Disease in a Dish Crispr-Edited HIPSC-CMS to Study the Cellular and Subcellular Mechanisms of HCM

Abstract

Genetic sequencing enables the pinpointing of specific genetic mutations correlated with disease. However, such associations do not describe mechanistic relationships between specific mutations and clinical phenotypes. To address this, we use human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as a model system to probe how mutations in β-myosin heavy chain (MHC) lead to disease. β-MHC, a major biochemical motor protein in heart muscle, is a common site of mutations implicated in hypertrophic cardiomyopathy Utilizing CRISPR editing of the MYH7 gene to introduce the G256E point mutation into the converter domain region of myosin, we have studied the effects that molecular level changes (nm) in protein structure have at the cellular level through simultaneous contractility and calcium handling measurements in hiPSC-CMs. These hiPSC-CMs were subjected to glucose starvation to mature and enrich the population of cardiomyocytes, and cultured on Matrigel-stamped 1500 μm2 island patterns to achieve single cells. We have also analyzed differences at the individual contractile organelle level through precise measures of myofibril contraction and relaxation kinetics using a custom-built apparatus. Our experimental results have been supplemented with molecular dynamics simulations of wild type and mutant β-MHC. Preliminary data from molecular dynamics simulations of β-myosin structure, suggests G256E alters the distributions of intra-molecular contacts made by loop 1 and distorts β-MHC structure near the nucleotide binding pocket as well as sites forming the interacting heads motif. Synthesis of computational, mechanical and structural studies will determine the mechanism of contractile dysfunction associated with β-MHC G256E. Development of these platforms for molecular and cellular level analysis serve as a template for future studies of other myofilament proteins and mutations to connect disease genotype with phenotype.