Archive for the ‘Cardiomyopathy’ Category

North Americans With Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy: Genomics of Ventricular arrhythmias, A-Fib, Right Ventricular Dysplasia, Cardiomyopathy – Comprehensive Desmosome Mutation Analysis

Posted in Cardiomyopathy, Cardiovascular Research, Computational Biology/Systems and Bioinformatics, Congenital Heart Disease, Electrophysiology, Frontiers in Cardiology and Cardiovascular Disorders, Genome Biology, HTN, Origins of Cardiovascular Disease, Pharmacogenomics, Population Health Management, Genetics & Pharmaceutical, Statistical Methods for Research Evaluation, tagged Arrhythmogenic right ventricular dysplasia, Cardiac dysrhythmia, Johns Hopkins School of Medicine, Johns Hopkins University, National Institutes of Health, University Medical Center Utrecht on December 18, 2013| Leave a Comment »

North Americans With Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy: Genomics of Ventricular arrhythmias, A-Fib, Right Ventricular Dysplasia, Cardiomyopathy – Comprehensive Desmosome Mutation Analysis

Reporter: Aviva Lev-Ari, PhD, RN

Genomics of Ventricular arrhythmias, A-Fib, Right Ventricular Dysplasia, Cardiomyopathy – Comprehensive Desmosome Mutation Analysis in North Americans With Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy

A. Dénise den Haan, MD, Boon Yew Tan, MBChB, Michelle N. Zikusoka, MD, Laura Ibañez Lladó, MS, Rahul Jain, MD, Amy Daly, MS, Crystal Tichnell, MGC, Cynthia James, PhD, Nuria Amat-Alarcon, MS, Theodore Abraham, MD, Stuart D. Russell, MD,David A. Bluemke, MD, PhD, Hugh Calkins, MD, Darshan Dalal, MD, PhD and Daniel P. Judge, MD

Author Affiliations

From the Department of Medicine/Cardiology (A.D.d.H., B.Y.T., M.N.Z., L.I.L., R.J., A.D., C.T., C.J., N.A.-A., T.A., S.D.R., H.C., D.D., D.P.J.), Johns Hopkins University School of Medicine, Baltimore, Md; Department of Cardiology, Division of Heart and Lungs (A.D.d.H.), University Medical Center Utrecht, Utrecht, The Netherlands; and National Institutes of Health, Radiology and Imaging Sciences (D.A.B.), Bethesda, Md.

Correspondence to Daniel P. Judge, MD, Johns Hopkins University, Division of Cardiology, Ross 1049; 720 Rutland Avenue, Baltimore, MD 21205. E-mail djudge@jhmi.edu

Abstract

Background— Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is an inherited disorder typically caused by mutations in components of the cardiac desmosome. The prevalence and significance of desmosome mutations among patients with ARVD/C in North America have not been described previously. We report comprehensive desmosome genetic analysis for 100 North Americans with clinically confirmed or suspected ARVD/C.

Methods and Results— In 82 individuals with ARVD/C and 18 people with suspected ARVD/C, DNA sequence analysis was performed on PKP2, DSG2, DSP, DSC2, and JUP. In those with ARVD/C, 52% harbored a desmosome mutation. A majority of these mutations occurred in PKP2. Notably, 3 of the individuals studied have a mutation in more than 1 gene. Patients with a desmosome mutation were more likely to have experienced ventricular tachycardia (73% versus 44%), and they presented at a younger age (33 versus 41 years) compared with those without a desmosome mutation. Men with ARVD/C were more likely than women to carry a desmosome mutation (63% versus 38%). A mutation was identified in 5 of 18 patients (28%) with suspected ARVD. In this smaller subgroup, there were no significant phenotypic differences identified between individuals with a desmosome mutation compared with those without a mutation.

Conclusions— Our study shows that in 52% of North Americans with ARVD/C a mutation in one of the cardiac desmosome genes can be identified. Compared with those without a desmosome gene mutation, individuals with a desmosome gene mutation had earlier-onset ARVD/C and were more likely to have ventricular tachycardia.

SOURCE:

Circulation: Cardiovascular Genetics.2009; 2: 428-435

Published online before print June 3, 2009,

doi: 10.1161/ CIRCGENETICS.109.858217

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Myocardial Damage in Cardiovascular Disease: Circulating MicroRNA-208b and MicroRNA-499

Posted in Biological Networks, Gene Regulation and Evolution, Cardiomyopathy, Cardiovascular Research, Cell Biology, Signaling & Cell Circuits, Chemical Genetics, Frontiers in Cardiology and Cardiovascular Disorders, Genome Biology, Genomic Testing: Methodology for Diagnosis, Myocardial metabolism, Myocardial ischemia, myocardial perfusion, Myocardial adenine nucleotide metabolism, Origins of Cardiovascular Disease, Personalized and Precision Medicine & Genomic Research, Population Health Management, Genetics & Pharmaceutical, tagged Cardiovascular disease, Conditions and Diseases, Heart Failure, Messenger RNA, MicroRNA on December 12, 2013| Leave a Comment »

Myocardial Damage in Cardiovascular Disease: Circulating MicroRNA-208b and MicroRNA-499

Reporter: Aviva Lev-Ari, PhD, RN

Circulating MicroRNA-208b and MicroRNA-499 Reflect Myocardial Damage in Cardiovascular Disease

Maarten F. Corsten, MD, Robert Dennert, MD, Sylvia Jochems, BSc, Tatiana Kuznetsova, MD, PhD, Yvan Devaux, PhD, Leon Hofstra, MD, PhD, Daniel R. Wagner, MD, PhD, Jan A. Staessen, MD, PhD, Stephane Heymans, MD, PhD and Blanche Schroen, PhD

Author Affiliations

From the Center for Heart Failure Research (M.F.C., R.D., S.J., S.H., B.S.), Cardiovascular Research Institute, Maastricht, The Netherlands; the Division of Hypertension and Cardiovascular Rehabilitation (T.K., J.A.S.), Department of Cardiovascular Diseases, University of Leuven, Leuven, Belgium and Department of Epidemiology, Maastricht University Medical Center, Maastricht, The Netherlands; Centre de Recherche Public–Santé, Luxembourg (Y.D., D.R.W.), Luxembourg; Maastricht University Medical Center (L.H.), Maastricht, The Netherlands; and Centre Hospitalier Luxembourg (D.R.W.), Luxembourg.

Correspondence to Blanche Schroen, PhD, Center for Heart Failure Research, Cardiovascular Research Institute Maastricht, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands. E-mail b.schroen@cardio.unimaas.nl

Drs Heymans and Schroen contributed equally to this work.

Abstract

Background— Small RNA molecules, called microRNAs, freely circulate in human plasma and correlate with varying pathologies. In this study, we explored their diagnostic potential in a selection of prevalent cardiovascular disorders.

Methods and Results— MicroRNAs were isolated from plasmas from well-characterized patients with varying degrees of cardiac damage:

(1) acute myocardial infarction,

(2) viral myocarditis,

(3) diastolic dysfunction, and

(4) acute heart failure.

Plasma levels of selected microRNAs, including heart-associated (miR-1, -133a, -208b, and -499), fibrosis-associated (miR-21 and miR-29b), and leukocyte-associated (miR-146, -155, and -223) candidates, were subsequently assessed using real-time polymerase chain reaction. Strikingly, in plasma from acute myocardial infarction patients, cardiac myocyte–associated miR-208b and -499 were highly elevated, 1600-fold (P<0.005) and 100-fold (P<0.0005), respectively, as compared with control subjects. Receiver operating characteristic curve analysis revealed an area under the curve of 0.94 (P<10⁻¹⁰) for miR-208b and 0.92 (P<10⁻⁹) for miR-499. Both microRNAs correlated with plasma troponin T, indicating release of microRNAs from injured cardiomyocytes. In viral myocarditis, we observed a milder but significant elevation of these microRNAs, 30-fold and 6-fold, respectively. Plasma levels of leukocyte-expressed microRNAs were not significantly increased in acute myocardial infarction or viral myocarditis patients, despite elevated white blood cell counts. In patients with acute heart failure, only miR-499 was significantly elevated (2-fold), whereas no significant changes in microRNAs studied could be observed in diastolic dysfunction. Remarkably, plasma microRNA levels were not affected by a wide range of clinical confounders, including age, sex, body mass index, kidney function, systolic blood pressure, and white blood cell count.

Conclusions— Cardiac damage initiates the detectable release of cardiomyocyte-specific microRNAs-208b and -499 into the circulation.

SOURCE:

Circulation: Cardiovascular Genetics. 2010; 3: 499-506

Published online before print October 4, 2010,

doi: 10.1161/ CIRCGENETICS.110.957415

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

Posted in Biological Networks, Gene Regulation and Evolution, Cardiomyopathy, Cardiovascular Research, Cell Biology, Signaling & Cell Circuits, Chemical Genetics, Computational Biology/Systems and Bioinformatics, Frontiers in Cardiology and Cardiovascular Disorders, Genome Biology, Genomic Testing: Methodology for Diagnosis, Medical and Population Genetics, Molecular Genetics & Pharmaceutical, Myocardial metabolism, Myocardial ischemia, myocardial perfusion, Myocardial adenine nucleotide metabolism, Origins of Cardiovascular Disease, Personalized and Precision Medicine & Genomic Research, Population Health Management, Genetics & Pharmaceutical, Translational Science, tagged Cardiology, Cardiovascular disease, MicroRNA, Takeshi Kimura on December 12, 2013| Leave a Comment »

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

Reporter: Aviva Lev-Ari, PhD, RN

Increased MicroRNA-1 and MicroRNA-133a Levels in Serum of Patients With Cardiovascular Disease Indicate Myocardial Damage

Yasuhide Kuwabara, MD, Koh Ono, MD, PhD, Takahiro Horie, MD, PhD, Hitoo Nishi, MD, PhD, Kazuya Nagao, MD, PhD, Minako Kinoshita, MD, PhD, Shin Watanabe, MD, PhD, Osamu Baba, MD, Yoji Kojima, MD, PhD, Satoshi Shizuta, MD, Masao Imai, MD,Toshihiro Tamura, MD, Toru Kita, MD, PhD and Takeshi Kimura, MD, PhD

Author Affiliations

From the Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan (Y. Kuwabara, K.O., T.H., H.N., K.N., M.K., S.W., O.B., Y. Kojima, S.S., M.I., T.T., T. Kimura); and Kobe City Medical Center General Hospital, Kobe, Japan (T. Kita).

Correspondence to Koh Ono, MD, PhD, Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, 54 Shogoin-kawahara-cho, Sakyo-ku, Kyoto, Japan 606-8507. E-mail kohono@kuhp.kyoto-u.ac.jp

Abstract

Background—Recently, elevation of circulating muscle-specific microRNA (miRNA) levels has been reported in patients with acute myocardial infarction. However, it is still unclear from which part of the myocardium or under what conditions miRNAs are released into circulating blood. The purpose of this study was to identify the source of elevated levels of circulating miRNAs and their function in cardiovascular diseases.

Conclusions—These results suggest that elevated levels of circulating miRNA-133a in patients with cardiovascular diseases originate mainly from the injured myocardium. Circulating miR-133a can be used as a marker for cardiomyocyte death, and it may have functions in cardiovascular diseases.

SOURCE:

Circulation: Cardiovascular Genetics. 2011; 4: 446-454

Published online before print June 2, 2011,

doi: 10.1161/ CIRCGENETICS.110.958975

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Stabilizers that prevent Transthyretin-mediated Cardiomyocyte Amyloidotic Toxicity

Posted in Cardiomyopathy, Cerebrovascular and Neurodegenerative Diseases, Chemical Biology and its relations to Metabolic Disease, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Frontiers in Cardiology and Cardiovascular Disorders, Human Immune System in Health and in Disease, Liver & Digestive Diseases Research, Metabolomics, Personalized and Precision Medicine & Genomic Research, Population Health Management, Genetics & Pharmaceutical, Proteomics, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, Technology Transfer: Biotech and Pharmaceutical, Translational Effectiveness, Translational Science, tagged cardiomyocyte toxicity, Cardiomyopathy, COX 1 & 2 inhibitors, elimination of adverse effects, ligand binding reactant, NSAIDS, population variation, stabilizers, Transthyretin (TTR), Transthyretin-related hereditary amyloidosis on December 2, 2013| Leave a Comment »

Stabilizers that prevent Transthyretin-mediated Cardiomyocyte Amyloidotic Toxicity

Reporter and curator: Larry H. Bernstein, MD, FCAP

http://pharmaceuticalintelligence.com/12-2-2013/larryhbern/Stabilizers that prevent transthyretin-mediated cardiomyocyte amyloidotic toxicity

Transthyretin is a small protein with a half-life of < 48 hours, synthesized by the liver, and a major transport protein for thyroxin. There are 80 variants known, and some variants that occur in the Portuguese, a small section of Japan, Sweden, and Brazil, are associated will primary amyloidosis, the only cure for which is liver transplantation. It causes fibrillary inclusions in the heart, but also affects the autonomic nervous system. Some of the major work on this has been done for many years in the laboratory of Jeffery W. Kelly, at the Skaggs Institute for Chemical Biology, the Scripps Research Institute. A recent publication is of considerable interest.

Potent Kinetic Stabilizers that Prevent Transthyretin-mediated Cardiomyocyte Proteotoxicity

Mamoun M. Alhamadsheh1,6,7, Stephen Connelly2,7, Ahryon Cho1, Natàlia Reixach3, Evan T. Powers3,4,5, Dorothy W. Pan1, Ian A. Wilson2,5, Jeffery W. Kelly3,4,5, and Isabella A. Graef1,*
Sci Transl Med. Author manuscript; available in PMC 2012 August 24.

1Department of Pathology, Stanford University Medical School, Stanford, California, USA

2Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, USA

3Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA

4Department of Chemistry, The Scripps Research Institute, La Jolla, California, USA

5The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA

6Department of Pharmaceutics & Medicinal Chemistry, University of the Pacific, Stockton, California, USA

Abstract

The V122I mutation that alters the stability of transthyretin (TTR) affects 3–4% of African Americans and leads to amyloidogenesis and development of cardiomyopathy. In addition, 10–15% of individuals over the age of 65 develop senile systemic amyloidosis (SSA) and cardiac

TTR deposits due to wild-type TTR amyloidogenesis. As no approved therapies for TTR amyloid cardiomyopathy are available, the development of drugs that prevent amyloid-mediated cardiotoxicity is desired. To this aim, we developed a fluorescence polarization-based HTS screen,

which identified several new chemical scaffolds targeting TTR. These novel compounds were potent kinetic stabilizers of TTR and

prevented tetramer dissociation,
unfolding and aggregation of both wild type and the most common cardiomyopathy-associated TTR mutant, V122I-TTR.

High-resolution co-crystal structures and characterization of the binding energetics revealed how these diverse structures bound to tetrameric TTR. Our study also showed that these compounds effectively inhibited the proteotoxicity of V122I-TTR towards human cardiomyocytes.
Several of these ligands stabilized TTR in human serum more effectively than diflunisal, which is one of the best known inhibitors of TTR aggregation, and may be promising leads for the treatment and/or prevention of TTR-mediated cardiomyopathy.

Author Contributions:

M.M.A. designed and performed most experiments, S.C. performed crystallographic structure determination, A.C peformed the serum TTR stabilization. N.R. performed the cell-based assays. E.T.P. analyzed the ITC data. D.W.P. helped with probe synthesis. I.A.W. supervised the crystallographic work. J.W.K. supervised the work, S.C., N.R., I.A.W. and J.W.K. edited the paper. I.A.G supervised the work, M.M.A. and I.A.G prepared the manuscript.

INTRODUCTION

The misassembly of soluble proteins into toxic amyloid aggregates underlies a large number of human degenerative diseases (1–3). TTR is one of more than 30 human amyloidogenic proteins whose misassembly can cause

a variety of degenerative gain-of-toxic-function diseases.

TTR is a tetrameric protein (54 kDa), secreted from the liver into the blood where, using orthogonal sites,

it transports thyroxine (T4) and
holo-retinol binding protein (4).

However, 99% of the TTR T4 binding sites remain unoccupied in humans

owing to the presence of two other T4 transport proteins in blood (3).

Familial TTR amyloid diseases, which are associated with one of more than 80 mutations in the TTR gene, include

the systemic neuropathies (familial amyloid polyneuropathy [FAP]),
cardiomyopathies (familial amyloid cardiomyopathy [FAC]), and
central nervous system amyloidoses (CNSA) (5–8).

Cardiac amyloidosis is most commonly caused by

deposition of immunoglobulin light chains or
TTR in the cardiac interstitium and conducting system.

It is a chronic and progressive condition, which can lead to arrhythmias, biventricular heart failure, and death (8–10). Two types of TTR-associated amyloid cardiomyopathies are clinically important.

Wild-type (WT) TTR aggregation underlies the development of senile systemic amyloidosis (SSA). Cardiac TTR deposits can be found in 10 to 15% of the population over the age of 65 at autopsy (10,11). Many of these patients are asymptomatic, but there is little doubt that SSA is an underdiagnosed disease.
In addition, a number of TTR mutations, including V122I, lead to amyloidogenesis and familial amyloid cardiomyopathy (FAC) (12–15). Population studies show that the V122I mutation is found in 3–4% of African Americans (~1.3 million people) and contributes to the increased prevalence of heart failure among this population segment (14,15).

The mutant TTR allele behaves as an autosomal dominant allele with age-dependent penetrance and

the frequency of cardiac amyloidosis from TTR in African-American individuals above age 60 is four times that seen in Caucasian-Americans of comparable age.

All of the TTR mutations associated with familial amyloidosis decrease tetramer stability, and

some decrease the kinetic barrier for tetramer dissociation (3, 16).
The latter is important because tetramer dissociation is the rate-limiting step in the TTR amyloidogenesis cascade (3).

Kinetic stabilization of the native, tetrameric structure of TTR by

interallelic trans suppression (incorporation of mutant subunits that raise the dissociative transition state energy) prevents

1. post-secretory dissociation and aggregation, as well as the related disease
2. familial amyloid polyneuropathy (FAP), by slowing TTR tetramer dissociation (17).

Occupancy of the TTR T4 binding sites with rationally designed small molecules is known to stabilize the native tetrameric state of TTR over the dissociative transition state,

raising the kinetic barrier,
imposing kinetic stabilization on the tetramer and
preventing amyloidogenesis (3, 16, 18).

Previous studies have focused on rational ligand design and as a result

most of the TTR stabilizers reported to date are halogenated biaryl analogues of T4,
many resembling non-steroidal anti-inflammatory drugs (NSAIDs).

Some of these compounds, such as the NSAID diflunisal, which is currently tested in clinical trials in FAP patients for its efficacy to ameliorate

peripheral neuropathy resulting from TTR deposition, (19) have anti-inflammatory activity (20, 21).

The pharmacological effects of NSAIDs are due to inhibition of cyclo-oxygenase (COX) enzymes (22). Inhibition of COX-1 can produce side effects such as

gastrointestinal irritation, leading to ulcers and bleeding (23).

Inhibition of COX-2 has been associated with an

increased risk of severe cardiovascular events, including heart failure,
particularly in patients with preexisting cardiorenal dysfunction (20, 21, 24, 25).

Therefore, heart and kidney impairment are exclusion criteria for participation of patients in the diflunisal clinical trials to treat TTR-mediated FAP (19). Genomic variations can

increase the sensitivity of individuals to adverse side effects of NSAIDs.

Serum concentrations of NSAIDs depend on CYP2C9 and/or CYP2C8 activity. CYP2C9 polymorphism might play a significant role in the profile of adverse side effects of NSAID and alleles that affect the activity of CYP2C9 are found at different frequency in subjects of Caucasian, African or Asian descent (26, 27). Hence, the long-term therapy with drugs that have inhibitory effect on COX activity to prevent TTR aggregation is especially problematic in patients who suffer from TTR-mediated cardiomyopathy. The design and development of drugs to treat/prevent FAC or SSA thus presents the challenge

not only to find compounds with a greater variety of chemical scaffolds that accomplish stabilization, but
do so without the adverse side effects due to inhibition of COX activity.

For these reasons, the development of a rapid and robust screen for compounds that bind to and stabilize TTR could be useful. To date, no high-throughput screening (HTS) methodology is available for the discovery of TTR ligands (28,29). Therefore, we developed a versatile

fluorescence polarization (FP) based HTS assay that can detect
binding of small molecules to the T4 binding pocket of TTR under physiological conditions.

RESULTS

Design and synthesis of the TTR FP probe

FP is used to study molecular interactions by monitoring changes in the apparent size of a fluorescently labeled molecule. Binding is measured by an increase in the FP signal, which is proportional to the decrease in the rate of tumbling of a fluorescent ligand upon association with macromolecules such as proteins (Fig. 1A). To synthesize a fluorescent TTR ligand 1, we initially started with the NSAID diflunisal analogue 2 (Fig. 1B) (30). The product of attaching a linker to 2, compound 3, had very low binding affinity to TTR (Kd1 >3290 nM, fig. S1A and fig. S1B).

The crystal structure of the diclofenac analog 4 showed that

the phenolic hydroxyl flanked by the two chlorine atoms is oriented out of the binding pocket into the solvent (31).

We reasoned that attaching a PEG amine linker to the phenol group of 4 would generate compound 5 which would bind to TTR (Fig. 1B and fig. S1C)

5 was coupled to fluorescein isothiocyanate (FITC) to produce the FITC-coupled TTR FP probe (1, Fig. 1B). The binding characteristics of the probe (Kd1 = 13 nM and Kd2 = 100 nM) were assessed with ITC (Fig. 2A).

Evaluation of the FP assay

The binding of 1 to TTR was evaluated to test its suitability for the FP assay with a standard saturation binding experiment. A fixed concentration of probe 1 (0.1 μM) was incubated with increasing concentrations of TTR (0.005 μM to 10 μM) and the formation of 1•TTR complex was quantified by the increase in FP signal (excitation λ 485 nm, emission λ 525 nm) relative to the concentration of TTR (Fig. 2B). The fluorescence polarization increased with the concentration of TTR until saturation was reached. A large dynamic range (70 – 330 mP) was measured for the assay. To validate the FP assay, we tested known TTR binders in a displacement assay (for detailed information see Supplemental Material). Compound 2 (Kapp = 231 nM, R2 = 0.997), Thyroxine (T4) (Kapp = 186 nM, R2 = 0.998) and diclofenac (Kapp = 4660 nM, R2 = 0.999) decreased the FP signal in a dose- dependent manner (Fig. 2C,  fig. S2B and S2C). The FP assay is a competitive displacement assay and therefore it provides apparent binding constants (Kapp). However, these apparent binding constants correlate well with the data obtained by ITC which measures direct interactions in solution and gives an actual (Kd) value.

Adaptation of the FP assay for HTS

Next, we optimized the FP assay for HTS and screened a ~130,000 small molecule library for compounds that displaced probe 1 from the T4 binding sites of TTR. The FP assay was performed in 384-well plates with low concentrations of probe 1 (1.5 nM) and TTR (50 nM) in a 10  μL assay volume. Detergent (0.01% Triton X-100) was added to the assay buffer to avoid false positive hits from aggregation of the small molecules. The assay demonstrated robust performance, with a, large dynamic range (~70–230 mP) and a Z′ factor (32, 33) in the range of 0.57–0.78 (fig. S3A and S3B).

Hits were defined as compounds, which resulted in at least 50% decrease in FP and demonstrated relative fluorescence between 70 and 130%. Many fluorescence quenchers and enhancers, which have less than 70% and greater than 130% total fluorescence relative to a control (compound without TTR), were excluded from the hit list. The excluded compounds have native fluorescence that is similar to fluorescein, which would interfere with the FP measurements and result in false positive hits. Two hundred compounds were designated as positive hits (0.167% hit rate). The top 33 compounds (compounds with lowest FP IC50) were assayed in a 10-point duplicate dose-response FP assay and displayed an IC50 (concentration that resulted in 50% decrease in the FP signal) between 0.277 and 10.957 μM (table S2).

Validation of the HTS hits

The top 33 compounds were retested with the FP assay (table S2) and with surface plasmon resonance (SPR) as another independent biophysical method. Solutions of the 33 hits were passed over immobilized, biotinylated TTR on a streptavidin coated chip. The binding of a small molecule to TTR on the sensor chip produces a SPR response signal (RU). The RU signal after addition of the top 33 compounds was measured and compared to a negative, solvent only, control. All compounds identified by the screen as hits were confirmed as TTR binders using SPR (fig. S4). We also found known TTR binders, such as NSAIDs (diclofenac, meclofenamic acid, and niflumic acid) and isoflavones (apigenin) in our screen (3, 34) (table S2). Among the best ligands (Fig. 2D) were the NSAID, niflumic acid, two catechol-O-methyl-tranferase (COMT) inhibitors, 3,5-dintrocatechol and Ro 41-0960 (35) and a number of compounds   not previously known to bind to TTR. The chemical structures of these ligands were confirmed by 1H NMR and high-resolution mass spectrometry(HRMS) and the chemical purity was determined to be >95% (fig. S5).

Inhibition of TTR amyloidogenesis by the HTS hits

To test whether the new TTR ligands (7.2 μM) could function as kinetic stabilizers, we measured their ability to inhibit TTR (3.6 μM) amyloidogenesis at 72 hrs at pH 4.4 (fig. S6) (29). All 33 compounds inhibited TTR aggregation (<50% fibril formation, table S2). Of these, 23 were very good (<20% fibril formation) and 11 were excellent (<2% fibril formation) TTR kinetic stabilizers (Fig. 3A). All of the potent TTR stabilizers, except niflumic acid, and the two COMT inhibitors 3,5-dintrocatechol and Ro 41-0960, were chemical entities with no previously reported biological activity. Since occupancy of only
one T4 binding site within TTR is sufficient for kinetic stabilization of the tetramer (3), we tested the most potent ligands at substoichiometric concentrations (2.4 fold molar excess of TTR relative to ligand) in a kinetic aggregation assay monitored over 5 days (Fig. 3B). Under these conditions ligands 7, 14, 15 and Ro 41-0960 dramatically slowed fibril formation and outperformed the known TTR stabilizer, diclofenac, which blocked only ~55% of TTR aggregation.

Evaluating the TTR ligands for COX-1 enzymatic inhibition and binding to thyroid hormone receptor

A successful clinical candidate against TTR amyloid cardiomyopathy should have minimal off-target toxicity due to the potential need for life-long use of these drugs. Specifically, the TTR ligands should exhibit minimal binding to COX and the nuclear thyroid hormone receptor (THR). Inhibition of COX is contraindicated for treating FAC patients, since COX inhibition can not only lead to renal dysfunction and blood pressure elevation, but may precipitate heart failure in vulnerable individuals (20, 21, 24, 25). Therefore, the most potent TTR ligands were evaluated for their ability to inhibit COX-1 activity, as well as, for binding to THR, in comparison with the NSAID niflumic acid. Although niflumic acid exhibited substantial (94%) COX-1 inhibition, three of the 12 new compounds evaluated (7, 6 and 10) displayed less than 1% inhibition of COX-1. Only one ligand (compound 8) showed significant (58%) and two compounds (6 and 10) minor (5%) binding to THR (Fig. 3C).

Characterization of the binding energetics to TTR

Many reported TTR ligands, including T4, bind TTR with negative cooperativity, which appears to arise from subtle conformational changes in TTR upon ligand binding to the first T4 site (3, 16, 36). We used ITC to determine the binding constants and to evaluate cooperativity between the two TTR T4 sites (Fig. 2A, Fig. 4A, Fig. 4B and fig. S1 and fig. S7). The ITC data for compounds 1, 7, 14, and Ro 41-0906 binding to TTR were fit to a two-site binding model and show that these potent ligands bind TTR with low nanomolar affinity. The dissociation constants for these ligands indicated that they bound TTR with negative cooperativity (table S3). Analysis of the free energies associated with ligand binding to TTR indicates that binding was driven both by burial of the hydrophobic ligand in the TTR binding site (which leads to the favorable binding entropies) and specific ligand-TTR interactions (which leads to the favorable binding enthalpies) (Fig. 2A, Fig. 4A, Fig.4B, and fig. S7B) (37). The binding of compounds 7 (Kd1 = 58 nM and Kd2 = 500 nM) and 14 (Kd1 = 26 nM and Kd2 = 1800 nM) to TTR did not cause major conformational changes to the TTR tetramer structure (Fig. 5).

Remainder of document is found at publication site, including Figures.

SOURCE

https://pubmed.ncbi.nlm.nih.gov/21865539/

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Calcium Signaling, Cardiac Mitochondria and Metabolic Syndrome

Posted in Calcium Signaling, Calmodulin Kinase and Contraction, Cardiomyopathy, Cardiovascular Research, Cell Biology, Signaling & Cell Circuits, Electrophysiology, Frontiers in Cardiology and Cardiovascular Disorders, Myocardial metabolism, Myocardial ischemia, myocardial perfusion, Myocardial adenine nucleotide metabolism, Pre-Clinical Animal Model Development, tagged Aviva Lev-Ari, Calcium, calcium calmodulin kinase, calcium efflux, calcium signaling, Calcium-Channel Blocker, cytosolic calcium, metabolic syndrome, mitochondria, Mitochondrial permeability transition pore, regulation of calcium homeostasis on November 9, 2013| Leave a Comment »

Calcium Signaling, Cardiac Mitochondria and Metabolic Syndrome

Larry H Bernstein: Author

and

Reporter: Aviva Lev-Ari, PhD, RN

Mitochondria, the cardiovascular system and metabolic syndrome

Start date: April 24, 2013
End date: April 24, 2013
Venue: London, UK / Kennedy Lecture Theatre, Institute of Child Health
Location: London, UK
Topics: – Mitochondrial ROS metabolism in the heart
– Mitochondrial permeability transition pore
– Mitochondria in vascular smooth muscle
– Therapeutic targets for cardiac disease

Invited speakers

This event has now passed – please visit our Conference calendar for future Abcam events

Confirmed speakers:

Paolo Bernardi, University of Padova, Italy
‘The mitochondrial permeability transition pore: A mystery solved?’

Susan Chalmers, University of Strathclyde Glasgow
‘Mitochondria in vascular smooth muscle: from regulation of calcium signals to control of proliferation’

Andrew Hall, UCL
‘The role of sirtuin 3 in cardiac dysfunction’

Derek Hausenloy, UCL
‘Mitochondrial dynamics as a therapeutic target for cardiac disease’

Guy Rutter, Imperial College London
‘Mitochondria and insulin secretion – links to diabetes’

Michael Murphy, MRC Mitochondiral Biology Unit, Cambridge
‘Exploring mitochondrial ROS metabolism in the heart using targeted probes and bioactive molecules’

Toni Vidal Puig, Institute of Metabolic Science, University of Cambridge
‘Adipose tissue expandability, lipotoxicity and the metabolic syndrome’

SOURCE

http://www.abcam.com/index.html?pageconfig=resource&rid=15722

It all happens in a heartbeat

Calcium signaling is instrumental for excitation-contraction coupling (ECC). The involvement of mitochondria in establishing rapid cytosolic calcium transients in this process remain debated.

Two models have emerged:

slow integration versus rapid and
ample beat-to-beat changes of

cytosolic calcium transients into the mitochondria matrix.

a brief outline of cardiac calcium signaling »

Mitochondrial Calcium transport mechanisms

Calcium influx can be mediated by:

Mitochondrial Calcium Uniporter (MCU)
Mitochondrial Ryanodine receptor type 1 (mRyR1)
Leucine-zipper-EF-hand-containing transmembrane protein 1 (LETM1)
Proposed uptake by UCP2 and 3 and Coenzyme Q10

Calcium efflux can be mediated by:

Na-dependent calcium extrusion pathway, mNCX1
Mitochondrial permeability transistion pore (mPTP)

Inhibiting Calcium signaling

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Leptin signaling in mediating the cardiac hypertrophy associated with obesity

Posted in Cardiomyopathy, Cell Biology, Signaling & Cell Circuits, Frontiers in Cardiology and Cardiovascular Disorders, Metabolomics, Myocardial metabolism, Myocardial ischemia, myocardial perfusion, Myocardial adenine nucleotide metabolism, Nutrition, Population Health Management, Genetics & Pharmaceutical, Population Health Management, Nutrition and Phytochemistry, Proteomics, Systemic Inflammatory Response Related Disorders, tagged heart, Hypertrophy, leptin, Obesity, Signal transduction, STAT3 on November 3, 2013| Leave a Comment »

Leptin signaling in mediating the cardiac hypertrophy associated with obesity

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

There has been a lot of interest in leptins and both insulin resistance and obesity for the last decade. The association between obesity and cardiac hypertrophy is also known, but what drives this association. We have covered heart disease from many aspects in a long series of articles. The next is a pleasure to take in.

Importance of leptin signaling and signal transducer and activator of transcription-3 activation in mediating the cardiac hypertrophy associated with obesity

Maren Leifheit-Nestler¹², Nana-Maria Wagner¹³, Rajinikanth Gogiraju¹,Michael Didié¹⁴, Stavros Konstantinides¹⁵, Gerd Hasenfuss¹ and Katrin Schäfer¹^*

¹Department of Cardiology and Pulmonary Medicine, Heart Research Center, Georg August University Medicine Goettingen, Robert Koch Strasse 40, D-37075, Göttingen, Germany

²Current address: Department of Pediatric Kidney, Liver and Metabolic Diseases, Hannover Medical School, Hannover, Germany

³Current address: Clinic for Anesthesiology and Intensive Care Medicine, University Medicine Rostock, Rostock, Germany

⁴Department of Pharmacology, Georg August University Medicine Goettingen, Goettingen, Germany

⁵Current address: Center for Thrombosis and Hemostasis, University Medicine Mainz, Mainz, Germany

J Translational Medicine: Cardiovascular, Metabolic and Lipoprotein Translation. 2013; 11:170. http://www.translational-medicine.com/content/11/1/170

h ttp://dx.doi.org/10.1186/1479-5876-11-170

This is an Open Access article distributed under the terms of the Creative Commons Attribution License
http://creativecommons.org/licenses/by/2.0

Abstract

Background

The adipokine leptin and its receptor are expressed in the heart, and

leptin has been shown to promote cardiomyocyte hypertrophy in vitro.

Obesity is associated with

hyperleptinemia

hypothalamic leptin resistance and

an increased risk to develop cardiac hypertrophy and heart failure.

However, the role of cardiac leptin signaling in mediating the cardiomyopathy associated with increased body weight is unclear, in particular, whether it develops subsequently to cardiac leptin resistance or overactivation of hypertrophic signaling pathways via elevated leptin levels.

Methods

The cardiac phenotype of high-fat diet (HFD)-induced obese wildtype (WT) mice was examined and compared to age-matched genetically obese leptin receptor (LepR)-deficient (LepR^db/db) or lean WT mice. To study the role of leptin-mediated STAT3 activation during obesity-induced cardiac remodeling,

mice in which tyrosine residue 1138 within LepR had been replaced with a serine (LepR^S1138) were also analyzed.

Results

Obesity was associated with hyperleptinemia and elevated cardiac leptin expression in both diet-induced and genetically obese mice.

Enhanced LepR and STAT3 phosphorylation levels were detected in hearts of obese WT mice, but not in those with LepR mutations, and
exogenous leptin continued to induce cardiac STAT3 activation in diet-induced obese mice.

Although echocardiography revealed signs of cardiac hypertrophy in all obese mice,

the increase in left ventricular (LV) mass and diameter was significantly more pronounced in LepR^S1138 animals.

LepR^S1138 mice also exhibited an increased activation of signaling proteins downstream of LepR, including Jak2 (1.8-fold), Src kinase (1.7-fold), protein kinase B (1.3-fold) or C (1.6-fold). Histological analysis of hearts revealed that the inability of leptin to activate STAT3 in LepR^db/db and LepR^S1138 mice

was associated with reduced cardiac angiogenesis as well as increased apoptosis and fibrosis.

Conclusions

Our findings suggest that hearts from obese mice continue to respond to elevated circulating or cardiac leptin, which

may mediate cardioprotection via LepR-induced STAT3 activation, whereas
signals distinct from LepR-Tyr1138 promote cardiac hypertrophy.

On the other hand, the presence of cardiac hypertrophy in obese mice with complete LepR signal disruption indicates that additional pathways also play a role.

Keywords:

Heart; Hypertrophy; Leptin; Obesity; Signal transduction; STAT3

Background

Obesity is frequently associated with elevated circulating leptin levels [1] and an increased risk to develop cardiac hypertrophy [2,3] or heart failure [4]. Clinical studies demonstrated a positive correlation between serum leptin levels and left ventricular (LV) mass or wall thickness [5,6], independent of blood pressure levels, suggesting a direct role for leptin in the pathogenesis of obesity-associated cardiomyopathy. Furthermore, leptin was shown to promote hypertrophy of isolated rat or human ventricular cardiomyocytes [7,8], and

this effect could be prevented using neutralizing antibodies [9].

Cardiac hypertrophy also develops in obese rodents fed high-fat diet (HFD)[10,11], and

studies in mice with (functional) leptin deficiency suggested that the cardiac hypertrophy developing in states of chronic hyperleptinemia
may result from the inability to transduce anti-hypertrophic and/or cardioprotective effects of the adipokine [12,13].

The effects of leptin on cell shortening and intracellular Ca²⁺ transients were abrogated in cardiomyocytes isolated from HFD-fed obese rats [14], but then others found

a preserved signal transduction in response to leptin in hyperleptinemic obese mice [15,16] or rats[17].

The leptin receptor (LepR) belongs to the family of cytokine type I receptors that signal via activation of

Janus kinase (Jak)-2 and
signal transducer and
activator of transcription (STAT)-3 [18].

Analysis of cardiomyocytes ex vivo revealed leptin promotes hypertrophy via activation of p38 and p42/44 MAP kinases as well as protein kinase B (Akt) [19,20]. But it is unknown whether STAT-3 activation downstream of LepR is required to transmit the cardiac effects of leptin and whether it may be involved in mediating protective (i.e. anti-apoptotic, anti-fibrotic or pro-angiogenic) signals, as previously reported in mice with cardiomyocyte-specific STAT-3 deletion [21,22].

In this study, we examined the cardiac phenotype of diet-induced (i.e. with hypothalamic leptin resistance) and genetically obese (i.e. with systemic leptin receptor deficiency) hyperleptinemic mice, developing with age or after continuous β-adrenergic stimulation. Moreover, we determined

the importance of leptin-mediated STAT-3 activation

for the development of cardiac hypertrophy in obesity

by analyzing mice with targeted mutation of the STAT3 binding site within LepR.

Methods

Animals

C57Bl6/J leptin receptor-deficient db/db (LepR^db/db; BKS.Cg Lepr^db/Lepr^db) mice and C57Bl6/J wildtype (WT) controls were obtained from Harlan Winkelmann, Germany. Mice heterozygous mutant for the LepR^S1138 allele (on the congenic B6.129/J background; 98- > 99% homozygous for C57Bl/6; [23]) were obtained from Professor Martin Myers (University of Michigan Medical School, Ann Arbor, USA) and bred at the animal facility of the University of Goettingen, Germany, to generate homozygous mutant obese LepR^S1138 mice. Age- and gender-matched WT (LepR^+/+) and heterozygous (LepR^S/+) littermates were used as controls. To induce obesity, 3 months-old mice were switched to high-fat diet (HFD; D12451) for 4 months, while controls were maintained on normal rodent chow (D12450B; both Research Diets Inc.). The composition of both diets is shown in Additional file 1: Table S1. To examine the cardiac response to hypertrophic stimuli other than leptin, osmotic minipumps (Alzet®; model 2002; Charles River Laboratories) were filled with isoprenaline hydrochloride (Sigma; 20 mg/kg body weight [BW] per day) and implanted for 14 days under the dorsal skinfold of 2 months-old, 2% isoflurane anesthetized mice. At the time of tissue harvest, mice were weighed followed by intraperitoneal anesthesia with a mixture of 2% xylazine (6 mg/kg BW) and 10% ketamine hydrochloride (100 mg/kg BW), and blood was drawn by cardiac puncture. Hearts were rapidly excised, the atria removed and ventricles immediately processed for protein isolation or cryoembedding, respectively. All animal care and experimental procedures had been approved by the institutional Animal Research Committee and complied with national guidelines for the care and use of laboratory animals.

Additional file 1: Table S1. Diet composition.
Format: DOC Size: 33KB

Serum analysis

Freshly drawn blood was allowed to clot at room temperature (RT) for 30 min, followed by centrifugation for 10 min at 3,000 rpm. The supernatant was stored at -80°C pending analysis of serum leptin levels using specific enzyme-linked immunoassays (ELISA; R&D Systems).

Echocardiography

Echocardiography was performed by a blinded examiner at the day before tissue harvest in mice under 1.5% isoflurane anesthesia using the VisualSonics Vevo 2100 system (Visualsonics) equipped with a 30 MHz center frequency ultrasound transducer, as previously described [24]. M-mode echocardiographical recordings were used to determine the end-diastolic and end-systolic LV diameter (EDD and ESD, respectively) and the ventricular wall thickness (WTh), corresponding to the mean of the anterior and posterior WTh. LV mass was calculated using the formula: 1.055 × ([AWTh + EDD + PWTh]³ – EDD³). Fractional shortening (FS) was calculated as (EDD – ESD)/EDD × 100. B-mode echocardiography images were used to calculate the heart weight, using the equation: 1.05 × (5/6) × ((Epi_syst × (L_syst + ((AWTh_syst + PWTh_syst)/2))) – (Area_syst × L_syst)).

Histology and immunohistochemistry

Histochemical analyses were performed on 5 μm-thick frozen cross sections through the LV. For each mouse, 4 sections (approx. 500 μm apart) and 4 randomly selected viewing fields (at 200-fold magnification) per section were analyzed and findings averaged. Cardiac fibrosis was determined after overnight incubation in Bouin’s fixative followed by Masson’s trichrome (MTC) stain. Monoclonal rat antibodies against mouse CD31 (Santa Cruz Biotechnology) were used to detect endothelial cells [24,25]. Their number was manually counted by a person blinded to the mouse genotype and expressed per mm² or cardiomyocyte, respectively.

Single cardiomyocytes were visualized by incubation with fluorescein-labeled wheat germ agglutinin (WGA; Molecular Probes), followed by determination of the cardiomyocyte cross-sectional area (CSA) using image analysis software (Image ProPlus). Per cross section, at least 10 randomly selected cardiomyocytes were evaluated and results averaged. Apoptosis was analyzed using the ‘In Situ Cell Death Detection kit’ (Roche). Cell nuclei were visualized using 4′,6-diamidino-2-phenylindole (DAPI; Sigma).

Immunoprecipitation and western blot analysis

Membranes were blocked in 1% bovine serum albumine (in TBS, containing 0.1% Tween-20) prior to incubation with antibodies against phosphorylated (p)-Akt (S473) and total Akt, p-Jak2 (Y1007/1008) and total Jak2, p-p38 (T180/Y182) and total p38, p-p42/44 (T202/Y204) and total p42/44, p-Src (Y416) and total Src, p-STAT3 (Y705) and total STAT3, or p-PKC (pan), respectively (all Cell Signaling Technologies), or against leptin (R&D Systems) and GAPDH (Biotrend), respectively. Protein bands were visualized using HRP-conjugated secondary antibodies (Amersham Biosciences), followed by detection with SuperSignal® West Pico Substrate (Pierce). For the analysis of LepR phosphorylation, 100 μg total heart tissue lysates were immunoprecipitated under rotation at 4°C with 2 μg anti-LepR antibody (against an internal domain present in the short and long isoforms of murine LepR; Santa Cruz Biotechnology) plus 50 μL nProtein A Sepharose™ 4 Fast Flow beads (GE Healthcare) followed by detection of phosphorylated tyrosines (p-Tyr [PY20]; Santa Cruz Biotechnology) or LepR. For the analysis of STAT3 phosphorylation in response to acute elevations of circulating leptin, mice were fasted overnight, injected with recombinant murine leptin (1 mg/kg BW i.p.) and hearts harvested 30 min later.

Statistical analysis

Quantitative data are presented as mean ± standard error of the mean (SEM). Normal data distribution was tested using the D’Agostino & Pearson omnibus normality test. When three or more groups were compared, ANOVA was employed, if samples were normally distributed, or Kruskal-Wallis test, if not. For post-hoc comparisons, ANOVA was followed by Bonferroni’s and Kruskal-Wallis by Dunn’s multiple comparison test. Differences before and after isoprenaline infusion were tested using Student’s t-test for paired means. Statistical significance was assumed when P reached a value less than 0.05. All statistical analyses were performed using GraphPad PRISM software, version 4.01 (GraphPad Software Inc).

Results

Clinical and experimental studies revealed that obesity is associated with LV hypertrophy [10,11], an important risk factor for the development of heart failure. As shown in Tables 1 and 2, WT mice fed HFD for 4 months (WT + HFD; mean body weight [BW], 44±1.9 g) to induce obesity exhibited a non-significant trend towards an increased mean heart weight, LV mass and WTh compared to age-matched lean controls fed normal chow (BW, 29±1.0 g). Marked LV hypertrophy was observed in 7 months-old obese LepR^db/db mice (Table 1 and 2), consistent with a previous report [12]. Longitudinal sections through hearts of WT, WT + HFD and LepR^db/db mice are shown in Figure 1A, representative M-mode echocardiography recordings in Figure 1B and cardiac cross-sections after WGA staining to delineate cardiomyocyte borders in Figure 1C. Of note, adiposity in mice with LepR deficiency was more pronounced compared to age-matched WT + HFD mice (Table 1; P < 0.001), in which obesity develops as result of hypothalamic resistance to chronically elevated leptin levels[26].

Figure 1.Cardiac phenotype of lean and obese WT, WT + HFD, LepR^S1138and LepR^db/dbmice.
(A)Representative H&E-stained longitudinal sections through hearts of 7 months-old mice are shown. Magnification, ×10.(B) Representative M-mode echocardiographic recordings.(C) Representative images of wheat germ agglutinin (WGA)-stained myocardial cross sections. The mean cardiomyocyte cross-sectional areas are given in Table 1.

Table 1.Body, visceral fat and heart weights in 7 months-old mice

Table 2.Echocardiographic parameter in 7 months-old mice

The presence of cardiac hypertrophy in LepR-deficient and, to a lesser extent also in diet-induced obese mice, suggests that it develops as a result of the heart’s inability to respond to elevated systemic (Table 1) and/or cardiac (Figure 2A) leptin levels. In this regard, Western blot analysis revealed increased levels of phosphorylated (p-) LepR (Figure 2B) and STAT-3 (Figure 2C) protein in hearts of HFD-induced obese mice (P < 0.05 vs. WT for both), whereas findings in LepR^db/db mice did not differ from those in lean controls or were reduced compared to those in WT + HFD mice (P < 0.05 for differences in LepR phosphorylation). Moreover, both lean and HFD-induced obese WT mice responded to a single i.p. injection of recombinant murine leptin with a significant increase in the cardiac STAT-3 phosphorylation (Figure 2D), suggesting a preserved cardiac leptin signal transduction in hyperleptinemic, diet-induced obese mice.

Figure 2.Cardiac leptin expression and signal transduction in lean and obese mice.
Protein was extracted from hearts of 7 months-old mice (n = 8 per group) and analyzed for the expression of (A) leptin, (B)phosphorylated LepR (using immunoprecipitation of LepR, followed by the detection of total phosphotyrosines and LepR) and (C) phosphorylated STAT3. (D) Cardiac STAT3 phosphorylation in response (30 min later) to a single injection of recombinant murine leptin (1 mg/kg BW i.p.) was examined in WT (n = 4) and WT + HFD (n = 6) mice. Results are expressed as -fold increase of controls (black bars) after normalization for total protein and GAPDH expression. The mean ± SEM as well as representative Western blot results are shown. *P < 0.05 and **P < 0.01 vs. WT mice; #P < 0.05 vs. WT + HFD mice.

To further study the role of leptin signaling in the development of cardiac hypertrophy and also to determine, whether the inability of leptin to activate STAT3 contributes to the cardiac maladaptation in obesity, we examined mice in which tyrosine (Tyr)1138 within LepR had been replaced by a serine (LepR^S1138). In these mice, leptin cannot signal via STAT3, but continues to be able to activate Jak2 and SH2 domain-containing adapter proteins. Western blot analysis revealed that p-LepR (Figure 2B) and p-STAT3 (Figure 2C) levels in hearts of LepR^S1138 mice did not significantly differ from those in WT and LepR^db/db mice. Similar to mice with complete LepR deficiency, lack of LepR-mediated STAT3 activation resulted in severe adiposity, although serum leptin levels were lower than those in LepR^db/db mice (P < 0.001; Table 1). Interestingly, obese LepR^S1138 exhibited a more pronounced increase in mean heart weights not only compared to lean or diet-induced obese WT mice, but also compared to LepR^db/db mice (P < 0.001 for all comparisons; Table 1), and differences persisted after normalization for body weight (P < 0.001) or tibia length (P < 0.001). Echocardiography confirmed increased LV mass (P < 0.01) or heart weights (P < 0.001) in LepR^S1138 mice compared to their LepR^db/dbcounterparts (Table 2; please also see Figure 1A-C). Moreover, hearts of LepR^S1138 mice exhibited elevated levels of phosphorylated Jak2 (P < 0.001 vs. WT; Figure 3A), Src kinase (P < 0.05 vs. WT, WT + HFD and LepR^db/db; Figure 3B), Akt (P < 0.001 vs. LepR^db/db; Figure 3C), PKC (P < 0.05 vs. WT and LepR^db/db, P < 0.01 vs. WT + HFD; Figure 3D) and p38 MAPK (P < 0.01 vs. LepR^db/db; Figure 3E), suggesting that an intact, Tyr1138-independent LepR activation in the presence of elevated leptin levels may have contributed to the pronounced cardiac hypertrophy present in these mice. On the other hand, cardiac levels of p-p42/44 MAPK did not significantly differ between the mouse groups (Figure 3F).

Figure 3.Hypertrophic signal transduction in hearts of lean and obese mice. Protein was isolated from hearts of 7 months-old WT (n = 15), WT + HFD (n = 12), LepR^S1138(n = 15) and LepR^db/db (n = 15) mice and analyzed for the expression of phosphorylated Jak2 (A), Src kinase (B), Akt (C), PKC (D), p38 (E) and p42/44 MAPK (F). Results are expressed as -fold increase of lean control mice (after normalization for total protein [with the exception of PKC] and GAPDH expression). The mean ± SEM as well as representative findings are shown. *P < 0.05, **P < 0.01 and ***P < 0.001 vs. WT mice; #P < 0.05 and ##P < 0.01 vs. WT + HFD mice; §P < 0.05, §§P < 0.01 and §§§P < 0.001 for the difference between LepR^db/db and LepR^S1138 mice.

M-mode echocardiography also revealed significantly increased enddiastolic LV diameters in LepR^S1138 mice (P < 0.01 vs. WT and LepR^db/db mice; Table 2; representative findings are shown in Figure 1B), suggesting that the observed (over-)activation of LepR signaling together with the inability to induce STAT3 may result in augmented hypertrophy and maladaptive cardiac remodeling. Of note, fractional shortening (FS) was not significantly altered in HFD-induced obese WT mice (P = n.s. vs. WT mice), but found to be increased in both LepR^db/db (P < 0.01 vs. WT and P < 0.001 vs. WT + HFD mice) and LepR^S1138mice (P < 0.05 vs. WT and P < 0.001 vs. WT + HFD mice). Histological analyses revealed significantly reduced numbers of CD31-positive capillary endothelial cells in LepR^db/db, and to a lesser extent also in LepR^S1138 mice (Figure 4A), whereas the number of TUNEL-positive apoptotic cells (Figure 4B) and the fibrotic tissue area (Figure 4C) were found to be significantly increased in hearts of both LepR^S1138 and LepR^db/db mice compared to lean and diet-induced obese WT mice.

Figure 4.Histological analysis of angiogenesis, apoptosis and fibrosis in hearts of lean and obese mice.
Serial cross sections through the LV of WT, WT + HFD, LepR^S1138 and LepR^db/db mice (n = 10 per group) were immunostained and the number of (A) CD31-positive endothelial cells and (B) TUNEL-positive apoptotic cell nuclei determined. Results are expressed per cardiomyocyte and/or mm².(C) The degree of cardiac fibrosis was quantified after Masson’s trichrome (MTC) staining. Results are expressed as % of total tissue area (at 200-fold magnification). The mean ± SEM as well as representative findings are shown. **P < 0.01 and ***P < 0.001 vs. WT; #P < 0.05, ##P < 0.01 and ###P < 0.001 vs. WT + HFD mice.

Figure 4.Histological analysis (unable to post)

To examine the specificity of leptin’s hypertrophic action in obesity, the cardiac response of young, i.e. 2 months-old WT (n = 12; body weight, 22 ± 0.9 g), LepR^S1138 (n = 9; 34 ± 1.1 g, P < 0.001 vs. WT) and LepR^db/db mice (n = 7; 40 ± 1.3 g; P < 0.001 vs. WT and P < 0.01 vs. LepR^S1138) to chronic isoprenaline infusion (20 mg/kg BW per day) was examined. Under basal conditions, similar findings as those in 7 months-old mice were observed, i.e. LepR^S1138 mice exhibited an increased heart weight (P < 0.05 vs. LepR^db/db; Figure 5A), LV mass (P < 0.01 vs. WT; Figure 5B) and mean WTh (P < 0.05 vs. WT; Figure 5C), whereas other changes, such as differences in fractional shortening (Figure 5D), ESD (Figure 5E) and EDD (Figure 5F) were not (yet) detected. On the other hand, all mouse groups responded to chronic β-adrenergic stimulation with significant cardiac hypertrophy, and no differences (with the exception of heart weight; Figure 5A) were observed between LepR^S1138 and LepR^db/db mice. Representative M-mode echocardiography tracings are shown in Figure 6 and summarized in Additional file 2: Table S2.

Figure 5.Echocardiography findings in young lean and obese mice before and after chronic β-adrenergic stimulation.
Isoprenaline-filled osmotic minipumps were subcutaneously implanted into 2 months-old WT (n = 12), LepR^S1138 (n = 9) and LepR^db/db (n = 7) mice to examine the cardiac response to a hypertrophic stimulus other than leptin. Echocardiography (A-F) was performed immediately before (open bars) as well as at the time of tissue harvest 14 days later (dotted bars). *P < 0.05, *P < 0.01 and ***P < 0.001 for differences vs. WT mice; §P < 0.05 for differences between LepR^db/db and LepR^S1138 mice. Significance levels for differences before and after isoprenaline stimulation (as determined using Student’s t test for paired means) are indicated within the graph.

Figure 6.Representative M-mode echocardiography recordings.

Additional file 2: Table S2. Echocardiographic parameter in 2 months-old mice before and 14 days after isoprenaline infusion.

Format: DOCX Size: 22KB

Discussion

The adipocytokine leptin may link obesity with cardiac hypertrophy, an important risk factor for the development of heart failure. Studies in humans [2,3] and rodents [10,11] have shown that obesity is associated with LV hypertrophy, and body mass index was identified as a strong and independent predictor of LV mass [2,3]. Importantly, cardiac hypertrophy is also observed in normotensive obese subjects [6], and plasma leptin levels are associated with increased myocardial wall thickness independent of BW or blood pressure elevations [5], suggesting a causal role for leptin in the pathogenesis of cardiac hypertrophy.

Although the major source of leptin is adipose tissue, cardiomyocytes are also capable of synthesizing leptin [27], and increased cardiac leptin levels have been reported in mice or rats following coronary ligation [13,18] or in patients with heart failure [28]. In this study, elevated circulating as well as cardiac leptin levels were detected in both diet-induced and genetically obese mice, which may have acted on cardiomyocytes as well as other, non-cardiomyocyte cells expressing leptin receptors [29]. Although leptin serum levels were higher than in previous publications [30], we explain this findings with the higher age of the mice, a factor previously found to be associated with increased circulating leptin levels [31]. Leptin has been shown to stimulate the hypertrophy of cardiomyocytes isolated from rats [7,20] or humans [8,19]. Moreover, chronic leptin infusion increased cardiac ANP expression after myocardial infarction (MI) in mice [32], whereas neutralizing LepR antibodies abrogated the hypertrophy of the surviving myocardium after coronary artery ligation in rats [33]. On the other hand and as confirmed in our analysis, cardiac hypertrophy also develops in leptin- and LepR-deficient mice and may be reversed by leptin substitution[12]. Caloric restriction experiments suggested that the anti-hypertrophic effects of leptin had occurred in addition to weight loss [12], which itself may preserve heart function and attenuate LV remodeling [34]. Thus, it is unclear whether the cardiac hypertrophy in obesity is the consequence of pro-hypertrophic effects of the adipokine or rather the result of a resistance towards leptin’s preventive effects on hypertrophic cardiac remodeling. Of note, since body weight is markedly elevated in the diet-induced and particularly, the genetically obese mice, the heart-to-body weight ratio decreases, even though the absolute heart weight is increased (but to a relatively lesser extent).

Obesity is associated with elevated circulating leptin levels and hypothalamic resistance to the weight-reducing effects of the adipokine, whereas the existence of a peripheral (e.g. cardiac) leptin resistance is controversial. For example, reduced cardiac LepR expression has been reported in HFD-fed rats[14], whereas others demonstrated unaltered cardiac STAT3 phosphorylation in diet-induced obese rodents following acute leptin administration [15–17]. Our findings also suggest that hearts from diet-induced obese mice continue to respond to leptin in the presence of chronically elevated leptin levels and that the observed elevation of serum and cardiac leptin may thus contribute to the development of cardiac hypertrophy in obesity. For example, hearts of hyperleptinemic obese WT mice (i.e. those with intact leptin receptors) exhibited signs of activated leptin signaling, including elevated levels of phosphorylated LepR and STAT3, while they were unchanged or reduced in mice with mutated or truncated forms of LepR (i.e. LepR^S1138 or LepR^db/db mice). Moreover, both lean and obese WT mice responded to a single leptin injection with increased cardiac STAT3 phosphorylation. Of note, we could not spatially dissect the cardiac responsiveness to leptin, since whole heart homogenates were examined. Possible explanations underlying the discrepancy between the present and some previous studies include the animal species, as the absence of a response to leptin in obesity has been so far primarily observed in rats[14]. In addition, age, sex and feeding status of the animals or the time of recombinant leptin administration may have influenced the results. Of note, previous studies in humans have reported the existence of individuals (up to 40%) exhibiting a blunted response to leptin [35], although it is unknown, whether such phenomenon also occurs in rodents.

Interestingly, hearts from LepR^S1138 mice exhibited a marked overactivation of STAT3-independent leptin signaling pathways, including Jak2, Src kinase, Akt or p38 MAPK, i.e. factors previously shown to mediate the pro-hypertrophic effects of the adipokine in cardiomyocytes [19,20]. Importantly, overactivation of leptin signaling in hearts of LepR^S1138 mice was accompanied by a pronounced cardiac hypertrophy, both at the organ and the single cardiomyocyte level, despite similar adiposity. Although leptin levels were found to be lower in LepR^S1138compared to LepR^db/db mice, as previously reported [23], leptin continues to be able to activate LepR signal transduction in these mice, for example via LepR-Tyr985. Similar echocardiographical findings were obtained in young (i.e. 2 months-old) and older (i.e. 7 months-old) mice, arguing against the development of cardiac hypertrophy secondary to hemodynamic or other metabolic changes associated with obesity, although we cannot exclude the possible contribution of a more pronounced hyperinsulinemia [23] to the development of cardiac hypertrophy in LepR^S1138 mice. On the other hand, hypertension had not been observed in ob/ob mice [12], and heart weight increase and concentric LV hypertrophy in obese mice and humans also occurs without systolic and diastolic blood pressure elevations [5,6,36].

Although a predominant cardiac expression of the short (i.e. without STAT3 binding site) over the long LepR isoform has been reported [7,29], previous studies have shown that stimulation of neonatal rat cardiomyocytes with leptin increased STAT3 phosphorylation, nuclear translocation and DNA binding activity [32]. Also, cardiac STAT3 activation after MI was blunted in leptin-deficient mice [13]. The observation that increased cardiac STAT3 phosphorylation in hyperleptinemic, diet-induced obese mice was reduced or almost completely abolished in LepR^S1138 or LepR^db/db mice suggests that cardiac STAT3 activation in obesity largely occurs downstream of elevated leptin levels and that other cytokines, also elevated in obesity and known to signal via Jak2-STAT3, may be of minor importance. On the other hand, the importance of leptin-mediated STAT3 activation in the heart and its contribution to cardioprotective signaling pathways in vivo have not been directly examined so far.

STAT3 has been implicated in cardioprotection after various injuries. For example, cardiomyocyte-specific STAT3 deletion results in dilatative cardiomyopathy, characterized by increased apoptosis and interstitial fibrosis as well as reduced myocardial capillary density [21,22]. Previous studies suggested that leptin may exert beneficial effects on the heart. For example, administration of leptin was associated with smaller infarct size after ischemia/reperfusion injury [37], whereas ischemic postconditioning failed to induce cardioprotection in mice lacking leptin or its receptor [38]. Also, leptin deficiency was associated with a worsened cardiac function and survival after coronary artery ligation, which could be improved by leptin repletion [13]. Regarding possible mechanisms, increased cardiac myocyte apoptosis was observed in hearts from leptin (receptor)-deficient mice [39,40]. Similar findings were obtained in vitro, showing that leptin protects cardiomyocytes against apoptotic cell death induced by serum starvation [41]. Our analyses also revealed significantly elevated numbers of apoptotic cells in hearts of obese LepR^S1138 and LepR^db/dbmice, consistent with a reduced activation of STAT3-responsive anti-apoptotic genes [40]. Although findings in mice with systemic defects in leptin signal transduction may have been confounded by the concomitant presence of obesity and associated metabolic and inflammatory alterations, adverse cardiac remodeling after MI [42] or lethal heart failure [43] were recently reported in mice with cardiomyocyte-specific LepR deletion. On the other hand, the beneficial effects of leptin-mediated STAT3 activation may not be restricted to cardiomyocytes. For example, we and others have shown that leptin promotes the angiogenic properties of endothelial (progenitor) cells [25,44], and cardiac angiogenesis was reduced in LepR^S1138 and LepR^db/db mice. In addition, hearts of obese LepR^S1138 and LepR^db/db mice exhibited increased interstitial fibrosis, which may have occurred secondary to increased cardiomyocyte loss, although previous studies have shown that leptin may also directly influence myocardial matrix metabolism [45]. On the functional level, the enhanced activation of pro-hypertrophic signaling pathways in the absence of STAT3-mediated cardioprotection may have contributed to the echocardiographic finding of LV cavity dilation in LepR^S1138 compared to LepR^db/db mice.

Conclusions

Taken together, our findings suggest that hearts from diet-induced obese mice continue to respond to chronically elevated leptin levels and that increased systemic and/or local leptin and enhanced cardiac LepR activation contribute the development of cardiac hypertrophy. On the other hand, chronic overactivation of hypertrophic signaling mediators together with an inabilitity to activate STAT3-dependent cardioprotective pathways may promote maladaptive cardiac remodeling. Of note, our findings also indicate that leptin signaling is not a prerequisite to develop cardiac hypertrophy in obesity and that additional pathways also contribute to the increase in LV mass associated with higher body weight.

References

Considine RV, Sinha MK, Heiman ML, Kriauciunas A, Stephens TW, Nyce MR, Ohannesian JP, Marco CC, McKee LJ, Bauer TL: Serum immunoreactive-leptin concentrations in normal-weight and obese humans.

N Engl J Med 1996, 334:292-295. PubMed Abstract | Publisher Full Text
Gottdiener JS, Reda DJ, Materson BJ, Massie BM, Notargiacomo A, Hamburger RJ, Williams DW, Henderson WG: Importance of obesity, race and age to the cardiac structural and functional effects of hypertension. The department of veterans affairs cooperative study group on antihypertensive agents.

J Am Coll Cardiol 1994, 24:1492-1498. PubMed Abstract |Publisher Full Text
Lauer MS, Anderson KM, Kannel WB, Levy D: The impact of obesity on left ventricular mass and geometry. The framingham heart study.

JAMA 1991, 266:231-236. PubMed Abstract | Publisher Full Text
Kenchaiah S, Evans JC, Levy D, Wilson PW, Benjamin EJ, Larson MG, Kannel WB, Vasan RS: Obesity and the risk of heart failure.

N Engl J Med 2002, 347:305-313. PubMed Abstract | Publisher Full Text
Paolisso G, Tagliamonte MR, Galderisi M, Zito GA, Petrocelli A, Carella C, de Divitiis O, Varricchio M: Plasma leptin level is associated with myocardial wall thickness in hypertensive insulin-resistant men.

Hypertension 1999, 34:1047-1052. PubMed Abstract |Publisher Full Text
Perego L, Pizzocri P, Corradi D, Maisano F, Paganelli M, Fiorina P, Barbieri M, Morabito A, Paolisso G, Folli F, Pontiroli AE: Circulating leptin correlates with left ventricular mass in morbid (grade III) obesity before and after weight loss induced by bariatric surgery: a potential role for leptin in mediating human left ventricular hypertrophy.

J Clin Endocrinol Metab 2005, 90:4087-4093. PubMed Abstract |Publisher Full Text
Rajapurohitam V, Gan XT, Kirshenbaum LA, Karmazyn M: The obesity-associated peptide leptin induces hypertrophy in neonatal rat ventricular myocytes.

Circ Res 2003, 93:277-279. PubMed Abstract | Publisher Full Text
Madani S, De Girolamo S, Munoz DM, Li RK, Sweeney G: Direct effects of leptin on size and extracellular matrix components of human pediatric ventricular myocytes.

Cardiovasc Res 2006, 69:716-725. PubMed Abstract | Publisher Full Text

Read Full Post »

Personalized Cardiovascular Genetic Medicine at Partners HealthCare and Harvard Medical School

Posted in Atherogenic Processes & Pathology, Biological Networks, Biomarkers & Medical Diagnostics, Cardiomyopathy, Cardiovascular Pharmacogenomics, Cardiovascular Research, Cell Biology, Chemical Biology and its relations to Metabolic Disease, Chemical Genetics, Computational Biology/Systems and Bioinformatics, Congenital Heart Disease, Electrophysiology, Frontiers in Cardiology and Cardiovascular Disorders, Genome Biology, Genomic Testing: Methodology for Diagnosis, HTN, Medical and Population Genetics, Molecular Genetics & Pharmaceutical, Myocardial Metabolism, Origins of Cardiovascular Disease, Personalized and Precision Medicine & Genomic Research, Pharmacogenomics, Pharmacotherapy of Cardiovascular Disease, Population Health Management, Proteomics, Regulated Clinical Trials: Design, Methods, Components and IRB related issues, Technology Transfer: Biotech and Pharmaceutical, Translational Research, Translational Science, tagged Aviva Lev-Ari, Children's Hospital Boston, DNA, DNA Sequencing, genetics, Harvard Medical School, Human Genome Project, Illumina, Partners HealthCare, Personalized medicine on February 25, 2013| 3 Comments »

Personalized Cardiovascular Genetic Medicine at Partners HealthCare and Harvard Medical School

Curator: Aviva Lev-Ari, PhD, RN

UPDATED on 5/4/2015

Goes to Clinic @MGH: Clinically validated versions of Exome Sequencing and Analysis using Broad-developed methods like Hybrid Capture, the Genome Analysis Toolkit (GATK), and MuTect

http://pharmaceuticalintelligence.com/2015/05/04/goes-to-clinic-mgh-clinically-validated-versions-of-exome-sequencing-and-analysis-using-broad-developed-methods-like-hybrid-capture-the-genome-analysis-toolkit-gatk-and-mutect/

Center for Personalized Genetic Medicine, Partners HealthCare and Harvard Medical School

The Partners HealthCare Center for Personalized Genetic Medicine offers technologies and technical support for the research activities of Partners investigators. Our objective is to help investigators advance their research programs and to provide the highest quality service, technical expertise, and leading technologies for genomics research. Our goal is to broaden the access to these technologies while offering the best customer service in the most cost conscious and time efficient manner possible.

We are organized into four principal service areas:

sequence analysis,
genotyping,
expression analysis, and
bioprocessing/sample management

Our platforms include next generation sequencing with Illumina HiSeq2000 and GA ii analyzers as well as Sanger sequencing using ABI 3730 XL sequence analyzers. Targeted custom genotyping is offered using Sequenom and Illumina GoldenGate panels as well as GWAS scale projects using Illumina Infinium and DNA methylation analysis using Illumina bead arrays. Expression analysis is available with capabilities for processing total RNA on either Affymetrix or Illumina arrays.

Through services from our BioSample Services Facility (BSF) and Partners Biorepository for Medical Discovery (PBMD) teams we provide a research platform for handling samples in a standardized manner to provide consistency from sample to sample. The BSF is able to assist investigators to configure projects utilizing your own samples or coupled with the PCPGM-PBMD we are able to support the integration of cohorts of samples selected from the PBMD into analysis on our genomics platforms.

http://pcpgm.partners.org/research-services

DNA Sequencing

The DNA Sequencing Group at the Partners HealthCare Center for Personalized Genetic Medicine has a strong history of producing high quality, dependable, and informative results for collaborators and clients. The DNA Sequencing Group participated in the Human Genome Project, building the STS-Based BAC map for Human Chromosome 12, and providing Chromosome 12 tiling path clones to the Baylor Human Genome Sequencing Center for sequencing.

The group sequenced 113 BACs for the Mouse Genome Project, contributing 24 megabases of finished mouse sequences to the published Mouse Genome, as well as providing draft sequences for unique strains of several bacterial genomes, including Pseudomonas aeruginosa, and Vibrio cholerae. More recently, the group participated in identifying mutations linked to numerous diseases, either in collaborations or by providing client laboratories with full service resequencing and analysis.

Services by Project Goals

Mutation Identification via Resequencing

This facility provides full-service resequencing of regions of interest in one or more genomic DNAs, including the following:

Discussion of the scope of the project and a cost quote
Identification of genes in the region of interest as needed, with the Investigator
Primer design using our automated system, to amplify desired regions
Primer ordering
QC of the primers on DNA standards, if required
PCR amplification of DNA provided by Investigator
PCR clean-up
Sequencing reactions
Sequencing reaction clean-up
Sequence application to the ABI 3730 XL Analyzer
Chromatograms are made available to Investigator over web (GIGPAD)
Data assembly and analysis using Phred Phrap and PolyPhred
One round of repeats and redesign if necessary
Report of variations found throughout sequence
Trouble shooting for 100% coverage if desired

Research Services

Fee-for-service sequencing
Fragment analysis / genotyping (Microsatellite Instability)

Technology Development

New technology testing and development
Collaborative Protocol development
Beta-test site for instrumentation and software

Clinical Diagnosis

Diagnostic test development
Sequencing for clinical diagnostics group

Genomic Sequencing Projects

Human
Rodent
Bacteria

http://pcpgm.partners.org/research-services/sequencing

Advancing Translational Genomics through Personalized Medicine Projects

The mission of the Partners HealthCare Center for Personalized Genetic Medicine (PCPGM) is to utilize genetics and genomics to promote and implement personalized medicine in caring for patients throughout the Partners HealthCare system and in health care nationally and globally.

The Personalized Medicine Project program was developed to support the clinical research efforts of junior Partners HealthCare investigators for translational genetics and genomic projects to advance personalized medicine. The goal of this program is to identify biological markers that can be used as potential predictive tests. This will be accomplished by:

Leveraging the Partners HealthCare Research Patient Data Registry (RPDR) and the Partners Biorepository for Medical Discovery (PBMD), centralized locations where Partners HealthCare patient data and/or samples are stored.
Identifying novel biological markers or new uses for existing markers.
Focusing on tests that could:
- improve diagnostic sensitivity or specificity;
- further stratify patient groups with a given diagnosis;
- predict improved clinical outcomes; or
- assist with selection of therapies or methods to manage disease.

http://pcpgm.partners.org/biorepository/pmprojectsrfp

Harvard Medical School Genetics Training Program

The Harvard Medical School (HMS) Genetics Training Program is one of the oldest and largest programs in the country. It was founded by Drs. John Littlefield at the Massachusetts General Hospital and Park Gerald at Children’s Hospital Boston in the early 1970’s. The program has trained scientists and clinicians who have become leaders in academic genetics, and has supported investigators who have made major contributions to the clinical practice of genetics and genetics research.

The HMS Genetics Training Program is accredited by the ABMG in all areas of training – Clinical Genetics, Biochemical Genetics, Cytogenetics, and Molecular Genetics. This provides an opportunity for our trainees to become active candidates for board certification in a discipline(s) of medical genetics in addition to receiving laboratory training. The training laboratories and clinics of the program are centered at HMS and its affiliated institutions including Brigham and Women’s Hospital (BWH), the HMS Department of Genetics, Beth Israel Deaconess Medical Center (BIDMC), Children’s Hospital Boston (CHB), Dana Farber Cancer Institute (DFCI), and Massachusetts General Hospital (MGH). The HMS Genetics Training Program provides trainees the opportunity to take advantage of the extraordinarily rich academic environment offered at HMS and its affiliated institutions as well as the greater Boston scientific community.

http://pcpgm.partners.org/educational-programs/harvard-medical-school-genetics-training-program

Cardiovascular Research Center @MGH

The Cardiovascular Research Center was founded in 1990, and occupies over 30,000 sq. ft. of laboratory space in both the Charlestown Navy Yard and the Richard B. Simches Research Building. Dr. Mark Fishman, now president of the Novartis Institutes for Biomedical Research, directed the Center from 1990 until 2002. From 2002-2005, Dr. Kenneth Bloch served as Interim Director and then in June 2005, the Massachusetts General Hospital welcomed Dr. Kenneth Chien as the new scientific director of the Cardiovascular Research Center. Prior to his MGH appointments, Dr. Chien directed the Institute for Molecular Medicine at the University of California at San Diego. An internationally recognized biologist specializing in cardiovascular science, he is a true pioneer in developing new therapeutic strategies to prevent the onset and progression of heart failure. Dr. Chien served as director until 2012.

Cardiovascular Research Center investigators have made many groundbreaking discoveries. Among these include:

• first identification of progenitor cells in the heart
• cloning of the first vertebrate cell death genes
• knocking out the genes that produce nitric oxide (NO), showing the importance of this molecule to atherosclerosis and stroke
• clinical use of NO to treat patients with pulmonary hypertension
• development of gene and cell transfer approaches to treat heart failure
• performance of the first large-scale genetic screen in a vertebrate (the zebrafish)
• identification of genes critical to cardiac pacemaking, rhythm, contractile function, and normal heart patterning
• discovery of a new methylase gene responsible for altering DNA structure during an individual’s lifetime

The Cardiovascular Research Center has taken great pride in the training of scientists with MDs and/or PhDs, as well as graduate students from a variety of Boston area institutions.

The Cardiovascular Research Center has two locations, one in the Charlestown Navy Yard and the other on the main campus’s Charles River Plaza complex in the Richard Simches Research Center.

Both the Simches and Navy Yard sites offer state of the art facilities, including tissue culture rooms, warm and cold rooms, histology rooms, autoclave facilities, hot labs, scope rooms and conference rooms. The Navy Yard lab has a topnotch zebrafish facility that is utilized by many scientists both inside and outside the Center, and a transgenic mouse core for both knock-ins and knock-outs. The Navy Yard facilities also contain echocardiogram equipment, specialized microscopes equipped with video capability for making movies, as well as a confocal microscope available to the Center researchers. The Simches lab houses the CVRC Stem Cell Biology + Therapy program, including a dedicated facility for human ES cell based technology, run by Dr. Chad Cowan, and future plans for high throughput screening facility to allow chemical screening in ESX cell based systems. Other cores available to researchers include a Cell Sorting and Flow Cytometry lab and a DNA sequencing core.

The Cardiology Laboratory for Integrative Physiology & Imaging lab is dedicated to large animal studies. An in house interventional cardiologist specializing in large animals performs the surgeries. In addition there are technicians that assist in the daily operations of the lab and can assist in experiment design and project implementation. This lab specializes in large animal imaging, CAT scans and catheter base manipulations. There is also an MRI imaging facility housed in the lab.

http://www2.massgeneral.org/cvrc/about.html

Genomics and Cardiovascular Medicine @MGH

	Translational Medicine: Genomics and Proteomics @MGH

The goal of the Translational Medicine Program is to harness the rich clinical cardiovascular population at the Massachusetts General Hospital to identify and validate novel genomic determinants of cardiovascular disease. Our goal is not to capture the entire cohort of cardiovascular patients presenting to Massachusetts General Hospital, but rather to focus our efforts on extremely well-phenotyped human models that are unique to cardiovascular disease. Of particular interest are “perturbational” studies in humans (e.g., cardiac exercise testing) that elicit robust phenotypes in affected individuals to serve as the springboard for analyses that span from genomics to proteomics and biochemical profiling. The Translational Medicine Program will involve a multidisciplinary group of investigators who contribute expertise in cardiovascular basic science, clinical cardiology, genetic/genomic epidemiology, bioinformatics, imaging, pathology, as well as clinical chemistry and mass spectrometry. While the Program in Translational Medicine will be physically located at the Massachusetts General Hospital Main Campus, the effort will leverage ongoing interdisciplinary collaborations with investigators at the Framingham Heart Study, the Broad Institute of M.I.T., Harvard University, and Harvard Medical School. Our goals are to:• Identify specific unmet needs in cardiovascular biomarker and pathway discovery (e.g., genomic markers of subclinical premature coronary artery disease, serum biomarkers of myocardial ischemia)• Match cutting-edge technologies with our unique patient cohorts for “first in man” studies• Establish the infrastructure necessary to phenotype patients with the targeted condition (from plasma samples, RNA, DNA, imaging, etc.) and enroll sufficiently sized cohort(s) with the requisite power to validate novel biomarkers.• Establish scientifically high priority research projects to target for independent funding.• Ultimately, develop novel therapeutic interventions.While efforts in translational investigation are already underway, this program will identify synergies between ongoing studies and catalyze new opportunities. Several of the ongoing projects that are anticipated to serve as cornerstones of this effort include:Proteomics and Metabolomics Studies (PI: Gerszten , Wang) Recent advances in proteomic and metabolic profiling technologies have enhanced the feasibility of high throughput patient screening for the diagnosis of disease states. Small biochemicals and proteins are the end result of the entire chain of regulatory changes that occur in response to physiological stressors, disease processes, or drug therapy. In addition to serving as biomarkers, both circulating metabolites and proteins participate as regulatory signals, such as in the control of blood pressure. Our ongoing studies have helped pioneer the application of novel mass spectrometry and liquid chromatography techniques to plasma analysis. In parallel with the profiling efforts, we have developed statistical software for functional pathway trend analysis and used it to demonstrate significant coordinate changes in specific pathways. Such analysis allows us to gain insight into the functionally relevant cellular mechanisms contributing to disease pathways and increases the likelihood that prospective biomarkers will be validated in other patient cohorts. Support for this effort would be synergistic with ongoing funding, including the recent appointment and support for Dr. Gerszten to lead a metabolomics initiative at the Broad Institute.Cardiovascular Genetics and Genomics Studies (PIs: Kathiresan, Newton-Cheh,Wang, and O’Donnell) Through the Human Genome Project and the International Haplotype Map project, researchers now have available the complete human genome sequence, a nearly complete set of common single nucleotide polymorphisms (SNPs), and a map of the patterns of correlation (“linkage disequilibrium”) among SNPs. Research on a large-scale is now possible to define associations of common, complex human cardiovascular diseases —such as myocardial infarction and sudden cardiac death—with genetic variants using candidate gene and genome-wide association studies, gene sequencing, and family-based linkage studies. Specific diseases and traits being studied by CVRC researchers include early-onset myocardial infarction, sudden cardiac death, blood lipids, blood pressure, electrocardiographic QT interval and blood hemostatic factor levels. These studies draw clinical material from the Massachusetts General Hospital and from collaborations with population-based epidemiologic cohorts such as the Framingham Heart Study. Like the metabolomics/proteomics work, these efforts build on the technologic and scientific expertise at the Broad Institute. Specifically, CVRC researchers leverage the Broad Institute’s expertise in large-scale genotyping, genomics, and statistical genetics. The collaboration between the Massachusetts General Hospital, the Framingham Heart Study, and the Broad Institute brings together resources that are unique to each institution to identify genes related to complex cardiovascular traits and to ultimately impact human health.Chemical Biology Program (PIs: Peterson and Shaw) Dr. Peterson’s group has championed the zebrafish as a tool for drug discovery. The zebrafish has become a widely used model organism because of its fecundity, its morphological and physiological similarity to mammals, the existence of many genomic tools and the ease with which large, phenotype-based screens can be performed. Because of these attributes, the zebrafish also provides opportunities to accelerate the process of drug discovery. By combining the scale and throughput of in vitro screens with the physiological complexity of animal studies, the zebrafish promises to contribute to several aspects of the drug development process, including target identification, disease modeling, lead discovery and toxicology. The Program in Translational Medicine will specifically support efforts to test novel pro-angiogenic factors (discovered as suppressors of the “gridlock” phenotype in zebrafish) on human cells such as circulating endothelial precursors.Dr. Shaw’s group is studying the cellular effects of human disease mutations in patient samples, by perturbing cells with a panel of thousands of drugs, and asking whether mutant versus wild-type cells react differently to a given biochemical (reminiscent of a genetic interaction screen). Dr. Shaw has demonstrated the feasibility of this approach using lymphoblast cell lines from a family affected by a monogenic form of diabetes (MODY1), and shown that glucocorticoid signaling differs between affected vs. unaffected patients. Because his studies incorporate the use of FDA-approved drugs, he can quickly identify both potentially “druggable” disease pathways as well as novel therapeutic agents. Further validation of these efforts in other monogenic disorders, such as LDL-receptor deficient patients is planned next. Ultimately this work will be extended to studies in complex genetic diseases.Director: Rob Gerszten, MDPrincipal Investigators: • Farouc Jaffer, MD, PhD • Sekar Kathiresan, MD • Chris Newton-Cheh, MD, MPH • Randall Peterson, PhD • Stanley Shaw, MD, PhD • Thomas Wang, MD

Genetic Basis of Cardiomyopathy

Original gene identification for Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy, Autosomal Dominant

McNally E, MacLeod H, Dellefave L. Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy, Autosomal Dominant. 2005 Apr 18 [Updated 2009 Oct 13]. In: Pagon RA, Bird TD, Dolan CR, et al., editors. GeneReviews™ [Internet]. Seattle (WA): University of Washington, Seattle; 1993-.

Summary

Disease characteristics. Autosomal dominant arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is characterized by progressive fibrofatty replacement of the myocardium that predisposes to ventricular tachycardia and sudden death in young individuals and athletes. It primarily affects the right ventricle; with time, it may also involve the left ventricle. The presentation of disease is highly variable even within families, and affected individuals may not meet established clinical criteria. The mean age at diagnosis is 31 years (±13; range: 4-64 years).

Available from:

http://www.ncbi.nlm.nih.gov/books/NBK1131/

Pan Cardiomyopathy Panel

@the Center for Personalized Genetic Medicine of Partners HealthCare and Harvard Medical School

The Pan Cardiomyopathy (PCM) Panel contains 51 cardiomyopathy genes including Titin (TTN), which encodes the largest human protein. This panel covers genes associated with HCM, DCM, RCM, LVNC, ARVC and CPVT and uses a combination of Next Generation Sequencing technology and conventional Sanger sequencing.

For illustrative reference, click to see one of our images or diagrams. Genes on Pan Cardiomyopathy Panels, Disease-Gene Associations, Gene Cellular Location.

Please select on the disease to read more: HCM,DCM, ARVC/CPVT, or LVNC.

Current Tests:

Pan Cardiomyopathy Panel – 51 genes

HCM Panel – 18 genes^§
DCM Panel – 27 genes^§
ARVC/CPVT Panel – 8 genes^§
LVNC Panel – 10 genes^§

^§Optional reflex to remaining genes

Storage Cardiomyopathy – please select a disease to learn more

Unexplained Hypertrophy Panel (LAMP2 & PRKAG2)
Fabry Disease – GLA Gene Sequencing
Transthyretin Amyloidosis – TTR Gene Sequencing

For any other single gene tests, please call the LMM at 617-768-8499 or lmm@partners.org.

For Variant Classification Rules – Lab for Molecular Medicine (LMM)

http://pcpgm.partners.org/sites/default/files/LMM/Resources/LMM_VariantClassification_05.26.11.pdf

For LMM Reference Sequences

http://pcpgm.partners.org/sites/default/files/LMM/Resources/LMMRefSeq-2.20.13.pdf

When to order which panel?

The Pan Cardiomyopathy panel may shorten the “testing odyssey” when a clear diagnosis has not been established. However, because many genes have not yet been associated with more than one cardiomyopathy, interpretation of novel variants may be more difficult when they are found in a gene that is not (yet) known to cause the patient’s cardiomyopathy. Please note: We are expecting an increase in “variants of unknown significance” and recommend careful consideration of the following factors when deciding whether to order the full panel or the disease specific sub-panels. The Pan Cardiomyopathy Panel may be best suited for patients who have already exhausted current testing options or whose clinical diagnosis is not yet clear. It may also be a good first line test for patients who have a family history where the number of living affected relatives would allow segregation analysis to establish or rule out pathogenicity for “variants of unknown significance (VUSs)”. Finally, the patient’s personal preferences should be considered as VUSs can cause anxiety.

Disease Backgrounds

Hypertrophic cardiomyopathy (HCM) is characterized by unexplained left ventricular hypertrophy (LVH) in a non-dilated ventricle. With a prevalence estimated to be ~1/500 in the general population, HCM is the most common monogenic cardiac disorder. To date, over 1000 variants have been identified in genes causative of HCM, most of which affect the sarcomere, the contractile unit of the cardiac muscle. In addition, defects in genes involved in storage diseases, such as LAMP2, PRKAG2 and GLA, typically cause systemic disease but may also result in predominant cardiac manifestations, which can mimic hypertrophic cardiomyopathy (HCM). For additional information about HCM, please visit GeneReviews.

Dilated cardiomyopathy (DCM) is characterized by ventricular chamber enlargement and systolic dysfunction with normal left ventricular wall thickness. The estimated prevalence of DCM is 1/2,500 and about 20-35% of cases have a family history showing a predominantly autosomal mode of inheritance. To date, over 40 genes have been demonstrated to cause DCM, encoding proteins involved in the sarcomere, Z-disk, nuclear lamina, intermediate filaments and the dystrophin-associated glycoprotein complex. Variants in some genes cause additional abnormalities: LMNA variants are frequently found in DCM that occurs with progressive conduction system disease. Variants in the TAZ gene cause Barth syndrome, an X-linked cardioskeletal myopathy in infants. In addition, variants in several genes (including LMNA, DES, SGCD, TCAP and EMD) can cause DCM in conjunction with skeletal myopathy. For additional information about DCM, please visit GeneReviews.

Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) is estimated to affect approximately 1/5,000 individuals in the general population, about half of which have a family history. The disease is characterized by replacement of myocytes by fatty or fibrofatty tissue, mainly in the right ventricle. The resulting manifestations are broad and include ventricular tachyarrhythmias and sudden death in young individuals and athletes. ARVC is typically inherited in an autosomal dominant fashion with incomplete penetrance and variable expressivity and to date, 5 ARVC genes (DSP, DSC2, DSG2, PKP2, TMEM43) have been identified, all but one (TMEM43) encode components of the desmosome. For more information about ARVC, please visit GeneReviews.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is typically characterized by exercise induced syncope due to ventricular tachycardia in individuals without structural heart disease. Two CPVT genes are known to date (RYR2 – autosomal dominant; CASQ2 – autosomal recessive). For more information about CPVT, please visit GeneReviews.

Left ventricular noncompaction (LVNC) has recently been established as a specific type of cardiomyopathy and is characterized by a spongy appearance of the left ventricular myocardium, resulting from an arrest in normal cardiac development. LVNC can be found in isolation or in association with other cardiomyopathies (HCM, DCM) as well as congenital cardiac abnormalities. The population prevalence is not known but LVNC is reported in ~0.014% of echocardiograms. LVNC is often familial and the genetic spectrum is beginning to emerge although it is not yet well defined. LVNC genes reported to date include ACTC, DTNA, LDB3, MYBPC3, MYH7, TAZ, and TNNT2 (Montserrat 2007, Klaassen 2008; Kaneda 2007, Zaragoza 2007; reviewed in: Maron 2006, Finsterer 2009). For more information about LVNC, please visit OMIM.org.

For any additional information, please contact us at 617-768-8500 or lmm@partners.org.

SOURCE:

http://pcpgm.partners.org/lmm/tests/cardiomyopathy

Genes: 51 genesMethodology: A combination of next generation sequencing technology and Sanger sequencingAnalytical Sensitivity:Substitutions: 100% (95%CI=98.5-100)Small InDels: 95% (95%CI=83-99)Clinical Sensitivity: See below.Additional Links:

Genetic Basis of Cardiomyopathy Booklet http://pcpgm.partners.org/sites/default/files/LMM/Resources/LMM_Cardiomyopathy_Booklet_2011.pdf

Cardiomyopathy

	Price	TAT	CPT Codes
Pan Cardiomyopathy Panel (51 Genes) – lmPCM-pnlAv2_L
	$3,950	8-12 wks	81479
HCM Panel (18 Genes) – lmPCM-pnlB_L
	$3,200	8-12 wks	81479
DCM Panel (27 Genes) – lmPCM-pnlCv2_L
	$3,850	8-12 wks	81479
ARVC/CPVT Panel (8 Genes) – lmPCM-pnlD_L
	$3,000	8-12 wks	81479
LVNC Panel (10 Genes) – lmPCM-pnlE_L
	$3,200	8-12 wks	81479
Remaining Pan Cardiomyopathy Genes (HCM Reflex) – lmPCM-pnlFv2_L
	$2,000	8-12 wks	81479
Remaining Pan Cardiomyopathy Genes (DCM Reflex) – lmPCM-pnlGv2_L
	$2,000	8-12 wks	81479
Remaining Pan Cardiomyopathy Genes (ARVC/CPVT Reflex) – lmPCM-pnlHv2_L
	$2,000	8-12 wks	81479
Remaining Pan Cardiomyopathy Genes (LVNC Reflex) – lmPCM-pnlIv2_L
	$2,000	8-12 wks	81479
Remaining Pan Cardiomyopathy Genes (Version 1 Reflex) – lmPCM-pnlL_L
	$750	8-12 wks	81479
Unexplained Cardiac Hypertrophy Panel (2 genes) – lmUCH-pnlA_L
	$1,500	3 wks	81479
ABCC9 Gene Sequencing – lmABCC9-a_L
	$1,800	3 wks	81479
ACTC Gene Sequencing – lmACTC-a_L
	$700	3 wks	81405
ACTN2 Gene Sequencing – lmACTN2-a_L
	$1,500	3 wks	81479
CSRP3 Gene Sequencing – lmCSRP3-a_L
	$900	3 wks	81479
CTF1 Gene Sequencing – lmCTF1-a_L
	$800	3 wks	81479
DES Gene Sequencing – lmDES-a_L
	$750	3 wks	81479
DSC2 Gene Sequencing – lmDSC2-a_L
	$1,150	3 wks	81479
DSG2 Gene Sequencing – lmDSG2-a_L
	$1,075	3 wks	81479
DSP Gene Sequencing – lmDSP-a_L
	$1,700	3 wks	81479
DTNA Gene Sequencing – lmDTNA-a_L
	$1,500	5-6 wks	81479
EMD Gene Sequencing – lmEMD-a_L
	$450	3 wks	81479
GLA Gene Sequencing – lmGLA-a_L
	$700	3 wks	81405
LAMP2 Gene Sequencing – lmLAMP2-a_L
	$700	3 wks	81405
LDB3 Gene Sequencing – lmLDB3-a_L
	$950	3 wks	81406
LMNA Gene Sequencing – lmLMNA-a_L
	$700	3 wks	81406
MYBPC3 Gene Sequencing – lmMYBPC3-a_L
	$1,500	3 wks	81407
MYH7 Gene Sequencing – lmMYH7-a_L
	$1,700	3 wks	81407
MYL2 Gene Sequencing – lmMYL2-a_L
	$700	3 wks	81405
MYL3 Gene Sequencing – lmMYL3-a_L
	$700	3 wks	81405
PKP2 Gene Sequencing – lmPKP2-a_L
	$1,500	3 wks	81479
PLN Gene Sequencing – lmPLN-a_L
	$400	3 wks	81479
PRKAG2 Gene Sequencing – lmPRKAG2-a_L
	$1,000	3 wks	81406
SCN5A Gene Sequencing – lmSCN5A-a_L
	$1,700	5-6 wks	81407
SGCD Gene Sequencing – lmSGCD-a_L
	$1,100	3 wks	81405
TAZ Gene Sequencing – lmTAZ-a_L
	$700	3 wks	81406
TCAP Gene Sequencing – lmTCAP-a_L
	$700	3 wks	81479
TMEM43 Gene Sequencing – lmTMEM43-a_L
	$700	3 wks	81479
TNNI3 Gene Sequencing – lmTNNI3-a_L
	$700	3 wks	81405
TNNT2 Gene Sequencing – lmTNNT2-a_L
	$1,000	3 wks	81406
TPM1 Gene Sequencing – lmTPM1-a_L
	$700	3 wks	81405
TTN Gene Sequencing – lmTTN-a_L
	$3,000	8-12 wks	81479
TTR Gene Sequencing – lmTTR-a_L
	$485	3 wks	81404
VCL Gene Sequencing – lmVCL-a_L
	$1,500	3 wks	81479

Congenital Heart Disease/Defects

	Price	TAT	CPT Codes
Congenital Heart Disease Panel A (GATA4, NKX2-5, JAG1) – lmCHD-pnlA_L
	$1,300	4 wks	81479
ELN (Elastin) Gene Sequencing – lmELN-a_L
	$1,300	4 wks	81479
GATA4 Gene Sequencing – lmGATA4-a_L
	$750	3 wks	81479
JAG1 Gene Sequencing – lmJAG1-a_L
	$1,100	3 wks	81407
NKX2-5 Gene Sequencing – lmNKX2-5-a_L
	$600	3 wks	81479

SOURCE:

http://pcpgm.partners.org/lmm/ordering/prices-CPTcodes_revised#Cardio_Header

Lakdawala NK, Funke BH, Baxter S, Cirino A, Roberts AE, Judge DP, Johnson N, Mendelsohn NJ, Morel C, Care M, Chung WK, Jones C, Psychogios A, Duffy E, Rehm HL, White E, Seidman JG, Seidman CE, Ho CY. Genetic Testing for Dilated Cardiomyopathy in Clinical Practice. J Card Fail 2012, In press.

Neri PM, Pollard SE, Volk LA, Newmark L, Varugheese M, Baxter S, Aronson SJ, Rehm HL, Bates DW. Usability of a Novel Clinician Interface for Genetic Results. J Biomed Informatics. 2012. In press.

Genomics @Brigham and Women’s Hospital and Harvard Medical School

The goal of The Cardiovascular Genome Unit (TCGU) is to foster interdisciplinary interaction between clinical investigators and scientists to comprehensively explore the era of human genomic research. In particular, our aim would be to identify, categorize and characterize the genes and genetic pathways of the vascular and cardiac tissues of the cardiovascular system during oncogenesis, normal function and the pathogenesis of cardiovascular diseases.

The Cardiovascular Genome Unit is responsible for indexing gene expression, profiling gene expression, identifying SNPs and generation of protein profiles from a wide variety of tissues representative of various anatomical regions as well as developmental and pathological stages in the cardiovascular system. This information resource emphasizes on cardiovascular disease and should aid in the discovery of disease causing genes, diagnostic and prognostic markers, drug targets, protein therapeutics and improved therapeutic strategies for cardiovascular disease.

Our laboratory is the curator of a genome-based resource for molecular cardiovascular medicine consisting of over 52,000 ESTs generated from nine heart and artery libraries, representing different developmental stages and disease states (Liew et al 1994, Hwang et al 1997, Dempsey et al 2000).

This comprehensive catalogue of cardiac and hematopoietic genes is an unmined molecular resource for microarray analysis and a genetic gold mine for the discovery of genes that may play a role in cardiovascular disorders. In order to exploit this raw data, we propose to develop cDNA microarrays consisting of known and novel sequence-tagged genes. The arrayed clones provide an excellent substrate for expression profiling of cardiovascular disease, for example heart failure or ischemic heart disease, leading the potential discovery of diagnostic as well as prognostic markers.

In order to accomplish the goals of the center, several cutting edge technologies are being employed.

The human cardiovascular research component of our labs.

One of the most efficient and effective strategies for the identification genes is the Expressed Sequence Tag (EST) approach. In this approach, randomly selected cDNA clones are subjected to automated sequencing (PCR or plasmid templates) to generate a partial sequence from either the 5’- or 3’-end termed an EST. This method allows for large-scale gene tagging and indexing from any tissue- or cell-type of interest. A comprehensive cardiovascular gene index could be developed using a variety of cardiovascular tissues representing different anatomical, developmental and pathological states.

Comparing transcript profiles between different development or disease states is a powerful way to gain insight into the genetic changes underlying these events. This is especially important when looking at complex systems, such as in development or disease (e.g. hypertension or atherosclerosis). There are several unique approaches to this problem, several of which are:

a) EST profile Comparison– After the production of a significant number of ESTs from 2 or more libraries, the frequencies of ESTs can be compared to identify those genes which are differentially expressed. However, normalized or subtracted cDNA libraries cannot be used for this and this method is most effective for finding large differences in expression.

b) cDNA Microarray Hybridization– The recent introduction of the cDNA microarray, a technology capable of analyzing the expression of thousands of genes simultaneously in a single experiment, may provide one of the best ways to delineate gene expression patterns. In the cDNA microarray, cDNA clones are spotted onto a glass slide matrix and hybridized with fluorescently labeled cDNA probes derived from total RNA pools of test and reference cells or tissues. The signal intensity for each probe is quantified and any differences between the two samples becomes readily apparent. Thus, the genetic changes underlying the phenotype of study can be identified at the level of a single gene.

c) Identification of Single Nucleotide Polymorphisms– SNPs are single-base heritable variations in the genome which occur once in approximately 1000 bases in the human genome and occur at a frequency of >1% in the human population. SNPs provide an important genetic resource useful for disease gene discovery. including the identification of disease susceptible genes. SNPs can be identified through comparison of EST sequences, DNA hybridization strategies and direct sequencing of genomic DNA. The generation of a SNP database for genes expressed in the cardiovascular system will provide a valuable resource to aid in disease gene discovery.

d) Quantitative determination of expressed genes– the up- and down- regulated genes are crucial to the phenotypic expression of any given cell. The frequency of gene expressed in development or disease state can be obtained from an EST approach using cDNA libraries as well as its intensity detected using microarrays. Such results can be verified through RT-PCR analysis from the tissue samples. A high through-put analysis of 96 samples can be performed by real-time PCR analyses.

Using our 10,000 element “CardioChip”, we elucidated over 100 differentially expressed genes in end-stage heart failure resulting from dilated cardiomyopathy. The results were published in

Am J Pathol. 2002 June; 160(6): 2035–2043.

Global Gene Expression Profiling of End-Stage Dilated Cardiomyopathy Using a Human Cardiovascular-Based cDNA Microarray

J. David Barrans,^*† Paul D. Allen,^‡ Dimitrios Stamatiou,^* Victor J. Dzau,^* and Choong-Chin Liew^*†

From Cardiovascular Genome Unit^*, the Department of Medicine, and the Department of Anesthesiology,^‡Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts; and the Department of Laboratory Medicine and Pathobiology,^†University of Toronto, Toronto, Ontario, Canada

Abstract

To obtain a genomic portrait of heart failure derived from end-stage dilated cardiomyopathy (DCM), we explored expression analysis using the CardioChip, a nonredundant 10,848-element human cardiovascular-based expressed sequence tag glass slide cDNA microarray constructed in-house. RNA was extracted from the left ventricular free wall of seven patients undergoing transplantation, and five nonfailing heart samples. Cy3- and Cy5-labeled (and reverse dye-labeled) cDNA probes were synthesized from individual diseased or nonfailing adult heart RNA, and hybridized to the array. More than 100 transcripts were consistently differentially expressed in DCM >1.5-fold (versus pooled nonfailing heart,P < 0.05). Atrial natriuretic peptide was found to be up-regulated in DCM (19-fold compared to nonfailing, P < 0.05), as well as numerous sarcomeric and cytoskeletal proteins (eg, cardiac troponin, tropomyosin), stress response proteins (eg, HSP 40, HSP 70), and transcription/translation regulators (eg, CCAAT box binding factor, eIF-1AY). Down-regulation was most prominently observed with cell-signaling channels and mediators, particularly those involved in Ca²⁺ pathways (Ca²⁺/calmodulin-dependent kinase, inositol 1,4,5-trisphosphate receptor, SERCA). Most intriguing was the co-expression of several novel, cardiac-enriched expressed sequence tags. Quantitative real-time reverse transcriptase-polymerase chain reaction of a selection of these clones verified expression. Our study provides a preliminary molecular profile of DCM using the largest human heart-specific cDNA microarray to date.

Dilated cardiomyopathy (DCM) is characterized clinically by left ventricular dilatation, wall thinning, and homogeneous dysfunction of the myocardium leading to congestive heart failure. Genetically, DCM seems to evolve through primary mutations in the genes of the sarcomeric proteins.¹ However, recent evidence suggests that, despite distinct pathways leading to divergent endpoint phenotypes of each disease, there may exist some overlapping genetic modifiers leading to a conversion of one to the other.² How this occurs is under question; to understand this, a better knowledge of the molecular pathways and intermediary regulators is required.

Global analysis of gene expression has proven to be a fruitful means of examining the overall molecular portrait of a particular event as well as seeking out novel candidate transcripts that may play a role in formulating the phenotype or genotype of interest. By using this strategy, multiple genes and pathways in complex disorders can be visualized simultaneously, allowing for a feasible platform from which to investigate new and interesting genes. Using expressed sequence tag technology, our laboratory has generated a compendium of genes expressed in the human cardiovascular system, with the ultimate goal of assembling the intricacies of development and of disease, particularly the pathways leading to heart failure.³ Through a computer-based in silico strategy, we have been able to identify—in a large scale—both known and previously unsuspected genetic modulators contributing to the growth of the myocardium from fetal through adult, and from normal to a perturbed hypertrophic phenotype. In contrast a gene-by-gene approach in elucidating the genes and mechanisms involved is time-consuming and cumbersome.

Recently, microarray technology has been used as a means of large-scale screening of vast numbers of genes—if not whole genomes—that possess differential expression in two distinct conditions. Although new and exciting developments have arisen in such fields as cancer⁴ and yeast,⁵ advances in understanding the complexity of cardiovascular disease,⁶ specifically DCM, have been limited. One recent study examined gene expression in two failing hearts using oligo-based arrays.⁷ Although the GeneChip^® (Affymetrix, Santa Clara, CA) offers a carefully controlled systematic method of analysis, its current lack of user flexibility in its design hinders novel gene discovery currently available in tissue-specific arrays. Our laboratory has taken advantage of our vast previously acquired resources and has constructed what we believe to be the first ever custom-made cardiovascular-based cDNA microarray, which we term the “CardioChip.”⁸ Its practicality and flexibility has allowed us to conceptualize the molecular events surrounding end-stage heart failure.

This report describes the most informative cDNA microarray-based analysis of end-stage heart failure derived from DCM currently available. Although we believe we have effectively demonstrated reproducibility and reliability of our technology (both for the entire array and for a selection of genes located on it), a larger n from our population would enhance the validity of our conclusions. Certainly, there exists no homogeneous heart failure genotype, especially among only seven DCM patients. Nonetheless, we have demonstrated a common expression pattern among our set of samples, from both microarray and QRT-PCR analysis. We are also limited by the genes (both in number and identity) present on this array. Although we are currently unable to spot every gene and gene cluster on our CardioChip, we have tried to draw from a diverse assortment of genes and gene pathways, both known and unknown. It must be emphasized that this investigation is not exhaustive; by no means does it attempt to fully characterize the molecular basis of heart failure. Its intention is to provide a preliminary portrait of global gene expression in complex cardiovascular disease using cDNA microarray and QRT-PCR technology, and to highlight the effectiveness of our ever-evolving platform for gene discovery. With even more patient samples and a CardioChip toward completeness, we will be in a better position to reap the important benefits from this initial work and expand our body of knowledge.

Am J Pathol. 2002 June; 160(6): 2035–2043.

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Liew CC, Takihara KY, Jandreski M, Liew J, Sole MJ. Structure and expression of human b-myosin heavy chain gene. In: Carraro U, editor. Sarcomeric and Non-sarcomeric Muscles: Basic and Applied Research Prospects for the 90s. Padova, Italy: Unipress
Padova; 1988. P 11-17.

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Dempsey A, Liew CC. Genes involved in normal cardiac development. In: Sheridan DJ, editor. Left Ventricular Hypertrophy. London: Churchill Communications Europe Ltd; 1998: p. 61-70.

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Global Gene Expression Profiling of End-Stage Dilated Cardiomyopathy Using a Human Cardiovascular-Based cDNA Microarray

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Maren Leifheit-Nestler¹², Nana-Maria Wagner¹³, Rajinikanth Gogiraju¹,Michael Didié¹⁴, Stavros Konstantinides¹⁵, Gerd Hasenfuss¹ and Katrin Schäfer¹^*