SummaryThick-filament sarcomere mutations are a common cause of hypertrophic cardiomyopathy (HCM), a disorder of heart muscle thickening associated with sudden cardiac death and heart failure, with unclear mechanisms. We engineered four isogenic induced pluripotent stem cell (iPSC) models of β-myosin heavy chain and myosin-binding protein C3 mutations, and studied iPSC-derived cardiomyocytes in cardiac microtissue assays that resemble cardiac architecture and biomechanics. All HCM mutations resulted in hypercontractility with prolonged relaxation kinetics in proportion to mutation pathogenicity, but not changes in calcium handling. RNA sequencing and expression studies of HCM models identified p53 activation, oxidative stress, and cytotoxicity induced by metabolic stress that can be reversed by p53 genetic ablation. Our findings implicate hypercontractility as a direct consequence of thick-filament mutations, irrespective of mutation localization, and the p53 pathway as a molecular marker of contraction stress and candidate therapeutic target for HCM patients.

Summary AMP-activated protein kinase (AMPK) is a metabolic enzyme that can be activated by nutrient stress or genetic mutations. Missense mutations in the regulatory subunit, PRKAG2, activate AMPK and cause left ventricular hypertrophy, glycogen accumulation and ventricular pre-excitation. Using human iPS cell models combined with three-dimensional cardiac microtissues, we show that activating PRKAG2 mutations increase microtissue twitch force by enhancing myocyte survival. Integrating RNA sequencing with metabolomics, PRKAG2 mutations that activate AMPK remodeled global metabolism by regulating RNA transcripts to favor glycogen storage and oxidative metabolism instead of glycolysis. Like patients with PRKAG2 cardiomyopathy, iPS cell and mouse models are protected from cardiac fibrosis, and we define a crosstalk between AMPK and post-transcriptional regulation of TGFβ isoform signaling that has implications in fibrotic forms of cardiomyopathy. Our results establish critical connections between metabolic sensing, myocyte survival and TGFβ signaling.

18860-837-2048 (t) | travis.hinson@jax.org 19 20 Thick filament sarcomere mutations are the most common cause of hypertrophic cardiomyopathy (HCM), 21 a disorder of heart muscle thickening associated with sudden cardiac death and heart failure, with unclear 22 mechanisms. We engineered an isogenic panel of four human HCM induced pluripotent stem cell (iPSc) 23 models using CRISPR/Cas9, and studied iPSc-derived cardiomyocytes (iCMs) in 3-dimensional cardiac 24 microtissue (CMT) assays that resemble in vivo cardiac architecture and biomechanics. HCM mutations 25 result in hypercontractility in association with prolonged relaxation kinetics in proportion to mutation 26 pathogenicity but not calcium dysregulation. RNA sequencing and protein expression studies identified 27 that HCM mutations result in p53 activation secondary to increased oxidative stress, which results in 28 increased cytotoxicity that can be reversed by p53 genetic ablation. Our findings implicate 29 hypercontractility as an early consequence of thick filament mutations, and the p53 pathway as a 30 molecular marker and candidate therapeutic target for thick filament HCM. 31 32 35 hypertrophy (LVH) with preserved systolic contractile function 2 . In young athletes, HCM manifests as a 36 common cause of sudden cardiac death; while in adults, HCM is associated with heart failure that may 37 progress to require cardiac transplantation 3 . Over the last few decades, the genetic basis of HCM has been 38 demonstrated by inheritance of autosomal dominant mutations in components of the force-producing 39 sarcomere 4 . About two-thirds of HCM patients harbor heterozygous mutations in one of two sarcomere 40 genes: b-myosin heavy chain (MHC-b is encoded by MYH7) or myosin-binding protein C (MYBPC3) 4 . 41 Along with titin, MHC-b and MYBPC3 are located in the thick filament where ATP hydrolysis by MHC-42b is coupled to force generation through interactions with the actin-rich thin filament ( fig.1A). A 43 prevailing model suggests that HCM mutations alter cardiac force generation through dysregulation of 44 calcium handling 5, 6 . Whether MYBPC3 and MYH7 mutations result in HCM by shared or heterogeneous 45 mechanisms also remains unclear. 46 Recent functional studies of thick filament HCM mutations in reconstituted sarcomere and 47 motility assays 7, 8 . Equally puzzling, contractile studies of single cardiomyocytes from MYH6-R403Q +/-1 mouse models, which recapitulate LVH and fibrosis in vivo 9 , have produced similarly conflicting results 2 for the identical mouse model and strain 10, 11 . Human patient-specific induced pluripotent stem cell (iPSc) 3 HCM models of MYH7-R663H (arginine 663 substituted with histidine) have recapitulated some features 4 of HCM including cellular enlargement and altered calcium handling 6 , but mechanical phenotypes of 5 1 expertise in confocal microscopy. We also thank Bo Reese for RNA sequencing technical expertise. We 2 thank Samantha Harris for her generous contribution of MYBPC3 antibody.3 4 Sources of Funding:5