patients appeared no different from the others, yet their stem cells seemed to show unique capacity for heart repair.
That observation drove them to  determine how to convert  nonreparative stem cells to become reparative. Doing so required determining precisely how the human heart naturally develops, at a subcellular level. That painstaking work was led by Atta Behfar, M.D., Ph.D., a cardiovascular researcher at Mayo Clinic in Rochester, Minn. With other members of the Terzic research team, Dr. Behfar identified hundreds of proteins involved in the process of heart development (cardiogenesis). The researchers then set out to identify which of these proteins are essential in driving a stem cell to become a cardiac cell. Using computer models,
  • they simulated the effects of eliminating proteins one by one from the process of heart development.
  • That method yielded about 25 proteins.
    • The team then pared that number down to 8 proteins that their data indicated were essential.
The research team was then able to develop the lab procedure that guides stem cells to become heart cells.
The treated stem cells were dubbed cardiopoietic, or heart creative. A proof of principle study about guided cardiopoiesis, whose results were published in the Journal of the American College of Cardiology in 2010, demonstrated that animal models with heart disease that had been injected with caridiopoietic cells had improved heart function compared with animals injected with untreated stem cells. Hailed as “landmark work,” by the journal’s editorial writer, the study showed it was indeed possible to teach stem cells to become cardiac cells. Stem cells from each patient in the cardiopoiesis group were successfully guided to become cardiac cells. The treated cells were injected into the heart wall of each of those patients without apparent complications.
“Ihis newprocessofcardiopoiesiswas achieved in 100 percent of cases, with a very good safety profile,” Dr.Terzic says. “We are enabling the heart toregainitsinitial structure and function,” Dr.Terzic says, “and we will not stop here.” The clinicaltrialfindingsareexpectedto be published in the Journal of the American College of Cardiology in 2013.  Meanwhile, research to improve the injection process and effectiveness is underway.

Stem Cells from Humans Repair Heart Damage in Monkeys

GEN News Highlights  May1, 2014

GPCR Insights Brighten Drug Discovery Outlook

Ken Doyle, Ph.D.

GEN Apr 15, 2014 (Vol. 34, No. 8)

Recent years have seen major advances in understanding the structure-function relationships of G protein-coupled receptors (GPCRs). This large superfamily of transmembrane receptors comprises over 800 members in humans.

GPCRs regulate a wide variety of physiological processes including

  • sensation (vision, taste, and smell),
  • growth,
  • hormone responses, and
  • regulation of the immune and
  • autonomic nervous systems.

Their involvement in multiple disease pathways makes GPCRs attractive targets for drug discovery efforts.

These multifaceted proteins will be the subject of “GPCR Structure, Function and Drug Discovery,” a Global Technology Community conference scheduled to take place May 22–23 in Boston. The conference is expected to cover a broad range of topics including biased signaling, membrane protein structures, GPCR signaling dynamics, computational approaches to disease.

According to Bryan Roth, M.D., Ph.D., Michael Hooker Distinguished Professor at the University of North Carolina, Chapel Hill,

  • drugs that can selectively target various downstream GPCR pathways hold the most promise.

Dr. Roth’s laboratory studies approximately 360 different GPCRs with therapeutic potential using massively parallel screening methods. His research focuses on “functional selectivity,” which he describes as

  • “the ligand-dependent selectivity for certain signal transduction pathways in one and the same receptor.”

Dr. Roth notes that structural data have demonstrated that GPCRs exist in multiple conformations: “The structures of the 5-hydroxytryptamine 2B receptor and the recent high-resolution delta-opioid receptor structure have provided evidence for conformational rearrangements that contribute to functional selectivity.” Drugs that take advantage of this selectivity by preferentially stabilizing certain conformations may have unique therapeutic utility.

“Generally, we look at G protein versus arrestin-based signaling, although it’s also possible to examine how drugs activate one G protein-mediated signaling pathway versus another.

 

fluorescently tagged Arrestin and GPRC of interest

fluorescently tagged Arrestin and GPRC of interest

 

 

 

 

 

 

 

  • β-Arrestins constitute a major class of intracellular scaffolding proteins that regulate GPCR signaling by preventing or enhancing the binding of GPCRs to intracellular signaling molecules. Laura Bohn, Ph.D., associate professor at Scripps Florida,  studies the roles that β-arrestins play in GPCR-mediated signaling.
  • a particular β-arrestin can play multiple, tissue-specific roles—shutting down the signaling of a receptor in one tissue while activating signaling in another.
  • different ligands can direct GPCR signaling to different effectors, which could result in different physiological effects,” comments Dr. Bohn. “Our challenge is in determining what signaling pathways to harness to promote certain effects, while avoiding others.”
Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling

Arrestin binding to active GPCR kinase (GRK)-phosphorylated GPCRs blocks G protein coupling

 

 

 

 

 

 

 

 

 

 

 

Using Designer Proteins

The multifunctional signaling abilities of β-arrestins has prompted large-scale study of their properties. Vsevolod Gurevich, Ph.D., professor of pharmacology at Vanderbilt University, studies

  1. the structure,
  2. function, and
  3. biology of arrestin proteins.

β-arrestins have three main functions.

  1. First, they prevent the coupling of GPCRs to G proteins, thereby blocking further G protein-mediated signaling (a process known as desensitization).
  2. Second, the binding of a GCPR releases the β-arrestin’s carboxy-terminal “tail” and promotes internalization of the receptor.
  3. Third, receptor-bound β-arrestins bind other signaling proteins, resulting in a second wave of arrestin-mediated signaling.

Dr. Gurevich’s laboratory studies β-arrestin biology through the use of three types of specially designed mutants—

  1. enhanced phosphorylation-dependent,
  2. receptor-specific, and
  3. signaling-biased mutants.

an enhanced mutant of visual β-arrestin-1 partially compensates for defects of rhodopsin phosphorylation in vivo,

“Several congenital disorders are caused by mutant GPCRs that cannot be normally phosphorylated because they have lost GPCR kinase (GRK) sites. Enhanced super-active arrestins have the potential to compensate for these defects, bringing the signaling closer to normal.”

  • Dr. Gurevich explains the strategy involved in creating designer β-arrestins: “We identify residues critical for individual β-arrestin functions by mutagenesis, using limited structural information as a guide.
  • We also work on getting more structural information. In collaboration with different crystallographers, we solved the crystal structures of all four vertebrate β-arrestin subtypes in the basal state, as well as the structure of the arrestin-1-rhodopsin complex.”
  • Dr. Gurevich believes that designer β-arrestins “are the next step in research and therapy, moving way beyond what small molecules can achieve.
  • The difference in capabilities between redesigned signaling proteins, including β-arrestins, and conventional small molecule drugs is about the same as that between airplanes and horse-driven carriages.”
  • Dr. Gurevich observes that redesigned signaling proteins face considerable obstacles in terms of gene delivery, but that the efforts are worth it. “Using designer signaling proteins, we can tell the cell what to do in a language it cannot disobey,” asserts Dr. Gurevich.

Synthesis and Antihypertensive Screening of Novel Substituted 1,2- Pyrazoline Sulfonamide Derivatives

Avinash M. Bhagwat , Anilchandra R. Bha , Mahesh S. Palled , Anand P. Khadke , Anuradha M. Patil, et al.

Am. J. PharmTech Res. 2014; 4(2).    http://www.ajptr.com/ 

Angiotensin II receptor antagonists, also known as angiotensin receptor blockers , AT1-receptor antagonists or sartans, are a group of pharmaceuticals which modulate the renin-angiotensin-aldosterone system. Their main use is in hypertension, diabetic nephropathy and congestiveheart failure. These substances are AT1-receptor antagonists which

  • block the activationof angiotensin II AT1 receptors.

Blockade of AT1 receptors directly causes

1 vasodilation,

2 reduces secretion of vasopressin,

3 reduces production and secretion of aldosterone, amongst other actions –

4 the combined effect of which is reduction of blood pressure.

Irbesartan is a safe and effectiveangiotensin II receptor antagonist with an affinity for the AT1 receptor that is more than 8,500times greater than its affinity for AT2 receptor. This agent has a higher bioavailability (60-80%) than other drugs in its class . In both Losartan and Irbesartan structures imidazole moiety is being present. A structure analog of losartan and Irbesartan are designed by incorporating the heterocycles like pyrazoline group. We felt it would be interesting to explore the possibilities of 1,2-pyrazoline derivatives for Angiotensin II receptor antagonistic activity.

The Irbesartan structure was a modified Losartan structure, which had all the identity of a Losartan molecule but with groups that would fit the hydrophobic cavity with a tetramethylene group and an alkyl side chain that would fit in the pocket in the AT1 receptor. The hydroxyl methyl group of Losartan being replaced with carbonyl group of Irbesartan. With a view to introduce a hydrogen bonding interaction with AT1 receptor, these structures were further modified with a view of retaining both hydrogen bonding characteristics and as well as lipophilic groups. Losartan and Irbesartan structure contains a diphenyl molecule & imidazole ring.

In Losartan and Irbesartan diphenyl molecule is attached to the nitrogen of the imidazole ring. It is interesting to to see the activity of compounds containing two phenyl rings attached at two different positions namely3,5 position of 1, 2-pyrazoline ring. The sulphonamide derivatives known for its diuretics activity which reduces renal hypertension. We use to synthesize sulphonamide and pyrazoline in one molecule to check its possible Angiotensin II receptor antagonist property. For this reason chalcones were synthesized reacted with hydrazine hydrate to yield the corresponding 1,2-pyrazoline derivatives which further condensed with sulphanilamide and formaldehyde by mannich condensation reaction.

Acute Toxicity Study (LD50)

This study was carried out in order to establish the therapeutic and toxic doses of the newly synthesized 1,2 pyrazoline derivatives. To establish LD50 of these compounds the method described by Miller & Tainter was employed.

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Diagnostic Value of Cardiac Biomarkers


Diagnostic Value of Cardiac Biomarkers

Author and Curator: Larry H Bernstein, MD, FCAP 

These presentations covered several views of the utilization of cardiac markers that have evolved for over 60 years.  The first stage was the introduction of enzymatic assays and isoenzyme measurements to distinguish acute hepatitis and acute myocardial infarction, which included lactate dehydrogenase (LD isoenzymes 1, 2) at a time that late presentation of the patient in the emergency rooms were not uncommon, with the creatine kinase isoenzyme MB declining or disappeared from the circulation.  The world health organization (WHO) standard definition then was the presence of two of three:

1. Typical or atypical precordial pressure in the chest, usually with radiation to the left arm

2. Electrocardiographic changes of Q-wave, not previously seen, definitive; ST- elevation of acute myocardial injury with repolarization;
T-wave inversion.

3. The release into the circulation of myocardial derived enzymes –
creatine kinase – MB (which was adapted to measure infarct size), LD-1,
both of which were replaced with troponins T and I, which are part of the actomyosin contractile apparatus.

The research on infarct size elicited a major research goal for early diagnosis and reduction of infarct size, first with fibrinolysis of a ruptured plaque, and this proceeded into the full development of a rapidly evolving interventional cardiology as well as cardiothoracic surgery, in both cases, aimed at removal of plaque or replacement of vessel.  Surgery became more imperative for multivessel disease, even if only one vessel was severely affected.

So we have clinical history, physical examination, and emerging biomarkers playing a large role for more than half a century.  However, the role of biomarkers broadened.  Patients were treated with antiplatelet agents, and a hypercoagulable state coexisted with myocardial ischemic injury.  This made the management of the patient reliant on long term followup for Warfarin with the international normalized ratio (INR) for a standardized prothrombin time (PT), and reversal of the PT required transfusion with thawed fresh frozen plasma (FFP).  The partial thromboplastin test (PPT) was necessary in hospitalization to monitor the heparin effect.

Thus, we have identified the use of traditional cardiac biomarkers for:

1. Diagnosis
2. Therapeutic monitoring

The story is only the beginning.  Many patients who were atypical in presentation, or had cardiovascular ischemia without plaque rupture were problematic.  This led to a concerted effort to redesign the troponin assays for high sensitivity with the concern that the circulation should normally be free of a leaked structural marker of myocardial damage. But of course, there can be a slow leak or a decreased rate of removal of such protein from the circulation, and the best example of this would be the patient with significant renal insufficiency, as TnT is clear only through the kidney, and TNI is clear both by the kidney and by vascular endothelium.  The introduction of the high sensitivity assay has been met with considerable confusion, and highlights the complexity of diagnosis in heart disease.  Another test that is used for the diagnosis of heart failure is in the class of natriuretic peptides (BNP, pro NT-BNP, and ANP), the last of which has been under development.

While there is an exponential increase in the improvement of cardiac devices and discovery of pharmaceutical targets, the laboratory support for clinical management is not mature.  There are miRNAs that may prove valuable, matrix metalloprotein(s), and potential endothelial and blood cell surface markers, they require

1. codevelopment with new medications
2. standardization across the IVD industry
3. proficiency testing applied to all laboratories that provide testing
4. the measurement  on multitest automated analyzers with high capability in proteomic measurement  (MS, time of flight, MS-MS)

nejmra1216063_f1   Atherosclerotic Plaques Associated with Various Presentations               nejmra1216063_f2     Inflammatory Pathways Predisposing Coronary Arteries to Rupture and Thrombosis.        atherosclerosis progression

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Adult Left Atrium: Reduction of Pitx2c Expression Promotes Atrial Fibrillation Inducibility and Complex Changes in Gene Expression

Aviva Lev-Ari, PhD, RN

PITX2c Is Expressed in the Adult Left Atrium, and Reducing Pitx2c Expression Promotes Atrial Fibrillation Inducibility and Complex Changes in Gene Expression

Paulus Kirchhof, MD*Peter C. Kahr*Sven KaeseIlaria Piccini, PhD, Ismail Vokshi, BSc, Hans-Heinrich Scheld, MD, Heinrich Rotering, MD, Lisa Fortmueller, MD (vet),Sandra Laakmann, MD (vet), Sander Verheule, PhD, Ulrich Schotten, MD, PhD,Larissa Fabritz, MD and Nigel A. Brown, PhD

Author Affiliations

From the Department of Cardiology and Angiology (P.K., P.C.K., S.K., I.P., L.F., S.L., L.F.) and the Department of Thoracic and Cardiovascular Surgery (H.-H.S., H.R.), University Hospital Muenster, Germany; Division of Biomedical Sciences (P.C.K., I.V., N.A.B.), St. George’s, University of London, United Kingdom; and the Department of Physiology (S.V., U.S.), Maastricht University, The Netherlands.

Correspondence to Nigel A. Brown, PhD, Division of Biomedical Sciences, St George’s, University of London, Cranmer Terrace, London, SW17 0RE, UK. E-mail  nbrown@sgul.ac.uk

* Drs Kirchhof and Kahr contributed equally to this work.

Abstract

Background— Intergenic variations on chromosome 4q25, close to the PITX2 transcription factor gene, are associated with atrial fibrillation (AF). We therefore tested whether adult hearts express PITX2 and whether variation in expression affects cardiac function.

Methods and Results— mRNA for PITX2 isoform c was expressed in left atria of human and mouse, with levels in right atrium and left and right ventricles being 100-fold lower. In mice heterozygous for Pitx2c (Pitx2c+/−), left atrial Pitx2c expression was 60% of wild-type and cardiac morphology and function were not altered, except for slightly elevated pulmonary flow velocity. Isolated Pitx2c+/−hearts were susceptible to AF during programmed stimulation. At short paced cycle lengths, atrial action potential durations were shorter in Pitx2c+/− than in wild-type. Perfusion with the β-receptor agonist orciprenaline abolished inducibility of AF and reduced the effect on action potential duration. Spontaneous heart rates, atrial conduction velocities, and activation patterns were not affected in Pitx2c+/− hearts, suggesting that action potential duration shortening caused wave length reduction and inducibility of AF. Expression array analyses comparing Pitx2c+/− with wild-type, for left atrial and right atrial tissue separately, identified genes related to calcium ion binding, gap and tight junctions, ion channels, and melanogenesis as being affected by the reduced expression of Pitx2c.

Conclusions— These findings demonstrate a physiological role for PITX2 in the adult heart and support the hypothesis that dysregulation of PITX2 expression can be responsible for susceptibility to AF.

SOURCE:

Circulation: Cardiovascular Genetics.2011; 4: 123-133

Published online before print January 31, 2011,

doi: 10.1161/ CIRCGENETICS.110.958058

 

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CaKMII Inhibition in Obese, Diabetic Mice leads to Lower Blood Glucose Levels

Reporter: Larry H Bernstein, MD, FCAP

This recent publication was reported in MedPage today. It is different than, but highly suggestive of our recent report about the Univesity of Iowa discovery of “Oxidized CaKMII inhibition” as a therapeutic target for atrial arrhythmia.

Oxidized Calcium Calmodulin Kinase and Atrial Fibrillation
Author: Larry H. Bernstein, MD, FCAP, and Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/10/26/oxidized-calcium-calmodulin-kinase-and-atrial-fibrillation/
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:
  • 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.
    • Established a critical role of ox-CaMKII in promoting AF
  • 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.
    • The protection from AF in MsrA TG mice appeared to be independent of pressor effects that are critical for the proarrhythmic actions.
  • 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.
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
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
These data to suggest that  in ox–the proarrhythmic effects of Ang II infusion depend upon an increaseCaMKII, sarcoplasmic reticulum Ca2+ leak and DADs.
Enhanced CaMKII-mediated phosphorylation of serine 2814 on RyR2
  • is associated with an increased susceptibility to acquired arrhythmias, including AF
Proarrhythmic actions of ox-CaMKII
  • require access to RyR2 serine 2814.
Mutant S2814A knock-in mice (lacking serine 2814) were highly resistant to Ang II mediated AF
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
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.
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 AF
Ang II and diabetes-induced CaMKII oxidation caused sinus node dysfunction by increased pacemaker cell death and fibrosis
 ox-CaMKII increases susceptibility for AF via increased diastolic sarcoplasmic reticulum Ca2+ release
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 the reader to the following related articles published in pharmaceutical Intelligence:
  1. Contributions to cardiomyocyte interactions and signaling
    Author and Curator: Larry H Bernstein, MD, FCAP  and Curator: Aviva Lev-Ari, PhD, RN
    https://pharmaceuticalintelligence.com/2013/10/21/contributions-to-cardiomyocyte-interactions-and-signaling/
  2. 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
    https://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/
  3. Part I. Identification of Biomarkers that are Related to the Actin Cytoskeleton
    Curator and Writer: Larry H Bernstein, MD, FCAP
    https://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-that-are-related-to-the-actin-cytoskeleton/
  4. 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
    https://pharmaceuticalintelligence.com/2013/08/26/role-of-calcium-the-actin-skeleton-and-lipid-structures-in-signaling-and-cell-motility/
  5. 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
    https://pharmaceuticalintelligence.com/2013/09/08/the-centrality-of-ca2-signaling-and-cytoskeleton-involving-calmodulin-kinases-and-ryanodine-receptors-in-cardiac-failure-arterial-smooth-muscle-post-ischemic-arrhythmia-similarities-and-differen/
  6. 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
    https://pharmaceuticalintelligence.com/2013/08/01/calcium-molecule-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/
  7. 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
    https://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/
  8. 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
    https://pharmaceuticalintelligence.com/2013/09/12/disruption-of-calcium-homeostasis-cardiomyocytes-and-vascular-smooth-muscle-cells-the-cardiac-and-cardiovascular-calcium-signaling-mechanism/
  9. 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
    https://pharmaceuticalintelligence.com/2013/09/16/calcium-channel-blocker-calcium-as-neurotransmitter-sensor-and-calcium-release-related-contractile-dysfunction-ryanopathy/
  10. 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
    https://pharmaceuticalintelligence.com/2013/09/10/synaptotagmin-functions-as-a-calcium-sensor-how-calcium-ions-regulate-the-fusion-of-vesicles-with-cell-membranes-during-neurotransmission/
  11. Genetic Analysis of Atrial Fibrillation
    Author and Curator: Larry H Bernstein, MD, FCAP ,  and Curator: Aviva-Lev Ari, PhD, RN
    https://pharmaceuticalintelligence.com/2013/10/27/genetic-analysis-of-atrial-fibrillation/
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 CaMKIId isoenzyme.

Diabetes: Mouse Studies Point to Kinase as Treatment Target

Published: Nov 24, 2013
By Kristina Fiore, Staff Writer, MedPage Today
Targeting a pathway that plays a major role in both hepatic glucose production and insulin sensitivity may eventually help treat type 2 diabetes, researchers reported.
In a series of experiments in mice, researchers found that inhibition of the kinase CaKMII — or even some of its downstream components — lowered blood glucose and insulin levels, Ira Tabas, MD, PhD, of Columbia University Medical Center in New York City, and colleagues reported online in Cell Metabolism.
The pathway is activated by glucagon signaling in the liver, and appears to have roles in both insulin resistance as well as hepatic glucose production in the liver.
In an earlier study, Tabas and colleagues showed that inhibiting the CaKMII pathway lowered hepatic glucose production by suppressing p38-mediated FoxO1 nuclear localization.
In the current study, they found CaKMII inhibition suppresses levels of the pseudo-kinase TRB3 to improve Akt-phosphorylation, thereby improving insulin sensitivity.
Thus this single pathway targets “two cardinal features of type 2 diabetes — hyperglycemia and defective insulin signaling,” the researchers wrote.
“When we realized we had one common pathway that was responsible for these two disparate processes that, in essence, comprises all of type 2 diabetes, we though it would be an ideal target for new drug therapy,” Tabas told MedPage Today.
Tabas and colleagues conducted several experiments to evaluate the CaKMII pathway.
In one experiment in obese mice, they found that no matter how CaKMII was knocked out, it led to lower blood glucose levels and lower fasting plasma insulin levels in response to a glucose challenge.
The improvements also occurred when they
  • knocked out downstream processes, including p38 and MAPK-activating protein kinase 2 (MK2).
“Thus liver p38 and MK2, like CaKMII, play an important role in the development of hyperglycemia and hyperinsulinemia in obese mice,” they wrote.
In further analyses, the researchers discovered deleting or inhibiting any of these three elements ultimately
  • improved insulin-induced Akt-phosphorylation in obese mice —
  • an important part of improving insulin sensitivity.
And unlike the effects on hepatic glucose production,
  • these changes didn’t occur through effects on FoxO1.
Instead, inhibiting the CaKMII pathway suppressed levels of the pseudo-kinase TRB3, which likely occurred because of
  • suppression of ATF4 — all of which led to an
  • increase in Akt-phosphorylation and insulin sensitivity.
Indeed, when mice were made to overexpress TRB3, the improvement in phosphorylation disappeared, “indicating that
  • the suppression of TRB3 by CaKMII deficiency is
  • causally important in the improvement in insulin signaling,
As a result, there “appear to be two separate CaKMII pathways”,
  1. one involved in CaKMII-p38-FoxO1 dependent hepatic glucose production, and
  2. the other involved in defective insulin-induced p-Akt,
The findings suggest the possibility of a drug that can target
  • both hyperglycemia and insulin resistance in type 2 diabetes
The authors have started developing such an agent. Although kinases can act very generally, Tabas said he and colleagues are working on
  • an allosteric version that will more specifically target MK2
  • by binding to a site that is unique to this enzyme.
He said this should help to avoid problems with drugs that targeted p38 but ultimately failed, with little efficacy and too many side effects.
The reason for this is now known at a very detailed level –
  • when you inhibit p38 by that mechanism, mainly by inhibiting MK2,
  • you avoid the adverse effects,
“When we realized all of this and had to make a choice [for further development], the obvious choice was MK2.”
  • CaKMII inhibitors are in development for heart failure and
  • MK2 inhibitors are being looked at as an alternative to p38 inhibitors for inflammatory diseases.
Tabas also said the drug may be valuable in treating prediabetes, since early data have suggested that
  • CaKMII is generally overactive in obese patients
  • who have not yet progressed to full blown diabetes, but is not overactive in lean people.
“One of the areas we’re most excited about in potential clinical use is in obese people before they get diabetic,” Tabas told MedPage Today. “There are hundreds of millions of people who are obese but not yet diabetic even though
  • they have the hallmarks that they’re going to get diabetes.”
This recent publication was reported in MedPage Today. [CaKMII overactivity in obesity]  Tabas noted that his group’s early human data “suggest that our pathway is turned on in prediabetes. If we can block that pathway before people get diabetes, it would even be better.”
The study was supported by the NIH, the American Heart Association, the German Center for Cardiovascular Research, the German Ministry of Education and Research, and the European Union.
Tabas and a co-author are among the founders of  Tabomedex Biosciences, which is developing MK2 inhibitors.
Primary source: Cell Metabolism
Source reference: Ozcan L, et al. “Activation of calcium/calmodulin-dependent protein kinase II in obesity mediates suppression of hepatic insulin signaling” Cell Metab 2013.

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Renal Function Biomarker, β-trace protein (BTP) as a Novel Biomarker for Cardiac Risk Diagnosis in Patients with Atrial Fibrilation

Curator: Aviva Lev-Ari, PhD, RN

Original Research | November 2013

β-Trace Protein and Prognosis in Patients With Atrial Fibrillation Receiving Anticoagulation Treatment

Juan Antonio Vílchez, BSc Pharm, PhD; Vanessa Roldán, MD, PhD; Sergio Manzano-Fernández, MD, PhD; Hermógenes Fernández, MD; Francisco Avilés-Plaza, MD, PhD; Pedro Martínez-Hernández, BSc Pharm, PhD; Vicente Vicente, MD, PhD; Mariano Valdés, MD, PhD; Francisco Marín, MD, PhD; Gregory Y. H. Lip, MD

From the University of Birmingham Centre for Cardiovascular Sciences (Drs Apostolakis and Lip), City Hospital, Birmingham, England; and the Division of Cardiovascular Medicine (Drs Sullivan and Olshansky), University of Iowa Hospitals and Clinics, Iowa City, IA.

Correspondence to: Gregory Y. H. Lip, MD, University of Birmingham Centre for Cardiovascular Sciences, City Hospital, Dudley Rd, Birmingham, B18 7QH, England; e-mail: g.y.h.lip@bham.ac.uk

Abstract

Background:  Atrial fibrillation (AF) is associated with a high risk of mortality and morbidity and it commonly coexists with chronic kidney disease. A biomarker of renal function, β-trace protein (BTP), has been implicated in the progression of cardiovascular disease. The aim of our study was to evaluate the association of BTP with adverse cardiovascular events, bleeding, and mortality in patients with AF.

Methods:  In a consecutive cohort of patients with nonvalvular AF receiving anticoagulation treatment, plasma BTP was determined using an automated nephelometer BN ProSpec System (Siemens) and related to estimated glomerular filtration rate (eGFR). We recorded adverse cardiovascular events (stroke, acute coronary syndrome, and acute pulmonary edema), major bleeding, and mortality.

Results:  We included 1,279 patients (48.6% men), aged 76 years (IQR, 71-81 years), who were followed up for 996 days (IQR, 802-1,254 days). During the follow-up, there were 150 cardiovascular events (annual rate, 3.99%), 57 embolisms (annual rate, 1.54%), and 114 major bleeding events (annual rate, 3.04%), and 161 patients died (annual rate, 4.32%). BTP levels were inversely associated with eGFR (P < .001). High BTP concentrations were significantly associated with embolic events (hazard ratio [HR], 4.64 [1.98-10.86]; P < .001), composite adverse cardiovascular events (HR, 1.93 [1.31-2.85]; P = .001), and mortality (HR, 2.08 [1.49-2.90]; P < .001), even after adjusting for CHAD2DS2-VASc (congestive heart failure, hypertension, age ≥ 75 years [doubled], diabetes mellitus, stroke [doubled], vascular disease, age 65 to 74 years, sex category) score and renal function. High BTP was associated with major bleeding events (HR, 1.88 [1.18-3.00]; P = .008), even after adjusting for the HAS-BLED (hypertension, abnormal renal/liver function, stroke, bleeding history or redisposition, labile international normalized ratio, elderly [> 65 years], drugs/alcohol concomitantly) score.

Conclusions:  We suggest that BTP, a proposed renal damage biomarker, may be a novel predictor of adverse cardiovascular events, major bleeding, and mortality in patients with AF. BTP may help refine clinical risk stratification in these patients.

SOURCE

http://journal.publications.chestnet.org/article.aspx?articleid=1730537

Editorials | November 2013

Predicting the Quality of Anticoagulation During Warfarin Therapy:The Basis for an Individualized Approach

Giuseppe Boriani, MD, PhD

From the Institute of Cardiology, Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna.

Correspondence to: Giuseppe Boriani, MD, PhD, Institute of Cardiology, Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Via Massarenti 9, 40138 Bologna, Italy; e-mail: giuseppe.boriani@unibo.it

Chest. 2013;144(5):1437-1438. doi:10.1378/chest.13-1285

In medicine, there is an emerging tendency toward individualized medicine, that is, an approach to medicine based on available evidence, but enriched by the awareness of the inherent limitations of any “one size fits all” approach. As a matter of fact, diseases show individual differences with regard to onset and course, and individuals show different responses to drugs and interventions, thus suggesting the rationale for an individualized approach to disease treatments, able to predict individual responses. The most sophisticated approach to individualization and tailoring of medicine is personalized medicine, a broad and rapidly advancing field of health care that is informed by each person’s unique clinical, genetic, genomic, and environmental information.1 Treatment with vitamin K antagonists (VKAs) has been one of the traditional settings for individualization of treatment. The concept of personalized medicine specifically applies to warfarin dosing, a setting where knowledge of the complex polymorphic variants in the gene encoding cytochrome 2C9 (CYP2C9) and of the genetic variants in the gene encoding vitamin K epoxide reductase complex 1 (VKORC 1) may help to predict the interindividual variability in warfarin pharmacokinetics and pharmacodynamics, as well as warfarin-associated events and costs.2 However, it is still uncertain and unproven whether management of warfarin dosing guided by pharmacogenetics may improve patient outcomes.3

Biomarker Can Predict Events in Afib Patients

Published: Nov 6, 2013 | Updated: Nov 7, 2013

By Todd Neale, Senior Staff Writer, MedPage Today
Reviewed by Zalman S. Agus, MD; Emeritus Professor, Perelman School of Medicine at the University of Pennsylvania and Dorothy Caputo, MA, BSN, RN, Nurse Planner

Beta-trace protein (BTP), a biomarker that has been associated with both kidney damage and an increased cardiovascular risk, may help identify high-risk atrial fibrillation patients, researchers found.

Among patients with atrial fibrillation who were on stable oral anticoagulant therapy, high plasma levels of the protein were associated with significantly elevated risks of embolic events, adverse cardiovascular events, death, and major bleeding, according to Gregory Lip, MD, of the University of Birmingham in England, and colleagues.

Also, adding information about BTP levels modestly improved the predictive ability of models that included two established risk scores — CHAD2DS2-VASc and HAS-BLED — as indicated by higher C-statistics, they reported in the Nov. 5 issue of CHEST.

“This raises the possibility that BTP may help refine the clinical risk stratification for thrombotic or hemorrhagic events and mortality in these patients,” they wrote.

BTP has been proposed has a marker of renal damage, and it has also been associated with inflammation, atherogenesis, angina, vasomotor reactivity, and hypertension. Previous studies have also identified a relationship between BTP and the progression of cardiovascular disease.

In the current study, Lip and colleagues explored whether BTP levels were related to outcomes in 1,279 patients with nonvalvular atrial fibrillation who were on stable oral anticoagulant therapy with an international normalized ratio (INR) of 2.0 to 3.0. Their average age was 76.

The median estimated glomerular filtration rate at baseline was 71.28 mL/min/1.73 m2; BTP levels and renal function were inversely related (P<0.001).

The BTP cut-offs with the best sensitivity and specificity for predicting each of the endpoints varied — 0.561 mg/L for adverse cardiovascular events, 0.556 mg/L for embolic events, 0.670 mg/L for mortality, and 0.573 mg/L for major bleeds.

During a median follow-up of 2.7 years, cardiovascular events occurred at a rate of 3.99% per year, embolisms at 1.54% per year, deaths at 4.32% per year, and major bleeds at 3.04% per year.

After adjustment for renal function and the CHAD2DS2-VASc risk score — which incorporates congestive heart failure, hypertension, age, diabetes, stroke, vascular disease, and sex — a BTP level above the cutoff was associated with increased risks of cardiovascular events (HR 1.93, 95% CI 1.31-2.85), embolic events (HR 4.64, 95% CI 1.98-10.86), and mortality (HR 2.08, 95% CI 1.49-2.90).

Also, after adjustment for the HAS-BLED risk score — which takes into account hypertension, abnormal renal and liver function, stroke, bleeding history or predisposition, labile INR, age over 65, and concomitant use of drugs and alcohol — a high BTP level was associated with a greater risk of major bleeding (HR 1.88, 95% CI 1.18-3.00).

“We suggest that BTP, a proposed renal damage biomarker, may be a novel predictor of adverse cardiovascular events, major bleeding, and mortality in patients with atrial fibrillation,” the authors wrote.

They acknowledged some limitations of the analysis, however, including possible selection bias because all of the patients were on stable oral anticoagulant therapy, the measurement of renal function and BTP levels at a single time point only, and the exclusion of patients with end-stage renal disease.

SOURCE

http://www.medpagetoday.com/Cardiology/Arrhythmias/42751

These are promising early results, but the data include plenty of limitations. As the article notes, the researchers themselves acknowledge that their work only looked at patients on a regular oral anticlotting drug at a certain point in time. Further research must include a broader class of patients to determine if BTP can be a reliable biomarker to help identify atrial fibrillation patients with an added risk of other health problems.

As hard as it might be to spot atrial fibrillation patients at risk of more problems, doctors struggle to definitively identify the condition in the first place and apply targeted treatments. The med tech industry, meanwhile, is trying to fill the gap. Topera, a 2013 Fierce 15 winner, recently won U.S. and EU approval for a 3-D device and mapping tool designed to better detect cardiac rhythm problems such as atrial fibrillation in order to enable more targeted and accurate treatment. In late August, St. Jude Medical ($STJ) snatched up Endosense, which makes a cutting-edge irrigated ablation catheter designed to treat atrial fibrillation, and rival companies are developing or promoting electrophysiology treatments and other devices for the condition.

 SOURCE

From: FierceBiomarkers <editors@fiercebiomarkers.com>
Reply-To: <editors@fiercebiomarkers.com>
Date: Wednesday, November 13, 2013 10:31 AM
To: AvivaLev-Ari@alum.berkeley.edu
Subject: | 11.13.13 | Investigators flag new biomarkers for atrial fib

Articles related to Diagnosis of Atrial Fibrilation published on this Open Access Online Scientific Journal include the following:

Genetic Analysis of Atrial Fibrillation, Larry H Bernstein, MD, FCAP  and Aviva-Lev Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/10/27/genetic-analysis-of-atrial-fibrillation/

Cardiac Contractility & Myocardium Performance: Ventricular Arrhythmiasand 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
https://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/

Oxidized Calcium Calmodulin Kinase and Atrial Fibrillation, Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/10/26/oxidized-calcium-calmodulin-kinase-and-atrial-fibrillation/

Genetics of Conduction Disease: Atrioventricular (AV) Conduction Disease (block): Gene Mutations – Transcription, Excitability, and Energy Homeostasis, Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/04/28/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, Justin D. Pearlman, MD, PhD, FACC and Aviva Lev-Ari, PhD, RN

Read Full Post »


Genetic Analysis of Atrial Fibrillation

Author and Curator: Larry H Bernstein, MD, FCAP  

and 

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
https://pharmaceuticalintelligence.com/2013/10/26/oxidized-calcium-calmodulin-kinase-and-atrial-fibrillation/

Jmjd3 and Cardiovascular Differentiation of Embryonic Stem Cells

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

https://pharmaceuticalintelligence.com/2013/10/26/jmjd3-and-cardiovascular-differentiation-of-embryonic-stem-cells/

Contributions to cardiomyocyte interactions and signaling
Author and Curator: Larry H Bernstein, MD, FCAP  and Curator: Aviva Lev-Ari, PhD, RN
https://pharmaceuticalintelligence.com/2013/10/21/contributions-to-cardiomyocyte-interactions-and-signaling/

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
https://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/

Part I. Identification of Biomarkers that are Related to the Actin Cytoskeleton
Curator and Writer: Larry H Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-that-are-related-to-the-actin-cytoskeleton/

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
https://pharmaceuticalintelligence.com/2013/08/26/role-of-calcium-the-actin-skeleton-and-lipid-structures-in-signaling-and-cell-motility/

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
https://pharmaceuticalintelligence.com/2013/09/08/the-centrality-of-ca2-signaling-and-cytoskeleton-involving-calmodulin-kinases-and-ryanodine-receptors-in-cardiac-failure-arterial-smooth-muscle-post-ischemic-arrhythmia-similarities-and-differen/

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
https://pharmaceuticalintelligence.com/2013/08/01/calcium-molecule-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/

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
https://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/

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
https://pharmaceuticalintelligence.com/2013/09/12/disruption-of-calcium-homeostasis-cardiomyocytes-and-vascular-smooth-muscle-cells-the-cardiac-and-cardiovascular-calcium-signaling-mechanism/

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
https://pharmaceuticalintelligence.com/2013/09/16/calcium-channel-blocker-calcium-as-neurotransmitter-sensor-and-calcium-release-related-contractile-dysfunction-ryanopathy/

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
https://pharmaceuticalintelligence.com/2013/09/10/synaptotagmin-functions-as-a-calcium-sensor-how-calcium-ions-regulate-the-fusion-of-vesicles-with-cell-membranes-during-neurotransmission/

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
https://pharmaceuticalintelligence.com/biomed-e-books/series-a-e-books-on-cardiovascular-diseases/volume-three-etiologies-of-cardiovascular-diseases-epigenetics-genetics-genomics/

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

3.2.2.1 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

3.2.2.2 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

3.2.4.2 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

Introduction

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.

Methods

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

ZSummary

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.

Results

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

Characteristics

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)

2.0×10−4

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

7 (20.0)

0.033

BMI, median (1st–3rd quartiles)

25.97 (24.27–28.66)

29.01 (27.06–32.11)

29.71 (26.72–35.10)

2.7×10−6

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

12 (21.1)

0.032

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

16 (45.7)

4.4×10−4

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

0 (0.0)

0.033

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)

0.065

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.

Dataset
Group

Principal
Component

Black

Blue

Brown

Cyan

Green

Green-
Yellow

Magenta

Mitral

1

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

2

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

3

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

CAD

1

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

2

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

3

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

LAF

1

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

2

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

3

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

Dataset
Group

Principal
Component

Midnight- Blue

Pink

Purple

Red

Salmon

Tan

Turquoise

Mitral

1

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

2

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

3

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

CAD

1

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

2

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

3

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

LAF

1

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

2

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

3

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

Gene

IMC

Gene

MM

Gene

IMC

Gene

MM

RCAN1 8.2

RCAN1

0.81

CPEB3

43.3

CPEB3

0.85
DNAJA4 7.7

DNAJA4

0.81

CPLX3

42.4

CPLX3

0.84
PDE8B 7.7

PDE8B

0.80

NEDD4L

40.8

NEDD4L

0.83
PRKAR1A 6.9

PRKAR1A

0.77

SGSM1

40.7

SGSM1

0.82
PTPN4 6.7

PTPN4

0.75

UCKL1

39.0

UCKL1

0.81
SORBS2 6.0

FHL2

0.69

SOSTDC1

37.2

SOSTDC1

0.79
ADCY6 5.7

ADCY6

0.69

PRDX1

35.5

RCOR2

0.78
FHL2 5.7

SORBS2

0.68

RCOR2

35.4

EEF2K

0.77
BVES 5.4

DHRS9

0.67

NPPB

35.3

PRDX1

0.76
TMEM173 5.3

LAPTM4B

0.65

LRRN3

34.6

MMP11

0.76

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.

Discussion

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.

Conclusions

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.

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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

http://circgenetics.ahajournals.org/content/6/4/362

CLINICAL PERSPECTIVE

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

and

Curator: Aviva Lev-Ari, PhD, RN

 

Introduction

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
https://pharmaceuticalintelligence.com/2013/10/21/contributions-to-cardiomyocyte-interactions-and-signaling/

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
https://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/

Part I. Identification of Biomarkers that are Related to the Actin Cytoskeleton
Curator and Writer: Larry H Bernstein, MD, FCAP
https://pharmaceuticalintelligence.com/2012/12/10/identification-of-biomarkers-that-are-related-to-the-actin-cytoskeleton/

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
https://pharmaceuticalintelligence.com/2013/08/26/role-of-calcium-the-actin-skeleton-and-lipid-structures-in-signaling-and-cell-motility/

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
https://pharmaceuticalintelligence.com/2013/09/08/the-centrality-of-ca2-signaling-and-cytoskeleton-involving-calmodulin-kinases-and-ryanodine-receptors-in-cardiac-failure-arterial-smooth-muscle-post-ischemic-arrhythmia-similarities-and-differen/

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
https://pharmaceuticalintelligence.com/2013/08/01/calcium-molecule-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/

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
https://pharmaceuticalintelligence.com/2013/08/28/cardiac-contractility-myocardium-performance-ventricular-arrhythmias-and-non-ischemic-heart-failure-therapeutic-implications-for-cardiomyocyte-ryanopathy-calcium-release-related-contractile/

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
https://pharmaceuticalintelligence.com/2013/09/12/disruption-of-calcium-homeostasis-cardiomyocytes-and-vascular-smooth-muscle-cells-the-cardiac-and-cardiovascular-calcium-signaling-mechanism/

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
https://pharmaceuticalintelligence.com/2013/09/16/calcium-channel-blocker-calcium-as-neurotransmitter-sensor-and-calcium-release-related-contractile-dysfunction-ryanopathy/

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
https://pharmaceuticalintelligence.com/2013/09/10/synaptotagmin-functions-as-a-calcium-sensor-how-calcium-ions-regulate-the-fusion-of-vesicles-with-cell-membranes-during-neurotransmission/

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;

http://circ.ahajournals.org/content/early/2013/09/12/CIRCULATIONAHA.113.003313
http://circ.ahajournals.org/content/suppl/2013/09/12/CIRCULATIONAHA.113.003313.DC1.html

http://dx.doi.org/10.1161/CIRCULATIONAHA.113.003313

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

 Abstract

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)

Introduction

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.

Methods

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.

Statistics

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.

Results

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.

 Discussion

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.
2007;53:1652-1657.
 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.
http://dx.doi.org/10.1161/CIRCULATIONAHA.113.003313

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.

http://circ.ahajournals.org/content/early/2013/09/12/CIRCULATIONAHA.113.003313
http://circ.ahajournals.org/content/suppl/2013/09/12/CIRCULATIONAHA.113.003313.DC1.html

http://dx.doi.org/10.1161/CIRCULATIONAHA.113.003313

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
https://pharmaceuticalintelligence.com/2013/10/21/contributions-to-cardiomyocyte-interactions-and-signaling/
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
http://circres.ahajournals.org/content/97/12/1314

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
http://circres.ahajournals.org/content/97/12/1213

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_520 CIRCULATIONAHA_oxCaMKII_AF_AR_Image_523
CIRCULATIONAHA_oxCaMKII_AF_AR_Image_539    CIRCULATIONAHA_oxCaMKII_AF_AR_Image_538
CIRCULATIONAHA_oxCaMKII_AF_AR_Image_535                  CIRCULATIONAHA_oxCaMKII_AF_AR_Image_534
CIRCULATIONAHA_oxCaMKII_AF_AR_Image_522       CIRCULATIONAHA_oxCaMKII_AF_AR_Image_496

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