We investigated whether Ca2+/calmodulin-dependent kinase II (CaMKII) and calcineurin (CaN) are involved in myocardial hypertrophy induced by tumor necrosis factor alpha (TNF-alpha). The cardiomyocytes of neonatal Wistar rats (1-2 days old) were cultured and stimulated by TNF-alpha (100 mu g/L), and Ca2+ signal transduction was blocked by several antagonists, including BAPTA (4 mu M), KN-93 (0.2 mu M) and cyclosporin A (CsA, 0.2 mu M). Protein content, protein synthesis, cardiomyocyte volumes, [Ca2+](i) transients, CaMKII delta(B) and CaN were evaluated by the Lowry method, [H-3]-leucine incorporation, a computerized image analysis system, a Till imaging system, and Western blot analysis, respectively. TNF-alpha induced a significant increase in protein content in a dose-dependent manner from 10 mu g/L (53.56 mu g protein/well) to 100 mu g/L (72.18 mu g protein/well), and in a time-dependent manner from 12 h (37.42 mu g protein/well) to 72 h (42.81 mu g protein/well). TNF-alpha (100 mu g/L) significantly increased the amplitude of spontaneous [Ca2+](i) transients, the total protein content, cell size, and [H-3]-leucine incorporation in cultured cardiomyocytes, which was abolished by 4 mu M BAPTA, an intracellular Ca2+ chelator. The increases in protein content, cell size and [H-3]-leucine incorporation were abolished by 0.2 mu M KN-93 or 0.2 mu M CsA. TNF-alpha increased the expression of CaMKII delta(B) by 35.21% and that of CaN by 22.22% compared to control. These effects were abolished by 4 mu M BAPTA, which itself had no effect. These results suggest that TNF-alpha induces increases in [Ca2+](i), CaMKIIdB and CaN and promotes cardiac hypertrophy. Therefore, we hypothesize that the Ca2+/CaMKII- and CaN-dependent signaling pathways are involved in myocardial hypertrophy induced by TNF-alpha.
