Study On Mitochondrial Mechanisms And The Associated Signaling In NPY-induced Cardiomyocyte Hypertrophy

Objectives:During the development of myocardial hypertrophy,it has been one of the difficult points to explore the specific molecular mechanisms of cardiomyocyte hypertrophy induced by different stimulating factors.Neuropeptide Y(NPY),the most widely known neuropeptide hormone in the cardiovascular system,is closely related to the development of various diseases.However,it is still unkonwn whether NPY can directly participate in the regulation of cardiomyocyte hypertrophy and injury through mitochondria-related pathways.The aim of this study is to determine the effects and related mechanisms in NPY?induced cardiomyocyte hypertrophy in primary cultured rat cardiomyocytes.We tried to explore the role of mitochondria in this pathological process and the potential signaling pathways as well,which might provide a theoretical basis for treatment of cardiovascular diseases and forensic identification of sudden cardiac death.Methods:(1)Primary cardiomyocytes were isolated from neonatal SD rats by the methods of blended enzyme(pancreatic enzyme and collagen ? type)digestion.Cell autonomous beating rate and purity were detected.(2)Primary cardiomyocytes were treated with different concentrations of NPY for 24 h and 48 h respectively,then the mRNA expressions of myocardial hypertrophy embryo gene,as well as morphological changes in cardiomyocytes were recorded.(3)Primary cardiomyocytes were treated with different concentrations of NPY for 24 h,changes of mitochondria oxidative stress,biogenesis and function were detected.(4)Primary cardiomyocytes were treated with different concentrations of NPY for 24 h,Western Blot were used to detecte the activation of Ca2+ and MAPK signaling pathways.(5)NAC,a removal agent of ROS,was applied to investigate the relationship between activated signaling pathways and intracellular ROS generation.(6)Specific inhibitors,including CsA(Ca2+/CaN),KN93(Ca2+/CaMK ?)and SB203580(p38 MAPK)were further used to determine the effects of activated Ca2+ nd MAPK signaling pathways on NPY-induced mitochondrial functional changes and cardiomyocyte hypertrophy.Results:(1)Culture and identification of neonatal rat cardiomyocytes.After 72-hours culture,the primary cardiomyocytes tended to be constant in the percentage of autonomous beating cells and autonomous beating rate.Immunofluorescence characterization revealed that more than 90%of cells were cTnT positive.Thus,the cells cultured in vitro for 72 hours were used for following experiments.(2)NPY induced cardiomyocyte hypertrophy.Results of real-time quantitative PCR showed that,after treatment with different concentrations of NPY(0,10,20,50,100 nM)for 24 h and 48 h respectively,the expression levels of ANP and BNP mRNA were significantly elevated in a dose-dependent and time-dependent manner.Results from immunofluorescence staining further confirmed that the cadiomyocytes morphology was changed,and the area of cardiomyocytes was markedly increased after stimulation of 10 nM and 100 nM NPY for 48 h.(3)The effect of NPY on mitochondrial oxidative stress,biosynthesis and membrane potential.? NPY increased mt-ROS and total RoS levels in primary cardiomyocytes.Results from MitoSOX Red Mitochondrial Superoxide Indicator staining showed that NPY dose-dependently increased mitochondrial ROS generation.The increase of mt-ROS induced by 10 nM NPY is about 2 times,and the increase of mt-ROS induced by 100 nM NPY is more obvious.Results from DCFH-DA fluorescence probe showed in comparison with control group,the total ROS fluorescence intensity also significantly elevated after NPY treatment in a dose-dependent manner.? NPY increased the expression levels of PGC-1?,NRF-1 and TFam in primary cardiomyocytes.Results of Western Blot showed that NPY induced a manifest elevated expression of mitochondrial biogenesis protein PGC-1? in a dose-dependent manner.Real-time quantitative PCR results further indicated that the mRNA expression levels of PGC-1?,NRF-1 and TFam were also increased after NPY treatment for 24 h.?NPY decreased mitochondrial membrane potential and cell viability in primary cardiomyocytes.After stimulation of NPY for 24 h,the mitochondrial membrane potential and cell viability in cardiomyocytes gradually decreased with the increase of NPY concentration.(4)NPY activates Ca2+ and p38 MAPK signaling pathways in primary cardiomyocytes.?NPY activates Ca2+/CaN and Ca2+/CaMK ? signaling pathways in primary cardiomyocytes.Western Blot results showed that after treatment with different concentrations of NPY for 24 h,expression levels of CaN,p-CaMK ? increased markedly,but no apparent changes were detected in t-CaMK ? protein,suggesting that NPY activates Ca2+/CaN and Ca2+/CaMK ? signaling pathways in primary cardiomyocytes.? NPY activates p38 MAPK signaling pathway in primary cardiomyocytes.Western Blot results showed that after treatment with different concentrations of NPY,protein expression levels of p-p38 elevated significantly,but no apparent changes were detected in t-p38 expressions,suggesting that NPY activates p38 MAPK signaling pathways in primary cardiomyocytes after treatment with 24 h.There were also no obvious changes in the expression levels of p-ERK,t-ERK,p-JNK and t-JNK proteins,suggesting that after treatment of NPY for 24 h,there were no significant changes in ERK and JNK signaling pathways in primary cardiomyocytes.(5)The activation of Ca2+ and p38 MAPK signal pathways induced by NPY is dependent on ROS generation in cardiomyocytes.Compared with the control group,NAC,a removal agent of ROS,significantly reversed the increased expression levels of CaN,p-CaMK ?,and p-p38 MAPK induced by 100nMNPY.(6)The activation of Ca2+ and p38 MAPK signal pathways is involved in NPY-induced mitochondrial injury and cardiomyocyte hypertrophy.? CsA,KN93,SB203580 relieved the mitochondrial injury and increased expression of PGC-1? induced by NPY.Resluts form Western Blot indicated that specific inhibitors as CsA(Ca2+/CaN),KN93(Ca2+/CaMK ?),SB203580(p38 MAPK)could markedly inhibit NPY-induced PGC-1? elevation.Meanwhile,compared with the control group,CsA,KN93,SB203580 significantly improved the reduction of mitochondrial membrane potential and cardiomyocytes viability caused by 100 nM NPY.? NAC,CsA,KN93,SB203580 relieved NPY-induced cardiomyocyte hypertrophy.Compared with the control group,NAC and CsA,KN93,SB203580 significantly relieved the elevation of NPY-induced ANP and BNP mRNA expression levels,as well as the increase in cardiomyocyte area.Conclusions:(1)NPY directly induced mitochondrial injury and cell hypertrophy in primary neonatal rat cardiomyocytes.(2)The activation of Ca2+ and p38 MAPK signal pathways induced by NPY was dependent on ROS generation in cardiomyocytes.(3)Mitochondrial oxidative stress,as well as further mandatory activation of mitochondrial biosynthesis might be one of the mechanisms for NPY-induced cariomyocyte hypertrophy.