The Role Of Endoplasmic Reticulum Stress In The Pathogenesy Of Diabetic Cardiomyopathy

PARTâ… Endoplasmic Reticulum Stress Is Involved in Myocardial Apoptosis of Sreptozocin-Induced Diabetic RatsBackgroundIn humans and animal models of diabetes,a heart muscle-specific disease in the absence of any vascular pathology has been described,and termed diabetic cardiomyopathy.The pathogenesis of diabetic cardiomyopathy is a chronic and complex process that is attributed to abnormal cellular metabolism and defects in organelles such as myofibrils, mitochondria,and sarcolemma.However,the mechanisms of diabetic cardiomyopathy are not fully known,and appropriate approaches to minimize these risks are still being explored.Apoptosis,as a regulated,energy-dependent,cell suicide mechanism has been reported to play a critical role in the development of diabetic cardiomyopathy.Endoplasmic reticulum(ER)is an organelle involved in the intrinsic pathway of apoptosis and it is involved in several important functions such as the folding of secretory and membrane proteins.Various conditions can disturb the functions of the ER and result in ER stress (ERS).These conditions include ER-Ca²⁺depletion,ischemia,hypoxia,exposure to free radicals,elevated protein synthesis,hyperhomocysteine,and gene mutation.Several signaling pathways are initiated to cope with ERS,which are designated as the unfolded protein response.One major pathway of UPR is to increase regulation of the expression of ER-localized molecular chaperons,such as glucose-regulated protein 78(GRP78), which can contribute to repairing unfolded proteins.The early UPR enhances cell survival by ensuring that the adverse effects of ERS are dealt with in a timely and efficient manner.When ERS conditions persist,initiation of apoptotic processes are promoted by transcriptional induction of CCAAT/enhancer-binding protein(C/EBP) homologous protein(CHOP/GADD153),the Caspase12-dependent pathway,and activation of the c-Jun NH2-terminal kinase(JNK)-dependent pathway.Evidence has demonstrated that apoptosis initiated by the ERS was involved in the pathogenesis of heart failure and diabetic kidney.The ERS has been found in cultured hepatocytes,monocytes,and smooth muscle cells with both glucose and glucosamine independent of increased O-linked glycosylation.Actually,hyperglycemia environment also induces several conditions that can invoke ERS.It was reported that the ER Ca²⁺ store and rates of Ca²⁺release and resequestration into ER were found depressed in diabetic rat myocytes because hyperglycemia inhibits the capacitative calcium entry,and there are direct correlations existing between hyperglycemia and oxidative stress and hyperglycemia and hyperhomocysteine.Ultrastructural analysis found ER swelling and dilation in the diabetic model myocardium. Dilated ER is a change in morphology that suggests ERS,but the relationship between ERS and diabetic cardiomyopathy is unknown.Objective1.To establish a DCM animal model.2.To observe the changes of cardiac structure and cardiac function in DCM model.3.To observe the expression of apoptotic cells in the myocardium.4.To observe the expression of ERS-relative factors in the myocardium in diabetic rats and controls.Methods1.Establishment of DCM animal modelTwenty-seven male Wistar rats were randomly divided into 2 groups:control group (n=10)and DCM group(n=20).Diabetes was induced by a single intraperitoneal injection of STZ(65 mg/kg body wt and dissolved in 0.1 mol/l citrate buffer,pH 4.2)in rats of DCM group.Control rats received citrate buffer alone.One week after injection of STZ,fasting plasma glucose levels were measured,and rats with plasma glucose at least two times higher than 16.7mmol/L were used.All rats were fed for 5 months after STZ or citrate buffer injections and had free access to standard rat diet and water.2.Echocardiogram examinationAt the beginning and at the end of the study,transthoracic echocardiogram was performed in diabetic and control animals.Rats were placed supine and the anterior chest wall was shaved.Echocardiograms were performed with a Hewlett-Packard Sonos 7500 sector scanner equipped with a 7.5-MHz phased-array transducer.Conventional images included 2-dimensional,M-mode,and continuous wave and pulsed Doppler images. 3.HE staining and Masson stainingHE staining was used to study the pathological changes and Masson staining was used to quantify the collagen content in this study.4.TUNEL stainingTUNEL staining was used to detect the expression of apoptotic cells.5.Real-time RT-PCRThe total RNA was extracted from left ventricles,right ventricles or atriums of control and diabetic rats.The mRNA expressions of GRP78/CHOP/Caspase12 were determined by real-time RT-PCR.6.ImmunohistochemistryImmunohistochemistry was used to detect the protein expression of GRP78.7.Western-blot analysisWestern-blot analysis was used to determine the protein expression of GRP78/CHOP/Caspase12/p-JNK.Results1.Establishment of the diabetic cardiomyopathy modelThe left ventricular systolic function parameters,fractional shortening(%),and ejection fraction of the diabetic animals were significantly reduced when compared with their week 1 levels and normal animals at 20 weeks.Left ventricular diastolic function variables expressed by the E-wave(early diastolic filling and early peak velocity)and A-wave(late atrial filling and atrial peak velocity)differed significantly in diabetic animals when compared with their week 1 levels and normal animals at 20 weeks.A significant decrease in the E-wave velocity,significant increase in the A-wave velocity, and a significant decrease in the E/A ratio was found,after 20 weeks in the diabetic groups,with a significant decrease in the fractional shortening and ejection fraction.The altered cardiac diastolic performance is thought to result from a reduced cardiac compliance.2.HE staining and Masson stainingWith hematoxylin-eosin staining and Masson staining,we found that diabetic cardiac muscle fibers were disordered,and many of them were collapsed.Diabetic myocardium showed fibrosis and extensive focal coalescent areas of ischemic myocyte degeneration in the subendocardial,subepicardial region,and papillary muscles of the myocardium.The above results certified that our diabetic rats suffered from diabetic cardiomyopathy. Localization of apoptosis by TUNEL assay and IHC analysis of GRP78 distribution3.TUNEL stainingTo assess whether diabetes results in apoptotic cell death in the diabetic myocardium,the tissue sections were labeled with an in situ TUNEL assay.Apoptosis was observed in both the cardiomyocyte and endothelium of the diabetic heart.Estimation of cardiac apoptosis revealed a nearly sevenfold increase in TUNEL-positive nuclei in diabetic hearts.4.RT-PCR analysis of GRP78,CHOP and Caspase12 expressionChanges in RNA expression of GRP78,CHOP,and Caspase12 were quantified by RT-PCR in control and diabetic rat hearts(Fig.5).Expressions of GRP78,CHOP,and Caspase12 were significantly increased in the diabetic heart(P＜0.05),paralleled with their enhanced protein expression.5.ImmunohistochemistryImmunochemistry studies showed that GRP78 was abundantly expressed in the myocardium from diabetic rats.In contrast,normal rats exhibited modest or weak immunoreactivity for this molecule.We also found that in diabetic myocardium,the increase of GRP78 positive ceils paralleled with the increase of apoptotic cells.6.Western blot analysis of GRP78,CHOP,Caspase12 and p-JNKThe densitometric analysis of bands for GRP78,CHOP,Caspase12 and p-JNKRevealed a significant(P＜0.05)increase in relative protein content in myocardium from diabetic rats in comparison with those from normal rats.This means CHOP was induced, and Caspase12 and JNK pathway were activated in the diabetic heart.Conclusions1.A DCM animal model was establishen by 5 months period of STZ-induced diabetes in male Wistar rats.2.Diastolic dysfunction is characteristic in this DCM model.3.Diabetic heart has higher expression of apoptosis than controls.4.Increase expression of GRP78 CHOP,Caspase12 and p-JNK in diabetic heart paralleled with the increase of apoptotic cells. PARTâ…¡Homocysteine Induces Programmed Cell Death in Cardiomyocytes through Activation of the Unfolded Protein ResponseBackgroundHcy is a toxic non-protein sulfur containing amino acids in humans.Hcy is metabolized either through remethylation or transsulfuration pathways and is nutritionally regulated. An elevated plasma Hcy level is denoted hyperhomocysteinemia(HHcy).It has been estimated that moderate HHcy occurs in 5-7%of the general population.Evidence now indicates that moderate HHcy is an important and independent risk factor for several disorders,including atherosclerosis,diabetes,fatty liver,immune activation,and neurodegenerations such as Alzheimer's and Parkinson's diseases.Hcy induced ER stress response has recently received much attention.Hcy causes ER stress by disrupting disulphide bond formation and causing misfolding of proteins traversing the ER.Hcy could increase the expression of ER stress response genes, including,but the exact cellular targets and other related gene expression induced by Hcy are still unknown.The physiological roles of the ER include regulation of protein synthesis,folding and targeting and maintenance of cellular calcium homeostasis.ER stress is a condition under which unfolded and misfolded proteins accumulate.ER stress triggers UPR,which is an intracellular signaling pathway,mediated by glucose-regulated protein 78(GRP78).The early UPR enhances cell survival by ensuring that the adverse effects of ER stress are dealt with in a timely and efficient manner.However,prolonged UPR following ER stress has severe consequences.It can lead to activation of several ER-resident protein kinases such as inositol-requiring enzyme 1(IRE1)and TNF receptor-associated factor 2(TRAF2),which activate ER-specific Caspase12,c-Jun N-terminal kinase(JNK)pathways and induce transcription factor C/EBP homologous protein(CHOP),resulting in programmed cell death. Although HHcy is now considered to be an independent risk factor for cardiovascular diseases,most studies focus on the effects of Hcy on endothelial cell and smooth muscle cell in atherosclerosis.Whether Hcy could contribute to development of cardiomyopathy, in which cardiomyocyte apoptosis is an important pathological feather,is obscure.Our preliminary study in vivo showed the plasma Hcy had direct ratio with ER stress hallmarkers,GRP78 and Caspase12,which resulted in the apoptosis of cardiomyocytes in diabetic rat hearts.This study was designed to investigate whether ER stress and activation of Caspase12 are implicated in the proapoptotic effects of Hcy in cardiomyocytes in vitro.We hypothesized Hcy can induce apoptosis through ER stress pathway in cultured cariomyocyte.Since induction of CHOP and activation of Caspase12 are ER-specific pathways,we therefore used these two pathways as markers of induction of the ER-specific death pathway.We also examined the activation of JNK pathway in this study,because several studies showed it played important role in ER stress induced cell death.This has allowed us to identify the induction of ER stress in the Hcy induced cardiomyocyte death.Objective1.To identify that Hcy can speed apoptosis of cardiomycytes.2.To determine whether ERS take part in the Hcy induced cardiomycytes apoptosis.Methods1.Cell cultureNeonatal cardiac cells were isolated from 1-to 2-d-old Wistar rat ventricles by digestion with trypsin-EDTA.Once the sequential digestions were terminated,the cells were pooled in DMEM supplemented with 10%fetal calf serum,and seeded in 90-mm etri dishes to allow selective adhesion of cardiac fibroblasts.Thereafter,cardiomyocytes were decanted from the plates and seeded in six-well culture plates.Cell cultures were incubated at 37℃in a humidified atmosphere of 5%CO₂/95%air.The majority of cultured cardiomyocytes (＞90%)began to contract spontaneously within 24-48 h of plating(30-50 beats/min). Cells were used on passages of 2 through culture for all experiments described herein. Before stimulation,cardiomyocytes were starved for 18-24 h in serum-free DMEM.2.Study design 1)The cardiomyocytes were incubated with Hcy(0.025,0.1,1mM)for 4 to 24 h,the harvested cells were used for TUNEL stain to detect the apoptosis cells.The time and dose point for maximal effect of Hcy was used in subsequent experiments.2)To demonstrate the relation between ER stress and cardiomyocyte apoptosis,after incubated with Hcy,the expression of GRP78,Caspase12,CHOP and p-JNK(mRNA and protein)was measured.3.Measurement of ApoptosisApoptosis was determined using a commercially available ApopTag Peroxidase kit that is based on the TUNEL(TdT-mediated nick end labeling)method.Briefly,after the appropriate treatment periods,cells were washed in PBS and fixed in 1% paraformaldehyde for 10 min at room temperature.The fixative was washed away and the cells were postfixed in precooled ethanol:acetic acid mixture(2:1 vol/vol)for 3 min at -20℃.After the endogenous peroxidase with 3.0%hydrogen peroxide in TBS was quenched,the cells were labeled with the reaction buffer containing TdT-digoxigenin-nucleotide and allowed to bind with peroxidase-conjugated antidigoxigenin.The color was developed with FITC.The cells were viewed under a 40 objective lens and the cells that fell within the 100 squares of an eye graticule were counted.Apoptotic nuclei showed one or more of the following changes:condensation, hyperchromasia,crescent shape,and apoptotic bodies.Apoptotic cells were expressed as a percentage of the total cells counted.4.RT-PCR analysisThe total RNA was estracted from collected cells.The Mrna expression of GRP78, Caspase12,CHOP and GAPDH were determined by real-time PCR.5.Western Blotting AnalysisThe total protein was extracted from collected cells.Western blot analysis was used to determine the protein of GRP78,Caspase12,CHOP and p-JNK.Results1.The Time and Dose Response of Cardiomyocyte Apoptosis to HcyThe TUNEL staining result showed that more apoptotic cells were observed in the Hcy treated cardiomyocytes than in the control cardiomyocytes,and the induction of apoptotic cells to Hcy treatment was time-and-dose-dependent and that the up-regulation of an ER stress-responsive gene resembled the apoptosis cells.2.The effect of Hcy stimulation to the expression of GRP78/CHOP/Caspase12/p-JNK. Hcy induced the up regulation of GRP78,CHOP and Caspase12 monitored by real time PCR and Western blot analysis,and p-JNK was also up-regulated in Western blot analysis,suggested that Hcy activated the UPR in cardiomyocytes,leading to cell death in vitro.Conclusions1.The induction of apoptotic cells to Hcy treatment was time-and-dose-dependent.2.Hcy stimulation leads to the increase of GRP78/CHOP/Caspase12/p-JNK expression.