Saito Tetsuya, Fukuzawa Jun, Osaki Junzo, Sakuragi Hitoshi, Yao Naoyuki, Haneda Takashi, Fujino Takayuki, Wakamiya Nobutaka, Kikuchi Kenjiro, Hasebe Naoyuki
First Department of Medicine, Asahikawa Medical College, 2-1-1-1 Midorigaoka-Higashi, Asahikawa 078 8510, Japan.
J Mol Cell Cardiol. 2003 Sep;35(9):1153-60. doi: 10.1016/s0022-2828(03)00234-7.
Calcineurin and calcium/calmodulin-dependent protein kinase (CaMK) II have been suggested to be the signaling molecules in cardiac hypertrophy. It was not known, however, whether these mechanisms are involved in cardiac hypertrophy induced by pressure overload without the influences of blood-derived humoral factors, such as angiotensin II. To elucidate the roles of calcineurin and CaMK II in this situation, we examined the effects of calcineurin and CaMK II inhibitors on pressure overload-induced expression of c-fos, an immediate-early gene, and protein synthesis using heart perfusion model. The hearts isolated from Sprague-Dawley rats were perfused according to the Langendorff technique, and then subjected to the acute pressure overload by raising the perfusion pressure. The activation of calcineurin was evaluated by its complex formation with calmodulin and by its R-II phosphopeptide dephosphorylation. CaMK II activation was evaluated by its autophosphorylation. Expression of c-fos mRNA and rates of protein synthesis were measured by northern blot analysis and by 14C-phenylalanine incorporation, respectively. Acute pressure overload significantly increased calcineurin activity, CaMK II activity, c-fos expression and protein synthesis. Cyclosporin A and FK506, the calcineurin inhibitors, significantly inhibited the increases in both c-fos expression and protein synthesis. KN62, a CaMK II inhibitor, also significantly prevented the increase in protein synthesis, whereas it failed to affect the expression of c-fos. These results suggest that both calcineurin and CaMK II pathways are critical in the pressure overload-induced acceleration of protein synthesis, and that transcription of c-fos gene is regulated by calcineurin pathway but not by CaMK II pathway.
钙调神经磷酸酶和钙/钙调蛋白依赖性蛋白激酶（CaMK）II被认为是心脏肥大中的信号分子。然而，尚不清楚这些机制是否参与了在没有血液衍生的体液因子（如血管紧张素II）影响的情况下由压力过载诱导的心脏肥大。为了阐明钙调神经磷酸酶和CaMK II在这种情况下的作用，我们使用心脏灌注模型研究了钙调神经磷酸酶和CaMK II抑制剂对压力过载诱导的即刻早期基因c-fos表达和蛋白质合成的影响。从Sprague-Dawley大鼠分离的心脏按照Langendorff技术进行灌注，然后通过提高灌注压力使其承受急性压力过载。通过钙调神经磷酸酶与钙调蛋白的复合物形成及其R-II磷酸肽去磷酸化来评估其激活情况。通过CaMK II的自身磷酸化来评估其激活情况。分别通过Northern印迹分析和14C-苯丙氨酸掺入来测量c-fos mRNA的表达和蛋白质合成速率。急性压力过载显著增加了钙调神经磷酸酶活性、CaMK II活性、c-fos表达和蛋白质合成。钙调神经磷酸酶抑制剂环孢素A和FK506显著抑制了c-fos表达和蛋白质合成的增加。CaMK II抑制剂KN62也显著阻止了蛋白质合成的增加，而它未能影响c-fos的表达。这些结果表明，钙调神经磷酸酶和CaMK II途径在压力过载诱导的蛋白质合成加速中都至关重要，并且c-fos基因的转录受钙调神经磷酸酶途径而非CaMK II途径调控。
