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 (TNFα) 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 TNFα decreased [Ca²⁺]_i in individual cardiomyocytes. However, TNFα did not reduce [Ca²⁺]_i after its increase by thapsigargin, (a SERCA2a inhibitor), indicating that TNFα attenuates Ca²⁺overload through Ca²⁺uptake by SERCA2a. TNFα did not reduce [Ca²⁺]_i, following its elevation when [Ca²⁺]_o levels were elevated in TNFα receptor knock-out mice. H-89, a protein kinase A (PKA) inhibitor, attenuated the protective effect of TNFα 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⁺]_iby TNFα. TNFα increased the phosphorylation of PKA in normoxic and hypoxic cardiomyoctes, indicating that the cardioprotective effect of TNFα against hypoxic damage was via PKA activation. Hypoxia decreased phosphorylated phospholamban levels; however, TNFα attenuated this decrease following hypoxia. It is suggested that TNFα activates phospholamban phosphorylation in hypoxic heart cultures via PKA to stimulate SERCA2a activity to limit Ca²⁺overload.