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(Circulation Research. 2009;105:316.)
© 2009 American Heart Association, Inc.

Molecular Medicine

CaMKII Negatively Regulates Calcineurin–NFAT Signaling in Cardiac Myocytes

Scott M. MacDonnell^*, Jutta Weisser-Thomas^*, Hajime Kubo, Marie Hanscome, Qinghang Liu, Naser Jaleel, Remus Berretta, Xiongwen Chen, Joan H. Brown, Abdel-Karim Sabri, Jeffery D. Molkentin, Steven R. Houser

From the Department of Physiology (S.M.M., J.W.-T., H.K., M.H., N.J., R.B., X.C., A.-K.S., S.R.H.), Cardiovascular Research Center, Temple University School of Medicine, Philadelphia, Pa; Cincinnati Children’s Hospital Medical Center (Q.L., J.D.M.), Ohio; and Department of Pharmacology (J.H.B.), University of California at San Diego, La Jolla.

Correspondence to Steven R. Houser, Temple University School of Medicine, Department of Physiology, Cardiovascular Research Center, 3400 N Broad St, Philadelphia, PA 19140. E-mail steven.houser{at}temple.edu

Rationale: Pathological cardiac myocyte hypertrophy is thought to be induced by the persistent increases in intracellular Ca²⁺ needed to maintain cardiac function when systolic wall stress is increased. Hypertrophic Ca²⁺ 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 Ca²⁺ 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_c 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 Ca²⁺ 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.

Key Words: CaMKII • calcineurin • NFAT • myocytes • heart disease