The Effect Of Neuropeptide Y On The Mitochondrial Structure And Energy Metabolism In Primary Cultured Neonatal Rat Cardiomyocyte

Objective The effects of NPY on the primary cultured neonatal rat cardiomyocyte were tested by detection or observation of its viability, the ATP content, mitochondrial structure, mitochondrial membrane potential and proliferator-activated receptor y coactivator-1alpha, which is a cellular study on stress-cardiomyopathy, to provide information for the setup of stress-cardiomopathy animal modelMethods 1.The culture and verification of primary cultured neonatal rat cardiomyocyte:After combined enzymatic digesting method had been used in getting primary neonatal rat cardiomyocyte, Brdu was added and different speed adherence methods were used for purifying neonatal rat cardiomyocyte. The autonomous throb of cardiomyocyte was real-time videoed, and its special markers were used in the immunohistochemical staining for verifying the purified cell was real neonatal rat cardiomyocyte.2. The functional test of primary cultured neonatal rat cardiomyocyte:After 24-hour stimulation of different dose of NPY, the viability of primary cultured neonatal rat cardiomyocyte and ATP content were determined by Cell Counting Kit-8 and ATP-dependent bio luminescence assay kit, respectively. 3.Observation of the morphology change of primary cultured neonatal rat cardiomyocyte: After stimulation of different dose of NPY, the change of mitochondrial membrane potential was observed under fluorescence microscope after JC-1 staining, and detected by JC-1 kit. The mitochondrial structure change was observed by transmission electron microscopy.4. The molecular test of primary cultured neonatal rat cardiomyocyte:After stimulation of different dose of NPY, the changes of PGC-1? mRNA and protein expression in primary cultured neonatal rat cardiomyocyte were determined by real-time PCR and western blotting, respectively. Furthermore, the block experiments of calcium ion pathway (CaN, CaMK?, P38MAPK) were tested to provide information for the further study in signal transduction pathway.Results 1.After being digested and purified, the pulsation of primary cultured neonatal rat cardiomyocyte was evident and the special markers (Alpha sarcomeric actin,?-SA and Cardiac troponin T,cTnT) were positive with immunohistochemical staining.2. After different dose of NPY stimulation, the viability of primary cultured neonatal rat cardiomyocyte was decreased, which indicates that NPY can depress its viability. Furthermore, the ATP content of primary cultured neonatal rat cardiomyocyte was decreased by NPY in dose-depend manner (P<0.01).3. After different dose of NPY stimulation, the changes of structure of mitochondria were observed under electron microscopy, including collapsed mitochondrial cristae, mitochondrial swell and rupture of mitochondrial membrane, in addition to a decrease in mitochondrial density and increased heterogeneity in size and shape etc. The mitochondrial membrane potential in mitochondria of primary cultured neonatal rat cardiomyocyte was induced.4. The levels of PGC-la mRNA and protein expression were up-regulated after beingtreated by different dose of NPY. In the selective blocking experiment, the expression is positive in pathway of P38MAPK (blocker SB203580).Conclusions NPY can suppress the viability and energy metabolism of primary cultured neonatal rat cardiomyocyte, and its mechanism may be the change of mitochondrial structure, decrease of the mitochondrial membrane potential, and up-regulation of the PGC-la mRNA and protein expression. Thus it can be used for the setup stress cardiomyopathy animal model in the future.