Abstract 916: Evidence of Cardiac Myosin Dephosphorylation by Two Distinct Pools of Myosin Light Chain Phosphatases

Abstract

Muscle myosins are hexameric proteins comprised of two each of the heavy chain, essential light chain, and regulatory light chain (RLC). In normal beating hearts, cardiac myosin RLC is phosphorylated, and the extents maintained at ~0.45 mol phosphate/mol RLC by the balanced activities of cardiac-specific myosin light chain kinases (cMLCK) and phosphatases (cMLCP). Hearts in failure have decreased cMLCK expression and RLC phosphorylation, indicating maintenance of near half-maximal RLC phosphorylation levels is necessary for normal function. However, how cMLCK and cMLCP activities are regulated is unclear. MLCP has been biochemically defined as a trimeric protein comprised of a regulatory myosin target subunit (MYPT1 in smooth muscles, and MYPT2 in striated muscles), a catalytic subunit PP1cβ, and a small subunit M20. Interrogations into the specificity and regulatory activity of smooth muscle MLCP using conditional MYPT1 knockout animals have provided evidence of MYPT1-independent RLC phosphatase activity, which contradicts long-standing dogma. Extending upon recent studies of PP1cβ knockout in smooth muscles, we knocked out PP1cβ from the cardiac muscle to test the hypothesis that a distinct MYPT-independent pool of PP1cβ activity directly contributes to cRLC dephosphorylation. Two weeks post gene-ablation, PP1cβ protein was reduced by >60%, and cRLC phosphorylation in beating hearts was increased to 0.6 mol phosphate/mol cRLC, without significant changes in MYPT1 or MYPT2 expression. Additionally, we measured MYPT-dependent and MYPT-independent PP1cβ phosphatase activities toward cRLC dephosphorylation in mouse cardiac myofibrils. These studies provide further evidence that cardiac RLC is dephosphorylated by two distinct pools of MLCP activities.