Regulation Of PKC To The Unloaded Contractile Function In Mouse Cardiomyocytes

Heart failure, which is also referred to as cardiac insufficiency, is one of the most challenging cardiovascular diseases in modern world, and it is also the last stage of some cardiovascular diseases. Chronic heart failure is a serious worldwide public health problem and clinic touchy disease with high disability rate and mortality. Therefore, researchers paid much more attention to its fundamental and clinical issues.Although the development of heart failure is a conclusive process, its mechanism is still unclear. Therefore, elucidating the molecular mechanism of heart failure is a more important problem in curing heart failure. Heart failure is characterized by progressively decreased heart function. Present researches indicated that the followings may be the underlying reasons for the decreased heart function: relative ischemia/hypoxia of the myocardium, changes in cardiomyocyte membrane ion channel, changes in cardiomyocyte sarcoplasmic reticulum, changes in cardiomyocyte contractile protein, changes in cytoskeletal protein and decreased number of cardiomyocytes. Recent researches indicated that decreased cardiomyocyte contractility played a considerately important role in the depressed heart function. Cardiomyocyte hypertrophy and decreased cardiomyocyte contractility are the two different stages of heart failure development. Accordingly, there seems to be amechanism that a certain molecule can regulate cardiomyocyte hypertrophy; meanwhile, it can regulate the cardiomyocyte contractile function. The latest researches hinted that PKC process such two-edge sword effect: mediating cardiomyocyte hypertrophy and participating in heart failure. Therefore, such effects of PKC deserve further and systematic investigation.To investigate the above-mentioned problems, our lab established a stable and reliable adult mouse cardiomyocyte isolation technique, which adopts classic Langendorff perfusion apparatus to perfuse the heart. Perfusion pressure is used to judge the end of digestion. Based on the model of cardiomyocyte, Edge-Detector system (Crescent USA) detects the contractile amplitude of cardiomyocyte, and Felix software (PTI, USA) collects and analyzes the unloaded contractile function of cardiomyocyte. The results are as follows:1. Establish a stable and reliable adult mouse cardiomyocyte isolation technique The cannulated heart was mounted on a Langendorff perfusion apparatus with constant flow and perfusion pressure was monitored. The flow rate was decided by the mouse age. The viability of freshly isolated cardiomyocytes was more than 70 %. The viability of cardiomyocytes reduced to 40 %-50 % after 4-h standing. More than 90% of rod-shaped cardiomyocytes were quiescent and had visible cross striations and sharp edges. These results indicate that high yield and high quality cardiomyocytes can be obtained. In addition, the standards of identifying cardiomyocyte quality are concise and are suitable to culture the cardiomyocytes or to studythe physiological function of cardiomyocytes.2. Amplitude of unloaded shortening in cardiomyocytes displays concentration-dependent changes and suppressed shortening after a temporal enhancement at 5.0Hz Cardiomyocytes were perfused with HEPES buffer. The amplitude of unloaded shortening in cardiomyocytes was9.72 + 0.43 % during 1.0 Hz stimulation, 11.28 ± 0.43 % at 2.0 Hz, 11.40 + 0.45 % at 5.0 Hz. When stimulated by 5.0 Hz, amplitude of shortening enhanced followed by suppressed shortening in almost all the cardiomyocytes.3. PMA, a PKC agonist, evokes negative inotropic effect in mouse cardiomyocytes Exposure of isolated cardiomyocytes to 50nmol/L, 100nmol/L and 200nmol/L PMA resulted in significant decreases in their contractile response to electrical stimulation by 20.40±2.56% ; 49.80±2.22%; 67.33±2.61%, respectively. Otherwise, PMA prolongs the shortening phase (P<0.05) and decreases shortening rate and relengthening rate (P<0.05).4. Staurosporine, a PKC inhibitor, evokes positive inotropic effect in mouse cardiomyocytes Exposure of cardiomyocytes to PKC inhibitor resulted in a large enhancement of the contractile response. In the presence of 200nmol/L Staurosporine, the mean peak shortening was increased about two-fold after 12 min incubation (179.15±1.84% compared with control 100%, P<0.01). The Staurosporine also displayed a progressive slowing of the contractile phase of the contraction (P<0.05), and there was a fast shortening rate and relengthening rate (P<0.01).5. 200nmol/L PMA and 200nmoI/L Staurosporine changes the amplitude-frequency relationship in mouse cardiomyocytes Apart from the decrease of the amplitude of contraction, 200nmol/L PMA caused the sustained shortening amplitude at 5.0 Hz frequency. However, 200nmol/L Staurosporine induces a larger enhancement in shortening at 5.0 Hz besides the increase of shortening amplitude.Conclusively, our lab established a technique of adult mouse cardiomyocytes isolation; meanwhile, we also observed that increasing the stimulating frequency caused the increased contractile amplitude. In present study, we have demonstrated that PKC agonist evoked negative inotropic effect and PKC inhibitor evoked positive inotropic effect in mouse cardiomyocytes. These findings indicate that PKC participate the regulation of...