ABSTRACT: Left ventricular non-compaction (LVNC), a rare but increasingly prevalent cause of cardiomyopathy stemming from developmental arrest of myocardial compaction, was identified as a primarily genetic, independent cardiomyopathy by the AHA in 2006 following debate due to its frequent association with variable degrees of myocardial dysfunction and other congenital heart defects (CHD). Symptom heterogeneity has likely left many cases undiagnosed, giving a lower estimation of its real impact as a cardiomyopathy. Congenital LVNC can progress in severity throughout life contributing to decompensated heart failure and sudden cardiac death. Little is known about the molecular mechanism(s) that lead to LVNC, but mutations in MYH7 (encoding human beta- cardiac myosin) have been implicated as a putative genetic cause. Human beta-cardiac myosin (M2β) mutations are known molecular triggers for heritable cardiomyopathies, typically impacting the motor protein’s intrinsic motor properties and/or auto-inhibited state. Studies of cardiomyopathy mutations in M2β have demonstrated that hypertrophic cardiomyopathy (HCM) is triggered by mutations that cause hyper-contraction and dilated cardiomyopathy (DCM) is triggered by mutations that cause hypo-contraction. However, very few studies have examined LVNC mutations and thus it is unclear how it falls on this genotype-phenotype spectrum. Examining LVNC cardiac myosin mutations would enlighten us about the unique etiology and reveal important information about myosin structure and function. We hypothesize that depressed monomeric ensemble force and/or an increase in autoinhibited state stabilization could trigger severe hypo-contraction and tissue level tension/force imbalances, leading to excessive trabeculation in LVNC. We conclude that additional biophysical LVNC M2β mutant studies both in purified protein and cellular systems are required to uncover common allosteric pathways altered in LVNC. Our goal is to investigate the impact of clinically identified MYH7 LVNC mutations M362R and L655M on protein structure-function, mechanochemistry, and ultimately pathogenic myocardial remodeling. M362R is in the loop 4 region of the motor domain known to play an important role in head-head interactions, tropomyosin positioning, and actin binding. L655M may disrupt a critical allosteric pathway leading to actin-activation of the power stroke and thus impair the transition into the force generating states. This proposal will characterize two LVNC mutations selected based on the isolation of LVNC from other forms of heritable cardiomyopathy and on their respective locations within myosin known to be important for performance. We will utilize transient kinetic, novel applications of FRET biosensors and analytical ultracentrifugation, and hiPSC-CM derived myofibril-based techniques to elucidate how these mutations impair myosin function leading to LVNC pathogenic remodeling of the heart.