| Hyperhomocysteinemia(HHcy)represents an independent risk factor for cardiovascular diseases.Even,moderately elevated plasma homocysteine(Hcy)levels increase the risk of heart failure,independent of classic cardiovascular risk factors.In general,the baseline of plasma Hcy in Chinese population is higher than that in Western countries population,which mainly due to dietary habit.In clinical,moderately elevated plasma Hcy levels is common in HHcy patients,whereas the pathological condition persistently exist because of lacking positive intervention,resulting in the irreversible damage of vessel and heart and promoting the occurrence of adverse cardiovascular events.Currently,numerous studies have indicated that prolonged HHcy causes vascular damage by inducing programmed cell death,thereby causing thrombogenesis and accelerating atherosclerosis.Our previous studies shown that mild and moderate HHcy could aggravate hypertention-induced left ventricular remodeling,thereby resulting in decreased cardiac function in patients.However,whether HHcy alonely can induce cardiomyocytes apoptosis and causes a decreased cardiac function is not well understand.Endoplasmic reticulum stress(ER stress)-induced cells apoptosis is the common pathological basis of various cardiovascular diseases.Various adverse factors such as hypertention,hyperlipidaemia,hyperlipidaemia,ischemia and anoxia can initate ER stress in cardiomyocytes;the plonged or/and severe ER stress can induce the expression of pro-apoptotic protein,thereby activating apoptotic signaling pathways and leading to cell death.Our previous studies shown that HHcy could deteriorate hypertension-induced cardiomyocytes ER stress,increases the expression of pro-apoptotic proteins CHOP and caspase-12,thereby activating the apoptotic signaling pathways which might modulate cardiomyocytes apoptosis and adverse left ventricular remodeling in rats.However,whether HHcy alonely can induce ER stress,and whether HHcy-induced ER stress directly modulates cardiomyocytes apoptosis are unclear.Mitochondria are essential for cardiomyocytes survival.In addition to pro-survival functions of ATP production and cellular metabolism,mitochondria play pivotal roles in cell death processes,during both apoptosis and necrosis.Mitochondria are involved both in the amplification of the external pathway and in the initiation of the intrinsic pathway.They serve as important sites of integration for anti-and pro-apoptotic signals for both pathways.However,in the process of cardiomyocytes apoptosis induced by Hcy,whether mitochondria play an important role is not well understood.In recent studies,the ER and mitochondria form a dynamic interconnected network.ER stress might play a functional role in modulating mitochondrial dysfunction;thereby they co-regulate organelle function and codetermine cellular fate,which are defined as ER-mitochondria axis.ER is an essential organelle for storing Ca2+,thus Ca2+ turbulence in ER lumen is a important triger of ER stress.When ER stress is prolonged and/or severe,Ca2+ in ER lumen will release to cytoplasm.Releases of Ca2+ from ER cytoplasm cause a sudden increase of Ca2+ in cytoplasm,which is the crucial irritant to mitochondrial stress.That may be the bond bridging ER stress and mitochondrial stress.However,studies of the major proposed molecular and cellular mechanisms underlying ER stress and mitochondrial stress are just underway.Whether HHcy can induce mitochondrial dysfunction in cardiomyocytes and relationship with ER stress are unclear.Homocysteine and taurine are both sulfur-containing amino acids sharing the same biosynthetic pathway,have been shown to play opposite role in the development of cardiovascular diseases.Taurine is the most intracellular sulfur-containing amino acids,especially in cardiomyocytes.It is suggested that appropriate taurine supplementation exerts therapeutical effect on hypertenstion,hyperlipidemia and hyperglycemia,thus plays a protective role in cardiovascular system.In clinical taurine has been claimed be beneficial to and has been used to patients that are unresponsive or resistant to digitalis or diuretics.The major cytoprotective mechanisms of taurine include maintaining calcium homeostasis,scavenging free radical,and ensuring the right folded proteins.In present,taurine relieves ER stress,improves mitochondrial function,thereby benefiting patients with brain disorder has attracted wide attention.Despite the accumulated knowledge,there are no published reports directly examining the relationship between taurine and Hcy-induced ER stress and mitochondrial dysfunction in cardiomyocyte.Therefore,the study was proformed on the basis of our previous works and the published reports.Firstly,in vivo study,we observed the adverse effect of HHcy on diac structure and function in rats,and investigated the functional role of ER stress and mitochondrial stress in the process.Secondly,in vitro study,we observed the toxic effect of Hcy on ER stress in H9C2 cardiomyocytes,and investigated whether ER stress induced by Hcy directly modulated cardiomyocyte apoptosis.Thirdly,in vitro study,we observed the toxic effect of Hcy on mitochondrial function in H9C2 cardiomyocytes,and investigated whether mitochondrial dysfunction induced by Hcy promoted cardiomyocyte apoptosis;mainwhile,preliminarily explored the signaling transduction mechanisms between ER stress and mitochondrial dysfunction induced by Hcy.Fourthly,in vivo and vitro study,the protective effect of taurine on cardiomyocytes exposed to Hcy and the mechanism underlying this effect were explored.Part 1: Animal ExperimentThe molecular mechanisms underlying the adverse effect of HHcy on cardiac structure and function,and the opposite effects of taurine in this process in ratsAims: This study was designed to examine the adverse effect of HHcy on cardiac structure and function in rats,and investigate the functional role of ER stress and mitochondrial stress underlying this process;meanwhile,investigate the protective role of taurine in cardiomyocytes exposed to HHcy.Methods: Thirty Wistar rats were randomly divided into three groups.The control groups: rats were fed with ordinary pellet feed and tap water.The hypermethionine groups: rats were fed with ordinary pellet feed containing 3% methionine and tap water.The taurine groups: rats were fed with ordinary pellet feed containing 3% methionine and tap water containing 2% w/v taurine.Each group of ten rats and sacrificed these rats in the end of 20 th week.Several indexes were evaluated,including heart rate,systolic arterial pressure,serum Hcy concentration,ultrasonic cardiogram,cardiac systolic and diastolic function,and the concentration of taurine in cardiac muscle tissue.Western blot was used to examin the expression of GRP78,CRT,CHOP,caspase-12,Bcl-2,Bax,caspase-3 and TAUT proteins.Immunohistochemistry was used to detect the expression of GRP78,CRT,Bcl-2 and Bax in cardiac muscle tissue.Apoptotic cardiac muscle cells were detected by means of TUNEL.The ultrastructure of cardiac muscle cells were observed by means of scanning electron microscope.Results: Our results showed that hypermethionine feeding significantly increased the Hcy concentration(p<0.01)and markedly decreased the taurine concentration of cardiac muscle tissue(p<0.01),resulted in an obvious decrease in cardiac function indexs,including LVEF,LVFS,LVSP and ądp/dtmax(p<0.05 or p<0.01)and an increase in LVEDP(p<0.05).Hypermethionine feeding significantly increased the expression of GRP78,CRT,CHOP,cleaved caspase-12,cleaved caspase-3 and Bax/Bcl-2 proteins(p<0.05 or p<0.01),whereas obviously decresed the expression of TUAT protein(p<0.01).Furthermore,hypermethionine feeding led to apoptotic cardiac muscle cells,and caused swelled mitochondria and disordered arrangement of myocardial fibers.Taurine intervention increaseed cardiac function indexs,including LVEF,LVFS,LVSP and ądp/dtmax(p<0.05 or p<0.01)and a decrease in LVEDP(p<0.05),downregulated the expression of GRP78,CRT,CHOP,cleaved caspase-12,cleaved caspase-3 and Bax/Bcl-2 proteins(p<0.05 or p<0.01),whereas upregulated the expression of TUAT(p<0.05).Taurine intervention also decreased apoptotic cardiac muscle cells and improved myocardial ultrastructure.Conclusion: HHcy can induced myocardial apoptosis,exert an adverse effect on cardiac structure and function,the activation of ER stress and mitochondrial stress might be the proposed molecular and cellular mechanisms underlying this process;and the adverse effect of HHcy could be prevented by taurine.Part 2: Cell ExperimentHcy-induced endoplasmic reticulum stress and mitochondrial stress modulated cardiomyocyte apoptosis,and taurine ameliorated the adverse effect of homocysteine in H9C2 cardiomyocyteAims: This study was designed to investigate the adverse effect of Hcy on ER stress and mitochondrial stress,explore the underlying relationship between ER stress,mitochondrial stress and cardiomyocyte apoptosis;to investigate the protective effect of taurine on cardiomyocyte and explore the related protective mechanisms of taurine in H9C2 cardiomyocyte.Methods: H9C2 cardiomyocytes were treated with increasing Hcy concentration or prolonged Hcy treatment duration.H9C2 cardiomyocytes were pretreated with 4-PBA,and were then co-treated with Hcy or thapsigargin.H9C2 cardiomyocytes were pretreated with salubrinal or transfected by specific si RNA against PERK gene,and were then co-treated with Hcy.H9C2 cardiomyocytes were pretreated with taurine,and were then co-treated with Hcy.CCK-8 was used to measure cell viability.Annexin V-FITC/PI double staining was used to detect cell apoptosis.Western blot was used to examin the expression of ER stress related proteins.Ca2+ fluorescent probe was used to detect the change of Ca2+ concentration in cytoplasm.The membrane potential-specific dye JC-1 was used to detect mitochondrial membrane potentiall(??m).DCFH-DA staining was used to detect cytoplasmic ROS.Mito SOX Red mitochondrial superoxide indicator was used to measure mitochondrial ROS production.Optical microscopy Scanning electron microscope was used to observe the ultrastructure of cardiomyocytes.Results:(1)Our results showed that Hcy treatment resulted in a decrease of cell viability and an increase of cell apoptotic rates in a time-and dose-manner.Hcy 1.0-4.0 m M treatment significantly decreased cell viability(p<0.01),whereas increased cell apoptotic rate(p<0.05,p<0.01).(2)Hcy 2.0 m M treatment can induce intracellular Ca2+ turbulence.(3)Lower Hcy treatment concentration or short-term treatment duration increased the expression of GRP78,p-e IF2?,p-IRE1 and p-PERK proteins,whereas decreased the expression of ATF6 p90 protein(p<0.05,p<0.01).Higher Hcy treatment concentration or plonged treatment duration resulted in a significant up-regulation of the expression of ATF4,CHOP,p-JNK and cleaved caspase-12 proteins(p<0.05,p<0.01).(4)Higher Hcy treatment concentration resulted in the collapse of the ??m,significantly up-regulated the expression of Bax/Bcl-2 protein ratio,cytoplasmic cytochrome C,cleaved caspase-3 and cleaved caspase-9,and induced an increased production of mitochondrial ROS and caused accumulating cytoplasmic ROS.(5)Compared with Hcy or thapsigargin treatment,pretreatment with 4-PBA resulted in a significant decrease of the expression of GRP78?cleaved caspase-12,p-JNK and CHOP proteins(p<0.05,p<0.01),and led to a significant decrease of Hcy or thapsigargin-induced cardiomyocytes apoptosis(p<0.01).(6)Compared with Hcy treatment,pretreatment with salubrinal resulted in a significant decrease of the expression of ATF4 and CHOP proteins(p<0.01),and decreased Hcy-induced cardiomyocytes apoptosis from 15.5% to11.7%(p<0.05).Compared with Hcy treatment,si-PERK resulted in a significant decrease of the expression of ATF4 and CHOP proteins(p<0.01),and decreased Hcy-induced cardiomyocytes apoptosis from 16.5% to 10.6%(p<0.05).(7)Blocking the PERK pathway partly alleviated the expression of mitochondrial stress related proteins Bax/Bcl-2 ratio and cleaved caspase-3 in H9C2 cardiomyocytes(p<0.01,p<0.05).(8)Taurine pretreatment obviously increased cell viability(p<0.01)and decrease cell apoptosis(p<0.01)compared with the Hcy threatment group.Taurine pretreatment significantly downregulated the expression of GRP78,CHOP,cleaved caspase-12,cleaved caspase-9 and Bax/Bcl-2 proteins compared with the Hcy treatment group(p<0.01),and obviously increased the expression of p-e IF2?,p-JNK,cleaved caspase-9 and cleaved caspase-3 proteins(p<0.05).Taurine pretreatment obviously increased the ??m,and decreased mitochondrial ROS production.Furthermore,Taurine pretreatment effectly alleviated the pathological phenomenon,including ER swelling,and mitochondrial swelling and fusion of H9C2 cardiomyocytes induced by Hcy.Conclusion:(1)Our results suggest that ER stress could be the crucial molecular mechanism mediating Hcy-induced cardiomyocyte apoptosis,and that the PERK signaling pathway played an important role in ER stress-modulated cardiomyocyte apoptosis.(2)Hcy induced mitochondrial stress,caused mitochondrial dysfunction,which might be promopt H9C2 cardiomyocytes death.Furthermore,intracellular Ca2+ turbulence induced by Hcy and PERK signaling pathway might be the bridge linking ER stress and mitochondrial dysfunction.(3)Taurine could prevent from Hcy-induced H9C2 cardiomyocytes apoptosis through ameliorating ER stress and mitochondrial dysfunction. |
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