Dissecting the mechanisms of how MYH7 S2 mutations lead to genetic hypertrophic cardiomyopathy

Familial hypertrophic cardiomyopathy (HCM) is a genetic cardiomyopathy affecting 1 in 500 US adults.1,2 Mutation in myosin heavy chain seven (MYH7), a sarcomeric thick filament protein, accounts for 20-40% of HCM cases.3,4 However, current understanding lacks a generalizable mechanism through which specific MYH7

variants result in HCM, and no specific disease-modifying therapy exists.5 The MYH7 S2 domain, host to numerous identified pathogenic variants, interacts with the C1C2 domain of cardiac myosin binding protein C (cMyBP-C).6 We have generated hiPSC-derived cardiomyocytes (hiPSC-CMs) from an HCM patient with MYH7

variant E848G. Our preliminary data suggest E848G disrupts the S2/C1C2 interaction and reduces MYH7 abundance. Thus, my central hypothesis is pathogenic MYH7 S2 variants that disrupt S2/C1C2 interaction and reduce MYH7 protein abundance result in contractile function; restoration of S2/C1C2 interaction

(Aim 1) or MYH7 abundance (Aim 2) may improve contractile function. My specific aims are to: (1) demonstrate disruption of MYH7 S2/C1C2 interaction results in contractile dysfunction; and (2) elucidate the mechanism of E848G-induced loss of MYH7 protein abundance and test whether restoring normal MYH7 protein

abundance can rescue contractile function. I will generate pathogenic MYH7 S2 variants with CRISPR/Cas9 for use in a mammalian two-hybrid approach to quantify dysfunction of the S2/C1C2 interaction. I will restore S2/C1C2 interaction disrupted by pathogenic MYH7 S2 variant by targeted mutation of the complementary amino

acid in C1C2, ideally recovering contractile function in corresponding engineered heart tissues (EHTs). These findings will be applied to MYH7 S2 variants of unknown significance to test for S2/C1C2 integrity and verify in hiPSC-CMs. This aim will establish a novel rapid method to functionally reclassify MYH7 S2 variants of unknown

significance. In the second aim, I will characterize hypocontractility in EHTs derived from MYH7-E848G hiPSC- CMs and correlate with observed loss in MYH7 protein abundance. I will use fluorescent recovery after photobleaching (FRAP) to capture the dynamics of MYH7 cycling in sarcomeres with and without the E848G

variant. Overexpression of MYH7 and silencing of the mutant allele will test the relationship between MYH7 abundance and contractile function. These findings will then be corroborated in other MYH7 pathogenic variants. This aim will establish a generalizable mechanism through which MYH7 variants lose MYH7 protein abundance

and consequently lose contractile function. In sum, these experiments will enable diagnostic tools for predicting pathogenicity of MYH7 S2 variants and therapeutic approaches to address contractile function. This project will take place in the highly supportive and collaborative environment of the University of Washington Department of

Medicine. With the mentorship of my Sponsor and Co-Sponsor (Dr. Daniel Yang and Dr. Charles E. Murry, respectively), this project will provide the training required for me to realize my goal of establishing an independent research laboratory at the crossroads of cellular biology, tissue engineering, and clinical application.