1 Actinins are actin cross-linkers that are expressed in nearly all cells and harbor mutations in 2 heritable diseases. We deployed proximity-dependent biotinylation to identify actinin proximity 3 proteins and RNA transcripts in human cardiomyocytes to reveal new functions of the actin 4 cytoskeleton. We identified 285 proximity protein partners including unexpected effectors of 5 RNA-binding and metabolism, and interrogated dynamic partners using a sarcomere assembly 6 model. Mammalian two-hybrid studies established that IGF2BP2, an RNA-binding protein 7 associated with type 2 diabetes, interacted with the rod domain of actinin through its K 8 Homology domain. This interaction was necessary for actinin localization and translation of 9 electron transport chain transcripts, and oxidative phosphorylation. Diminished IGF2BP2 in 10 cardiac microtissues impaired contractile function in accord with diminished metabolic functions. 11 This actinin proximity protein and RNA study expands our functional knowledge of the actin 12 cytoskeleton, and uncovers new modes of metabolic regulation through interactions with RNA-13 binding proteins. 14 15 Main 16 Actinin proteins are ubiquitous spectrin family members that cross-link actin, and are important 17 for a myriad of cellular functions including cell adhesion, migration, contraction, and signaling 1 . 18 The four human actinin isoforms have distinct expression profiles (non-muscle ACTN1 and 19 ACTN4; cardiac muscle ACTN2; and skeletal muscle ACTN3), but share homologous amino 20 acid sequences that are organized into three structural domains-an actin-binding (AB) domain 21 composed of two calponin-homology domains, a central rod domain containing four spectrin-like 22 repeats (SR), and a calmodulin-like domain (CaM) containing two EF hand-like motifs 2 . While it 23is thought that actinin evolved initially to regulate the early eukaryotic actin-based cytoskeleton 3 , 24 it has acquired more elaborate functions in vertebrates, including a mechanical role in the 25 sarcomere, a specialized contractile system required for striated muscle function 3, 4 . Moreover, 21 microtissues with heart failure-associated mutations, twitch force was unaltered by Actinin-BirA* 22 expression (extended data Fig. 1b). We confirmed appropriate localization of Actinin-BirA* to the 23 Z-disk by both co-localization immunofluorescence (Fig.1d) and HA-immunoprecipitation assays 24 (Fig.1e). Actinin-BirA* interacted with the Z-disk protein titin-cap (TCAP), but not the sarcomere 25 M-line protein myomesin [22][23][24][25] , which supported appropriate localization. With the knowledge that 26 Confidential Manuscript 6 Actinin-BirA* functions similarly to unmodified actinin, we next optimized biotin supplementation

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