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.