CaMKII Negatively Regulates Calcineurin–NFAT Signaling in Cardiac Myocytes

Abstract

Rationale: Pathological cardiac myocyte hypertrophy is thought to be induced by the persistent increases in intracellular Ca 2+ needed to maintain cardiac function when systolic wall stress is increased. Hypertrophic Ca 2+ binds to calmodulin (CaM) and activates the phosphatase calcineurin (Cn) and CaM kinase (CaMK)II. Cn dephosphorylates cytoplasmic NFAT (nuclear factor of activated T cells), inducing its translocation to the nucleus where it activates antiapoptotic and hypertrophic target genes. Cytoplasmic CaMKII regulates Ca 2+ handling proteins but whether or not it is directly involved in hypertrophic and survival signaling is not known. Objective: This study explored the hypothesis that cytoplasmic CaMKII reduces NFAT nuclear translocation by inhibiting the phosphatase activity of Cn. Methods and Results: Green fluorescent protein–tagged NFATc3 was used to determine the cellular location of NFAT in cultured neonatal rat ventricular myocytes (NRVMs) and adult feline ventricular myocytes. Constitutively active (CaMKII-CA) or dominant negative (CaMKII-DN) mutants of cytoplasmic targeted CaMKII δc were used to activate and inhibit cytoplasmic CaMKII activity. In NRVM CaMKII-DN (48.5±3%, P <0.01 versus control) increased, whereas CaMKII-CA decreased (5.9±1%, P <0.01 versus control) NFAT nuclear translocation (Control: 12.3±1%). Cn inhibitors were used to show that these effects were caused by modulation of Cn activity. Increasing Ca 2+ increased Cn-dependent NFAT translocation (to 71.7±7%, P <0.01) and CaMKII-CA reduced this effect (to 17.6±4%). CaMKII-CA increased TUNEL and caspase-3 activity ( P <0.05). CaMKII directly phosphorylated Cn at Ser197 in CaMKII-CA infected NRVMs and in hypertrophied feline hearts. Conclusion: These data show that activation of cytoplasmic CaMKII inhibits NFAT nuclear translocation by phosphorylation and subsequent inhibition of Cn.