Phospholemman Overexpression Inhibits Na+-K+-ATPase in Adult Rat Cardiac Myocytes: Relevance to Decreased Na+ pump Activity in Post-Infarction Myocytes

摘要

Messenger RNA levels of phospholemman (PLM), a member of the FXYD family of small single-span membrane proteins with putative ion-transport regulatory properties, were increased in postinfarction (MI) rat myocytes. We tested the hypothesis that the previously observed reduction in Na⁺-K⁺-ATPase activity in MI rat myocytes was due to PLM overexpression. In rat hearts harvested 3 and 7 days post-MI, PLM protein expression was increased by 2- and 4-fold, respectively. To simulate increased PLM expression post-MI, PLM was overexpressed in normal adult rat myocytes by adenovirus-mediated gene transfer. PLM overexpression did not affect the relative level of phosphorylation on serine⁶⁸ of PLM. Na⁺-K⁺-ATPase activity was measured as ouabain-sensitive Na⁺-K⁺ pump current (Ip). Compared to control myocytes overexpressing green fluorescent protein alone, Ip measured in myocytes overexpressing PLM was significantly (P<0.0001) lower at similar membrane voltages, pipette Na⁺ ([Na⁺]pip) and extracellular K⁺ concentrations ([K⁺]o). From −70 to +60 mV, neither [Na⁺]pip nor [K⁺]o required to attain half-maximal Ip was significantly different between control and PLM myocytes. This phenotype of decreased Vmax without appreciable changes in Km for Na⁺ and K⁺ in PLM overexpressed myocytes was similar to that observed in MI rat myocytes. Inhibition of Ip by PLM overexpression was not due to decreased Na⁺-K⁺-ATPase expression since there were no changes in either protein or messenger RNA levels of either α1 or α2 isoforms of Na⁺-K⁺-ATPase. In native rat cardiac myocytes, PLM co-immunoprecipitated with α-subunits of Na⁺-K⁺-ATPase. Inhibition of Na⁺-K⁺-ATPase by PLM overexpression, in addition to previously reported decrease in Na⁺-K⁺-ATPase expression, may explain altered Vmax but not Km of Na⁺-K⁺-ATPase in postinfarction rat myocytes.