Tumor Necrosis Factor-α Decreases the Phosphorylation Levels of Phospholamban and Troponin I in Spontaneously Beating Rat Neonatal Cardiac Myocyte

The tumor necrosis factor (TNF)αlevel is elevated in patients with advanced heart failure, and the phosphorylation of contractile regulatory proteins is reduced in the human heart. We hypothesized that TNFαaffects the phosphorylation of proteins involved in regulating contraction; phospholamban (PLB), myosin light chain 2 (MLC2) and troponin I (TnI). Spontaneously beating rat neonatal cardiac myocytes, prelabelled with [32P]orthophosphate, were treated with TNFαfor 30 min, and stimulated with isoproterenol for 5 min.32P-labelled myofibrillar proteins were isolated by 15% SDS-PAGE. Baseline phosphorylation levels of PLB, TnI and an unknown 23 kDa phosphoprotein were decreased by TNFαin a dose-dependent manner. Moreover, TNFαattenuated the phosphorylation levels of PLB and TnI increased by a concentration of 0.01μ isoproterenol, but not by 1μ of isoproterenol. Although TNFαhad no effect on the cAMP content or cAMP-dependent protein kinase activity in the presence or absence of isoproterenol, an inverse relationship was observed between the concentration of TNFαand the cGMP content in cardiac myocytes, and treatment with TNFαresulted in a concentration-dependent increase in type 2A protein phosphatase activity. The observation that TNFαdecreases phosphorylation levels of PLB and TnI in cardiac myocytes suggests that the reduction of these protein phosphorylation levels is partially responsible for alterations of intracellular Ca2+-cycling and the force of contraction in TNFα-treated cardiac myocytes. Furthermore, TNFαreduces myocyte contraction and protein phosphorylation states possibly via cAMP-independent mechanisms, at least in part, by the activation of type 2A protein phosphatase.