Tumor necrosis factor alpha protects heart cultures against hypoxic damage via activation of PKA and phospholamban to prevent calcium overload.

This study aims to elucidate the mechanisms by which tumor necrosis factor alpha (TNF alpha) provides protection from hypoxic damage to neonatal rat cardiomyocyte cultures. We show that when intracellular Ca2+ ([Ca2+](i)) levels are elevated by extracellular Ca2+ ([Ca2+] o) or by hypoxia, then TNF alpha decreased [Ca2+](i) in individual cardiomyocytes. However, TNF alpha did not reduce [Ca2+](i) after its increase by thapsigargin, (a SERCA2a inhibitor), indicating that TNF alpha attenuates Ca2+ overload through Ca2+ uptake by SERCA2a. TNF alpha did not reduce [Ca2+](i), following its elevation when [Ca2+](o) levels were elevated in TNF alpha receptor knock-out mice. H-89, a protein kinase A (PKA) inhibitor, attenuated the protective effect of TNF alpha when the cardiomyoctyes were subjected to hypoxia, as determined by lactate dehydrogenase (LDH) and creatine kinase (CK) released and from the cardiomyocytes. Moreover, when the levels of [Ca2+](i) were increased by hypoxia, H-89, but not KN93, (a calmodulin kinase II inhibitor), prevented the reduction in [Ca2+](i) by TNF alpha. TNF alpha increased the phosphorylation of PKA in normoxic and hypoxic cardiomyoctes, indicating that the cardioprotective effect of TNF alpha against hypoxic damage was via PKA activation. Hypoxia decreased phosphorylated phospholamban levels; however, TNF alpha attenuated this decrease following hypoxia. It is suggested that TNF alpha activates phospholamban phosphorylation in hypoxic heart cultures via PKA to stimulate SERCA2a activity to limit Ca2+ overload.