PP1cβ dephosphorylates cardiac myosin by MYPT‐dependent and independent mechanisms

Abstract

In beating hearts, cardiac muscle myosin is phosphorylated at its regulatory light chain (RLC) at 0.4 mol phosphate/mol RLC. This level of constitutive phosphorylation is necessary for normal cardiac function, and is maintained by balanced activities of the dedicated cardiac muscle‐specific myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). The MLCP is best characterized in the smooth muscle, and is comprised of the regulatory myosin phosphatase target subunit MYPT1, a catalytic subunit PP1cβ, and an accessory subunit of unknown function M20. In smooth muscles in vivo, MYPT1 and PP1cβ co‐stabilize each other and are both necessary for normal smooth muscle contractions. In the cardiac muscle, the ubiquitously‐expressed MYPT1 and the striated muscle‐specific MYPT2 are both expressed in the cardiomyocyte, but the contributions to physiological regulation of cardiac myosin dephosphorylation by PP1cβ are unclear.Using a conditional knockout of PP1cβ, we tested the hypothesis that the main catalytic subunit for cardiac MLCP is PP1cβ, and that its activity toward phosphorylated cardiac RLC in vivo is dependent on regulation by striated muscle‐specific MYPT2. We measured the effects of the knockout on the expression levels of other regulators of cardiac RLC phosphorylation, and a cardiac myofibrillar phosphatase assay was developed to measure MYPT‐regulated and MYPT‐independent phosphatase activities toward phosphorylated cardiac myosin. We report that 1) PP1cβ is the main isoform expressed in the cardiac myocyte, 2) cardiac muscle pathogenesis in PP1cβ knockout animals involve upregulation of PP1cα and MYPT1 in activated myofibroblasts, 3) the protein stability of endogenous mouse cardiac MYPT1 and MYPT2 are not dependent on the expression of PP1cβ, and 4) phosphorylated cardiac myosin in intact myofibrillar preparations are dephosphorylated by both myosin‐targeted and soluble MYPT‐independent PP1cβ activities. These results contribute to defining properties of the cardiac MLCP in vivo.