Familial hypertrophic cardiomyopathy (HCM) is the leading cause of sudden cardiac death in young people, including trained athletes, and is the most common inherited heart defect. In this proposal, we will generate human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from patients with HCM. The specific aims are as follow:
Specific Aim 1: Generate iPSCs from patients with HCM and healthy controls.
Specific Aim 2: Determine the extent of disease by performing molecular and functional analyses of hiPSC-CMs.
Specific Aim 3: Rescue the molecular and functional phenotypes using zinc finger nuclease (ZFN) technology.
Over the past year, we have now derived iPSCs from a 10-patient family cohort with the MYH7 mutation. Established iPSC lines from all subjects were differentiated into cardiomyocyte lineages (iPSC-CMs) using standard 3D EB differentiation protocols. We found hypertrophic iPSC-CMs exhibited features of HCM such as cellular enlargement and multi-nucleation beginning in the sixth week following induction of cardiac differentiation. We also found hypertrophic iPSC-CMs demonstrated other hallmarks of HCM including expression of atrial natriuretic factor (ANF), elevation of β-myosin/α-myosin ratio, calcineurin activation, and nuclear translocation of nuclear factor of activated T-cells (NFAT) as detected by immunostaining. Blockade of calcineurin-NFAT interaction in HCM iPSC-CMs by cyclosporin A (CsA) and FK506 reduced hypertrophy by over 40%. In the absence of inhibition, NFAT-activated mediators of hypertrophy such as GATA4 and MEF2C were found to be significantly upregulated in HCM iPSC-CMs beginning day 40 post-induction of cardiac differentiation, but not prior to this point. Taken together, these results indicate that calcineurin-NFAT signaling plays a central role in the development of the HCM phenotype as caused by the Arg663His mutation.
Reporting Period:
Year 2
Familial hypertrophic cardiomyopathy (HCM) is the leading cause of sudden cardiac death in young people, including trained athletes, and is the most common
inherited heart defect. In this proposal, we will generate and characterize human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from patients with HCM. The
specific aims are as follow:
Specific Aim 1: Generate iPSCs from patients with HCM and healthy controls.
Specific Aim 2: Determine the extent of disease by performing molecular and functional analyses of hiPSC-CMs.
Specific Aim 3: Rescue the molecular and functional phenotypes using zinc finger nuclease (ZFN) technology.
Over the past year, we have characterized the pathological phenotypes from iPSCs derived from a 10-patient family cohort with the MYH7 mutation.
We've differentiated all stablished iPSC lines from all subjects into cardiomyocyte using a modified protocol from that published by Palacek in PNAS 2011. This protocol increased the yield of cardiomyocytes significantly to consistently greater than 70% beating cardiomyocytes. We then tested the electrophysiological properties of iPSC-CMs from control and patients with HCM and found that both control and patient iPSC-CM display atrial, ventricular and nodal-like electrical waveforms by whole cell patch clamping. However, by day 30, a large subfraction (~40%) of the HCM iPSC-CM exhibit arrhythmic waveforms including delayed after-depolarizations (DADs) compared with control (~5.1%). In addition we found that treatment of HCM hiPSC-CM with positive inotropic agents (beta-adrenergic agonist - isoproterenal) for 5 days caused an earlier increase in cell size by 1.7 fold as compared to controls and significant increase in irregular calcium transients. Furthermore, we found that HCM iPSC-CMs exhibited frequent arrhythmia due to their increased intracellular calcium level by 30% at baseline. These HCM iPSC-CM also exhibited decreased calcium release by the sarcoplasmic reticulum. These findings emphasize the role of irregular calcium recycling in the pathogenesis of HCM. To confirm that the regulation of myocyte calcium is the key to HCM pathogenesis, we treated several lines from multiple HCM patients with calcium channel blocker (verapamil/diltiazem) and found that this treatment significantly ameliorated all aspects of the HCM phenotype including myocyte hypertrophy, calcium handling abnormalities, and arrhythmia. These finding supports the use of calcium channel blockers in patients with HCM and encourages further clinical studies in HCM patients using these agents.
Reporting Period:
Year 3
Familial hypertrophic cardiomyopathy (HCM) is the leading cause of sudden cardiac death in young people, including trained athletes, and is the most common
inherited heart defect. In this proposal, we will generate human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from patients with HCM. The
specific aims are as follow:
Specific Aim 1: Generate iPSCs from patients with HCM and healthy controls.
Specific Aim 2: Determine the extent of disease by performing molecular and functional analyses of hiPSC-CMs.
Specific Aim 3: Rescue the molecular and functional phenotypes using zinc finger nuclease (ZFN) technology.
Over the past year, we have characterized iPSC-CMs from a 10-patient family cohort with the MYH7 mutation using standard 3D EB differentiation protocols.
We found normal and hypertrophic iPSC-CMs were predictive as in vitro model for arrhythmia screening using microelectroarrays and single cell patch-clamping
analysis. For example, we found that administration of catecholamine drug norepinephrine causes the formation of torsade de point which is a lethan arrhythmia.
This recapitulates the phenotype in patients with HCM receiving catecholamine drugs. We also found increase in torsade formation when the iPSC-CMs are treated
with hERG blockers that are also known to cause increases in arrhythmia in HCM patients. We believe the use of hiPSC-CM from healthy individuals and patients with
genetic heart disease can help predict the potential arrhythmic risk in existing or new drug agents that are undergoing FDA evaluation.
We have also generated HCM mutations in lines of normal iPSC to determine whether these mutant lines will exhibit HCM phenotype. This would satisfy the Koch's postulate
with regards to the role of the mutant DNA sequence on HCM manifestation. We found, using TALEN and piggyBac transposon technologies that genome edited can be generated
to carry R663H mutation in the MYH7 gene and that these genome edited iPSC-CM recapitulated the HCM phenotype associated with the R663H mutation such as sarcomere
disassembly and intracellular calcium abnormalities as well as contractile arrhythmias. We have also corrected mutant HCM human iPSC from patients with MYH7 R663H mutation
and show that these corrected iPSC-CM exhibit normal sarcomeric phenotype with smaller cell size and reduced calcium transient irregularities.
Grant Application Details
Application Title:
Elucidating Molecular Basis of Hypertrophic Cardiomyopathy with Human Induced Pluripotent Stem Cells
Public Abstract:
Familial hypertrophic cardiomyopathy (HCM) is the leading cause of sudden cardiac death in young people, including trained athletes, and is the most common inherited heart defect. Until now, studies in humans with HCM have been limited by a variety of factors, including variable environmental stimuli which may differ between individuals (e.g., diet, exercise, and lifestyle), the relative difficulty in obtaining human cardiac samples, and inadequate methods of maintaining human heart tissue in cell culture systems. Cellular reprogramming methods that enable derivation of human induced pluripotent stem cells (hiPSCs) from adult cells, which can then be differentiated into cardiomyocytes (hiPSC-CMs), are a revolutionary tool for creating disease-specific cell lines that may lead to effective targeted therapies.
In this proposal, we will derive hiPSC-CMs from patients with HCM and healthy controls, then perform a battery of functional and molecular tests to determine the presence of cardiomyopathic disease and associated abnormal molecular programs. With these preliminary studies, we believe hiPSC-CMs with HCM phenotype will dramatically enhance the ability to perform future high-throughput drug screens, evaluate gene and cell therapies, and assess novel electrophysiologic interventions for potential new therapies of HCM. Because HCM is not a rare disease but rather the leading cause of inherited heart defects, we believe the findings here should have broad clinical and scientific impact toward understanding the molecular and cellular basis of HCM.
Statement of Benefit to California:
Familial hypertrophic cardiomyopathy (HCM) is the leading cause of sudden cardiac death in young people and is the most common inherited heart defect. In this study, we will generate hiPSC-derived cardiomyocytes from patients with HCM, then perform a number of functional, molecular, bioinformatic, and imaging analyses to determine the extent and nature of cardiomyopathic disease. We believe hiPSC-CMs with HCM phenotype will dramatically enhance the ability to perform future high-throughput drug screens, evaluate gene and cell therapies, and assess electrophysiologic interventions for potential novel therapies of HCM. The experiments outlined are pertinent and central to the overall mission of CIRM, which seeks to explore the use of stem cell platforms to yield novel mechanistic insights into the molecular and cellular basis of disease. Because HCM is not an orphan disease, but rather the leading cause of sudden cardiac death in young people, we believe the research findings will benefit the state of California and its citizens.
