Risk Alleles in Protein Quality Control Genes as Modifiers of Hypertrophic Cardiomyopathy - Patients with hypertrophic cardiomyopathy (HCM) experience a high symptomatic burden, heart failure and lethal arrhythmias. While the HCM has been recognized as a disease of the sarcomere for >30 years, the heterogeneity of disease expression is substantial and not fully explained by these primary genetic variants. Recent genome wide association studies (GWAS) for HCM have uncovered common genetic variants as risk alleles for HCM. Many of these are conversely protective alleles for dilated cardiomyopathy (DCM), supporting the concept of polygenic regulation of contractile function. However, the mechanisms by which they exert their effects are unknown. Here, we focus on a subset of GWAS loci that strongly implicate protein quality control as a modifier of HCM. Three of the top 10 loci associated with HCM reside within or near genes that encode proteins of the heat shock protein 70 (HSP70) chaperone network: BAG3, HSPB7, and DNAJC18. Prior studies, and our own preliminary data, indicate that these cochaperones are involved in sarcomeric protein maintenance. We hypothesize that risk alleles in BAG3, HSPB7 and DNAJC18 are strong genetic modifiers of sarcomeric HCM, acting through distinct molecular mechanisms to coordinate sarcomere protein dynamics and cardiac contractility. Our approach involves focused human genetic association studies and functional studies in human model systems. In Aim 1, we will determine the impact of risk alleles in BAG3, HSPB7, and DNAJC18 on disease expression and clinical outcomes in patients with HCM, stratified by sarcomere genotype and genetic similarity. Combining cases and controls from several sources, including the Sarcomeric Human Cardiomyopathy Registry (SHaRe), Penn Medicine BioBank, TOPMED and All of Us, we will perform a case-control study to determine associations of BAG3, DNAJC18 and HSPB7 risk alleles with HCM, a case-only analysis to determine association with clinical outcomes, and additive and epistasis modeling to determine interaction effects. In Aim 2, the role of each co-chaperone in regulating sarcomeric protein dynamics and contractility will be explored through genetic knockdown experiments. We will determine effects of each variant on transcript/protein abundance and splice isoforms in cardiomyopathic human hearts. In parallel, we will perform genome editing of each variant in human stem cell-derived cardiac myocytes (hiPSC-CMs) and measure cellular phenotypes, including contractility. For the BAG3 coding variant, Cys151Arg, we will use proximity labeling to determine how this variant affects BAG3 localization and its interactome. Finally, we will use patient-derived hiPSC-CMs that have reduced levels of MyBP-C to determine whether chaperone manipulation stabilizes wild-type MyBP-C to rescue haploinsufficiency. Successful completion of these aims will impact the field by gaining an understanding of the clinical and biological relevance of HSP70 risk alleles to HCM. Our team is composed of highly experienced investigators in cardiomyopathy, cardiovascular genetics, and bioinformatics and is well poised to achieve these goals.