Abstract 16773: Ablation of Cardiac Myosin Binding Protein-C in Human iPSC- Engineered Cardiac Tissue Model Causes Increased Calcium Sensitivity and Accelerated Contractile Kinetics

Abstract

Hypertrophic cardiomyopathy (HCM) is an inherited cardiac disease that can cause sudden cardiac arrest, diastolic dysfunction and heart failure. Mutations in cardiac myosin binding protein-C (cMyBP-C), a regulator of contractility, are among the most prevalent causes of HCM. cMyBP-C truncation mutations may affect as many as 1 in 1,600 Americans. Though murine models of cMyBP-C ablation have aided in understanding the role of cMyBP-C in health and disease, they are limited by differences in cardiac physiology between mice and humans. iPSC technology now allows researchers to study the effect of HCM mutations in iPSC-derived cardiomyocytes from HCM patients and controls, however differences in genetic background between patients and controls and clonal variation remain confounders. We hypothesized that cMyBP-C ablation in human cardiomyocytes would primarily alter cardiac contractility. CRISPR/Cas9 was used to introduce frame shift mutations in cMyBP-C in the well characterized DF19-9-11T human iPSC line. Human iPSCs were differentiated into cardiomyocytes and then cast into an integrated 3D engineered cardiac tissue (ECT) model for functional testing against non-targeted isogenic controls. Homozygous cMyBP-C ablation significantly accelerated contraction (CT 100 = 219±3 vs. 235±5ms; p = 0.02) and early relaxation (RT 50 = 144±2 vs. 167±4ms; p <0.001), while late relaxation was slowed (RT 50-90 = 127±2 vs. 112±2ms; p <0.001; n=6 for each group). These accelerated contractile kinetics were accompanied by a significantly blunted response to adrenergic stimulation and an increase in Ca 2+ sensitivity. Heterozygous cMyBP-C ablation resulted in acceleration in relaxation kinetics (RT 50 = 148±7 vs. 167±4ms; p = 0.04 and RT 50-90 = 96±4 vs. 112±2ms; p = 0.002), without significantly affecting the rate of contraction. The accelerated kinetics of contraction and early relaxation in cMyBP-C null ECT provides important human evidence that cMyBP-C acts as a brake on cardiac contractility. These data provide strong evidence that the human model system can be used to study the function of cMyBP-C and provide a platform to test therapies that may rectify the contractile defects caused by cMyBP-C haploinsufficiency.