Elucidation of molecular mechanism of mechanical stretch-induced cardiac hypertrophy

我们之前通过使用可变形的硅胶培养皿报道，心肌细胞的拉伸引起蛋白激酶的磷酸化级联反应，如细胞外信号调节激酶（ERKs）和90-kD核糖体S6激酶，随后蛋白质合成增加，ERKs的激活对肥厚反应至关重要。为了进一步阐明拉伸诱导的导致心肌细胞ERK活化的信号转导通路，推测上游激酶如Src的作用。抑制蛋白法检测Ras、Raf-1激酶（Raf-1）和蛋白激酶C （PKC）。将CSK活性突变体（最近分离的抑制Src家族激酶的酪氨酸激酶）、显性阴性Ras突变体或显性阴性Raf-1突变体共转染到ha标记ERK2的新生大鼠心肌细胞中。转染后，在可变形硅胶培养皿上培养的心肌细胞拉伸20%，拉伸8分钟。用抗ha单克隆抗体免疫沉淀ha标记的ERK2，用髓鞘碱性蛋白作为底物检测ERK2的活性。心肌细胞的拉伸迅速增加ERK2的活性。CSK或显性阴性Ras与ha标记的ERK2共转染对拉伸诱导的ERK2活化没有抑制作用，而显性阴性Raf-1共转染完全抑制机械拉伸诱导的ERK2活化。此外，当PKC被calphostin C （10^<-6>M持续60分钟）或TPA长时间暴露（10^<-7>M持续24小时）抑制时，机械拉伸诱导的Raf-1和ERK激活几乎完全被抑制。PKC激活剂如TPA （10^<-7>M）可激活培养心肌细胞中的Raf-1和ERK。这些结果表明，PKC，而不是Src或Ras，通过Raf-1在机械应力诱导的ERK激活中起重要作用。

We have previously reported by using deformarable silicone dishes that stretching of cardiac myocytes evokes the phosphorylation cascade of protein kinases such as extracellular signal-regulated kinases (ERKs) and 90-kD ribosomal S6 kinase followed by an increase in protein synthesis, and that activation of ERKs is critical for hypertrophic responses. To further elucidate stretch-induced signal transduction pathways leading to ERK activation in cardiac myocytes, the role of presumable upstream kinases such as Src.Ras, Raf-1 kinase (Raf-1) and protein kinase C (PKC) was examined by using inhibitory proteins. Active mutant of CSK (the recently isolated tyrosine kinase which inhibits Src family kinases), dominant-negative Ras mutant or dominat-negative Raf-1 mutant was co-transfected into cardiac myocytes of neonatal rats with HA-tagged ERK2. After transfection, cardiac myocytes cultured on deformable silicone dishes were stretched by 20% for 8 min. HA-tagged ERK2 was immunoprecipitated using an anti-HA monoclonal antibody and the activity of ERK2 was assayd by using myelin basic protein as a substrate. Stretching of cardiac myocytes rapidly increased the activity of ERK2. Co-transfection of either CSK or dominant-negative Ras with HA-tagged ERK2 showed no inhibitory efects on ERK2 activation by stretch, while the co-transfection of dominant-negative Raf-1 completly inhibited mechanical stretch-induced ERK2 activation. Futhermore, when PKC was inhibited by calphostin C (10^<-6>M for 60 min) or long exposure of TPA (10^<-7>M for 24 hours), Raf-1 and ERK activation induced by mechanical stretch was almost completely surpressed. PKC activators such as TPA (10^<-7>M) activated Raf-1 and ERK in cultured cardiac myocytes. These results suggest that PKC,but not Src or Ras, plays a vital role in mechanical stress-induced ERK activation through Raf-1.