Vincent Fanny, Duquesnes Nicolas, Christov Christo, Damy Thibaud, Samuel Jane-Lise, Crozatier Bertrand
Unité INSERM U400, Créteil, France.
Cardiovasc Res. 2006 Jul 1;71(1):97-107. doi: 10.1016/j.cardiores.2006.03.012. Epub 2006 Mar 24.
Myocardial stretch activates a number of interconnected pathways including the protein kinase C (PKC) pathway that in turn activates mitogen activated protein kinases (MAPK), leading to gene expression stimulation and ventricular hypertrophy. A role of calcineurin has also been shown during hypertrophy. The goal of our study was to look for a possible interconnection between PKC and calcineurin in myocardial stretch.
Neonatal rat cardiomyocytes were cultured for 5 days and a 15% stretch was applied. Expression of MAPK and PKC-epsilon was evaluated by Western blot analysis. The specific role of PKC-epsilon was evaluated by transfection of cardiomyocytes with a specific inhibitor peptide. Calcineurin and PKC-epsilon complex formation and co-localization were evaluated by co-immunoprecipitation and by immunolocalization.
The PKC isoform involved in stretch-induced ERK and JNK activations was PKC-epsilon. We show here that calcineurin is also found to be involved in the stretch response and that calcineurin and PKC-epsilon co-operate at 2 levels during stretch. First, stretch-induced translocation of PKC-epsilon from the cytosolic to the membrane fraction was inhibited by calcineurin inhibitors, indicating that calcineurin was necessary for PKC-epsilon activation induced by stretch. A second level of interaction was the formation of a calcineurin-PKC-epsilon complex, which increased during stretch. Immunofluorescent studies indicated that, after stretch, calcineurin and PKC-epsilon were co-localized at the level of the perinuclear membrane. These results may have a major relevance in vivo since we also found similar PKC-epsilon-calcineurin complexes in the phase of thoracic aortic stenosis in rats during which heart failure develops.
Calcineurin appears to be necessary for stretch-induced PKC-epsilon activation after which the phosphatase and the kinase are co-localized in a complex at the level of the perinuclear membrane where they may finely regulate the phosphorylation of their target proteins.
心肌牵张激活许多相互关联的信号通路，包括蛋白激酶C（PKC）通路，该通路进而激活丝裂原活化蛋白激酶（MAPK），导致基因表达受刺激和心室肥厚。钙调神经磷酸酶在心肌肥厚过程中的作用也已得到证实。我们研究的目的是探寻PKC与钙调神经磷酸酶在心肌牵张过程中可能存在的联系。
将新生大鼠心肌细胞培养5天，施加15%的牵张力。通过蛋白质印迹分析评估MAPK和PKC-ε的表达。用特异性抑制肽转染心肌细胞来评估PKC-ε的具体作用。通过免疫共沉淀和免疫定位评估钙调神经磷酸酶与PKC-ε复合物的形成及共定位情况。
参与牵张诱导的细胞外信号调节激酶（ERK）和应激活化蛋白激酶（JNK）激活的PKC同工型是PKC-ε。我们在此表明，钙调神经磷酸酶也参与牵张反应，且在牵张过程中钙调神经磷酸酶与PKC-ε在两个水平上协同作用。首先，钙调神经磷酸酶抑制剂抑制了牵张诱导的PKC-ε从胞质向膜部分的转位，表明钙调神经磷酸酶是牵张诱导PKC-ε激活所必需的。相互作用的第二个水平是形成钙调神经磷酸酶-PKC-ε复合物，该复合物在牵张过程中增加。免疫荧光研究表明，牵张后，钙调神经磷酸酶和PKC-ε在核周膜水平共定位。这些结果在体内可能具有重要意义，因为我们在大鼠胸主动脉狭窄导致心力衰竭发生的阶段也发现了类似的PKC-ε-钙调神经磷酸酶复合物。
钙调神经磷酸酶似乎是牵张诱导PKC-ε激活所必需的，之后磷酸酶和激酶在核周膜水平共定位于一个复合物中，在那里它们可能精细调节其靶蛋白的磷酸化。
