Studying The Role Of CamkⅡδ Isozyme In Myocardial Fibrosis And Its Molecule Mechanism

Objective: To observe the role of calcium - calmodulin-dependent protein Kinase(CaMK)δisozyme in rat cardiac fibroblasts proliferation and collagen synthesis secreation induced by electric field stimulation (EFS) or G protein-coupled receptor activation ; approaching the role of calcium - calmodulin-dependent protein kinase (CaMK) in myocardial fibrosis (MF) and their molecular mechanism.Methods: Using cultured cardiac fibroblasts from neonatal 1-3 days rat (3 generation); either electrical field stimulation (EFS) or G protein-coupled receptor activation with angiotensin II were explored to induce cardiac fibroblasts proliferation as evidenced by Cell counting and MTT; Using pharmacological approach, we further observe if CaMKâ…¡inhibitor (KN93, AIP) inhibited cardiac fibroblasts proliferation. Meanwhile the content of TGFβ1 and TNF-a in cell supernatant were determined by immunoassay; Using Western blot,RT-PCR expression of MMp-2,MMp-,CaMKⅡδB, CaMKⅡδC,collagenâ… ,â…¢mRNA ,MMP-1mRNA in cardiac fibroblasts. were detected.Results: 1. Both 10-7mol/l Angâ…¡and 10V1.0Hz EFS enhanced cardiac fibroblasts proliferation and they addititively enhanced cardiac fibroblasts proliferation. More importantly, CaMKII inhibitors (KN93 and AIP) inhibited these effects; 2. Angâ…¡induced cardiac fibroblasts proliferation in a dose-dependent manner; 3. EFS induced cardiac fibroblasts proliferation in a voltage -dependent manner; 4. Both 10-7mol/l Angâ…¡and 10V1.0Hz EFS enhanced excretion of TGFβ1 and TNF-a in cardiac fibroblasts, which were inhibited by KN93 or AIP. 5. Both 10-7mol/l Angâ…¡and 10V1.0Hz EFS increased CaMKIIδB ,δC mRNA expression in cardiac fibroblasts which were inhibited by either KN93 or AIP.;6. Both 10-7mol/l Angâ…¡and 10V1.0Hz EFS enhanced collagenâ… ,â…¢mRNA expression while KN93 and AIP inhibited the effect; 7. Both 10-7mol/l Angâ…¡and 10V1.0Hz EFS enhanced MMP-1,2,9 mRNA, MMp-2,MMp-9 protein expression in cardiac fibroblasts which were suppressed by either KN93 or AIP.Conclusion: Either EFS or G protein-coupled receptor (Angâ…¡) involved in myocardial fibrosis; the mechanism might be related to cytokines (TGFβ1å'ŒTNF-a)and extracellular matrix (MMp-1,2,9 ,collagenâ… ,â…¢)secreation caused by calcium - calmodulin-dependent protein kinaseâ…¡(CaMK)δB, C up-regulation.