MacDonnell Scott M, Weisser-Thomas Jutta, Kubo Hajime, Hanscome Marie, Liu Qinghang, Jaleel Naser, Berretta Remus, Chen Xiongwen, Brown Joan H, Sabri Abdel-Karim, Molkentin Jeffery D, Houser Steven R
Department of Physiology, Cardiovascular Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Circ Res. 2009 Aug 14;105(4):316-25. doi: 10.1161/CIRCRESAHA.109.194035. Epub 2009 Jul 16.
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.
This study explored the hypothesis that cytoplasmic CaMKII reduces NFAT nuclear translocation by inhibiting the phosphatase activity of Cn.
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(deltac) 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.
These data show that activation of cytoplasmic CaMKII inhibits NFAT nuclear translocation by phosphorylation and subsequent inhibition of Cn.
病理性心肌细胞肥大被认为是由于收缩期壁应力增加时，为维持心脏功能而导致细胞内Ca(2+)持续升高所诱发。肥大的Ca(2+)与钙调蛋白（CaM）结合并激活磷酸酶钙调神经磷酸酶（Cn）和CaM激酶（CaMK）II。Cn使细胞质中的活化T细胞核因子（NFAT）去磷酸化，诱导其转位至细胞核，在细胞核中它激活抗凋亡和肥大相关的靶基因。细胞质CaMKII调节Ca(2+)处理蛋白，但它是否直接参与肥大和存活信号传导尚不清楚。
本研究探讨细胞质CaMKII通过抑制Cn的磷酸酶活性来减少NFAT核转位这一假说。
利用绿色荧光蛋白标记的NFATc3来确定NFAT在培养的新生大鼠心室肌细胞（NRVMs）和成年猫心室肌细胞中的细胞定位。细胞质靶向CaMKII（deltac）的组成型激活（CaMKII-CA）或显性负性（CaMKII-DN）突变体用于激活和抑制细胞质CaMKII活性。在NRVM中，CaMKII-DN增加了NFAT核转位（48.5±3%，与对照组相比P<0.01），而CaMKII-CA降低了NFAT核转位（5.9±1%，与对照组相比P<0.01）（对照组：12.3±1%）。使用Cn抑制剂表明这些效应是由Cn活性的调节引起的。增加Ca(2+)增加了Cn依赖的NFAT转位（至71.7±7%，P<0.01），而CaMKII-CA降低了这种效应（至17.6±4%）。CaMKII-CA增加了TUNEL和半胱天冬酶-3活性（P<0.05）。在CaMKII-CA感染的NRVMs和肥大的猫心脏中，CaMKII直接使Cn的Ser197位点磷酸化。
这些数据表明，细胞质CaMKII的激活通过磷酸化并随后抑制Cn来抑制NFAT核转位。
