| BackgroundDiabetic cardiomyopathy (DCM) describes the diabetes-associated changes in the structure and function of the myocardium that is not directly attributable to other diseases such as coronary artery disease or hypertension. DCM which was first introduced in1972by Rubler is characterized by myocardial dilatation and hypertrophy, as well as a decrease in the systolic and diastolic function of the left ventricle. The pathogenesis of DCM is a chronic and complex process which is attributed to abnormal cellular metabolism and defects in organelles such as endoplasmic reticulum, mitochondria, and myofibril, and then lead to the cardiocytes hypertrophy and apoptosis at last. Apoptosis, as a regulated, energy-dependent, cell suicide mechanism has already been reported to play a critical role in the progression of DCM.Apoptosis is essentially a programmed cell death which can activate the innate mechanism through a series of signaling pathways and at last lead to the death of cell itself. Both apoptosis and proliferation can regulate the self-renewing of the cells and keep the homeostasis of the internal environment. Although the hypertrophy, proliferation and apoptosis of cardiocytes may be found in the early period of DCM, cardiac function can still maintain normal. But with the development of DCM, the apoptosis of cardiocytes will exceed the levels of proliferation, and thus leading to the massive loss of cardiocytes and the serious cardiac dysfunction. Apoptosis is regulated by a series of complex chain reaction pathways. One of the most important patheways is caspase-mediated apoptosis pathway including death receptor pathway, mitochondrial pathway and newly found endoplasmic reticulum (ER) stress associated pathway.ER is a central organelle entrusted with lipid synthesis, calcium homeostasis, protein folding and maturation. Most secreted and membrane proteins of eukaryotic cells are translocated into the lumen of ER. And the ER lumen provides a specialized environment for posttranslational modification and folding of many proteins. Various factors that interfere with ER function lead to accumulation of unfolded proteins, including oxidative stress, ischemia and disturbance of calcium homeostasis. This process is called ER stress and further activates the unfolding protein response (UPR). The UPR initially tries to restore normal function of the cell by halting protein translation and activating the signaling pathways that lead to increasing the production of molecular chaperones involved in protein folding. If these objectives are not achieved within a certain time lapse or the disruption is prolonged, the UPR tries to turn on apoptotic pathway.The X-box binding protein1(XBP1) was originally identified as a basic region-leucine zipper (bZIP) protein capable of binding to the cis-acting X box present in the promoter regions of human major histocompatibility complex gene. In mammalian cells, XBP1is a key signal transducer in the endoplasmic reticulum (ER) stress response. In response to ER stress, XBP1mRNA undergoes IRE1-dependent splicing and results in the conversion of a261amino acid unspliced XBP1protein (XBP1-u) to a376amino acid spliced XBP1protein (XBP1-s). XBP1-s, a potent transactivator, translocates into the nucleus where it binds to its target sequence to regulate UPR gene expression. Transient ER stress activation mediated by XBP1splicing may increase proliferation of cells, while sustained activation leads to apoptosis. Under the condition of DCM, insulting factors of ER stress are prone to increase, including hyperglycemia, oxidative stress and cellular electrolyte disorder. And the persistent activation of ER stress will result in the cardiocyte apoptosis at last. However, the mechanism of cardiocyte apoptosis induced by ER stress and whether XBP1can play a key role on regulating ER stress in this process are still poor understanding.Valsartan, a selective AT1receptor antagonist, has been widely used to step down blood pressure and to modify other cardiovascular actions of Ang â…¡. It has been reported recently that valsartan can lower the levels of blood glucose significantly through improving glucose utilization in streptozotocin (STZ)-induced diabetic rats. Therefore, we establish the DCM rat model treated with valsartan and the aim of our research is to explore the role of valsartan on improving cardiac remodeling in the pathogenesis of DCM. Meanwhile, the relationship between the protective effect of valsartan and the expression of XBP1has also been investigated. Our research will provide a new theoretical support on understanding the pathological mechanism of DCM and may be helpful in designing novel therapeutic strategies against cardiocyte apoptosis mediated by ER stress.Objectives1. To establish the DCM rat model, and then demonstrate the increase of apoptotic myocardial cells in the progression of DCM.2. To investigate the activation of ER stress in the myocardium of DCM.3. To elucidate the involvement and the signaling pathway of XBP1in the process of myocardial apoptosis in DCM.4. To evaluate the effect and the mechanism of valsartan on preventing myocardial apoptosis in DCM, and then observe the relationship between the treatment of valsartan and the expression of XBP1.Methods1. Establishment of diabetic rat modelAfter one week of acclimatization,50male Wistar rats (8weeks age, body weight 260-280g) were divided into two groups randomly, normal control (n=10) and diabetes mellitus (DM) group (n=40). The rats in DM group were given a single intraperitoneal injection with streptozotocin (STZ, dissolved in citrate buffer, pH4.5) at the dose of65mg/kg, while the rats in normal control group were given the same dosage of citrate buffer (pH4.5). Seven days post-STZ injection, blood samples were obtained from the rat tail vein after12hours of fasting, and the rats with glucose concentration higher than16.7mmol/L (300mg/dl) were chosen into DCM group. Those did not reach the standard of glucose concentration were removed. The rats in DCM group were divided into the two subgroups, DCM group which were treated with saline and DCM plus valsartan treatment group were treated with valsartan at dosage of30mg/kg/d. Normal control group were treated with saline throughout the entire experiment. All rats were killed after another16weeks.2. Biological measurements of each groupAll the rats have their body weight and tail blood pressure measured per2weeks. Except for being detected per4weeks regularly, the levels of blood glucose were also checked at the time points of one week after STZ injection and the end of the experiment.3. Echocardiogram examinationAt the beginning and the end of the study, echocardiogram was used to evaluate the cardiac function. Ejection fraction (EF), E/A, left ventricular internal dimension in systole (LVIDs), left ventricular internal dimension at diastole (LVIDd), interventricular septum thickness (IVST), left ventricular posterior wall (LVPW), fractional shortening (FS), deceleration time of the E wave (EDT) and isovolumic relaxation time (IVRT) were calculated.4. Histopathologic staining of myocardiumAfter being killed, the materials of the DCM rats were drawn from tissue and then fixed, dehydrated, paraffin-embedded and sliced in turn. Hematoxylin and eosin (H&E), tricrome Masson and Sirius red staining were used to observe the cardiac morphology and the distribution of collagen.5. TUNEL staining Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL) staining was used to detect the apoptosis of myocardial cells in the DCM rats.6. ImmunofluorescenceParaffin sections of the hearts were chosen to observe the expression of glucose-regulated protein78kDa (GRP78), a biomarker which could represent the activation of ER stress, through immunofluorescence analysis. Data were analyzed by use of ImagePro-Plus software.7. Western blot analysisTotal proteins of the renal samples were extracted and conducted10%-15%SDS-PAGE. Then Western blot analysis was used to measure the protein expression levels of GRP78, CHOP, XBP1-s, Puma, cleaved caspase3and cytochrome c.8. Real time RT-PCR and semi-quantitative RT-PCR analysisReal time RT-PCR was used to detect the mRNA levels of GRP78and CHOP. Meanwhile, semi-quantitative RT-PCR was used to detect the expression of XBP1-s mRNA.Results1. General features of the experimental ratsThroughout the whole experiment,3rats was not up to the standards of diabetes,5diabetic rats died and might be caused by diabetic ketoacidosis, infection or other diabetic complications. Finally42rats survived and10from normal control group,15from DCM group and17from DCM plus valsartan treatment group.Among the entire experiment, the rats in normal control group had the body weight added remarkably, react cute and have a colorful white fur. The rats in DCM group emerged symptoms of more drink, polyuria, more food and the loss of weight. In addition, spirit languishment and matt fur were also found. The status of rats was better in DCM plus valsartan treatment group than the DCM group.2. The measurement of biological indicatorsThere were no significant differences in aspects of blood glucose, body weight and tail blood pressure at the beginning of the experiment (P>0.05). Compared to the normal control group, the concentrations of glucose blood in DCM group were higher than16.7mmol/L persistently (P<0.05). Meanwhile, with the extending of time, body weight were gradually reduced and blood pressure were gradually increased in DCM group (P<0.05). At the end of experiment, DCM plus valsartan treatment group had a lower levels of blood glucose and blood pressure, while had a higher level in the body weight than DCM group (P<0.05).3. Echocardiogram examinationAt the beginning and the end of the study, echocardiogram was performed among groups. The Echocardiogram examination indexes include LVIDs, LVIDd, EF, E/A, FS, EDT, IVRT, IVST and LVPW. At the beginning, all of indexes in the three groups had no difference (P>0.05). While at the end of experiment, compared with normal control group, DCM group had a significant increase on the value of LVIDs, LVIDd, FS, IVST, LVPW, IVRT'and EDT'(PO.05) and a remarkable decrease on the value of EF and E/A (PO.05). Compared with DCM group, the results of LVIDs, LVIDd, FS, IVST, LVPW, IVRT'and EDT'in DCM plus valsartan treatment group fell dramatically (P<0.05), while the value of EF and E/A increased significantly (P<0.05).4. Changes of myocardial morphology and histologyH&E staining:The cardiocytes in normal control group arranged regularly, and the staining of cytoplasm was homogeneous. While in DCM group, cardiocytes were hypertrophic, disordered and interstitial fibring. Compared with DCM group, the hypertrophy, irregular arrangement and interstitial fibrosis of cardiocytes had been ameliorated significantly in DCM plus valsartan treatment group (P<0.05).Masson staining:Myocardial collagenous fibers of the rats in normal control group arranged in order. Collagen deposition was obviously increased in the myocardium of rats in DCM group than normal control group. And compared with DCM group, cardiac fibrosis in DCM plus valsartan treatment group alleviated obviously (P<0.05). Sirius red staining:Type â… collagen is in colour red or orange and type â…¢ collagen is usually in colour green under the polarization microscope. Much more collagen â… and â…¢ existed in the myocardium of rats in DCM groups than normal control group, which decreased significantly in the DCM plus valsartan treatment group (P<0.05).5. Detection of cardiocytes apoptosis levelsTUNEL staining was used to investigate the levels of myocardial apoptosis. Meanwhile, the biomarkers of apoptosis such as Puma, cleaved caspase3and cytochrome c were detected by the use of Western blot. And the results showed that compared with normal control group, the levels of myocardial apoptosis in DCM group increased significantly, which decreased obviously in the DCM plus valsartan treatment group (P<0.05).6. Expression of GRP78and CHOP in myocardiumThrough immunofluorescence, Western blot, the expression of GRP78and CHOP in myocardium was detected. Our data showed that compared with normal control group, the expression of GRP78and CHOP in DCM group up-regulated significantly, which reduced obviously in the DCM plus valsartan treatment group (P<0.05). These results were also confirmed by real time RT-PCR (P<0.05), and thus suggesting the activation of ER stress in DCM and the inhibitory role of valsartan on ER stress.7. Activation of XBP1in myocardiumThe data of Western blot and semi-quantitative RT-PCR demonstrated that compared with normal control group, the expression of XBP1-s in DCM group increased significantly, which decreased obviously in the DCM plus valsartan treatment group (P<0.05). Our research suggested that the expression of XBP1was activated in the process of ER stress and the treatment of valsartan could inhibit the activation of XBP1.Conclusions1. In the present study, we obtained DCM rats successfully by using a single intraperitoneal injection with STZ, which provided a reliable animal model for the research of DCM.2. Cardiocytes apoptosis and myocardial remodeling were obviously in the progression of DCM, and apoptosis may play a key role in this process.3. The increased expression of GRP78and CHOP in myocardium demonstrated the activation of ER stress in DCM.4. The expression of XBP1was up-regulated in the process of DCM, which suggested the involvement of XBP1mediated ER stress in this process.5. Valsartan could reduce the cardiocytes apoptosis and ameliorate myocardial remodeling through inhibiting activation of XBP1-s in DCM rats. BackgroundDiabetic cardiomyopathy (DCM) is one of the most important complications of diabetes mellitus, which is characterized by the hypertrophy and apoptosis of cardiocytes and interstitial fibrosis. In the early stage of DCM, the main change of cardiocytes is hypertrophy. But with the development of the disease, apoptosis which will result in massive reduction of cardiocytes become dominant, and then lead to the occurrence of DCM.Unfolding protein response (UPR) involved in the process of endoplasmic reticulum stress (ER) is closely related to the metabolism of glucose and lipid in diabetes mellitus. Under the status of dysbolism, as the main organelles of protein and lipid synthesis, the overload of ER can trigger UPR and then result in the happening of ER stress. ER stress is a key point in the glycolipid metabolic disorders. Meanwhile, the energy metabolism disorder is one of the leading causes of cardiocyte apoptosis in the progression of DCM. Therefore, ER stress may play a crucial role in cardiocyte apoptosis.The X-box binding protein1(XBP1) is a key signal transducer in the ER stress response. The persistent activation of XBP1will lead to the occurrence of apoptosis finally. C/EBP homologous protein (CHOP) is an important transcription factor of basic region-leucine zipper (bZIP). CHOP can be expressed in various kinds of mammalian cells and participate in many pathophysiological processes such as cellular metabolism, proliferation and apoptosis. CHOP pathway is one of the three chief signaling pathways of ER stress which can induce the cellular apoptosis. CHOP proximal promoter contains XBP1-s binding sites, which suggests that the activation of XBP1-s may induce the transcription and expression of CHOP gene. However, whether the activation of CHOP transcription is related to XBP1-s in the progression of DCM is still poor understanding.As the "plant of power", mitochondrion is also one of the most important organelles which can mediate the apoptosis of cells. CHOP may induce the apoptosis through mitochondrion-dependent apoptosis pathway. Mitochondrion-dependent apoptosis pathway is regulated by Bcl-2family members, while p53up-regulated modulator of apoptosis (Puma) is just a newfound member of pro-apoptosis BH3-only Bcl-2family. In the normal condition, the expression of Puma is very low. While the stress responses such as DNA damage happen, the expression level of Puma can be up-regulated significantly through both p53-dependent and-independent pathways. The increased level of Puma can activate mitochondrion-dependent apoptosis pathway and then lead to the apoptosis of cells. Therefore, Puma plays a key mediated role in the process of cellular apoptosis. However, whether or not CHOP and Puma can be up-regulated in the ER stress and which upstream factors can activate of Puma transcription still need more further investigation.Basing on above discussion, we propose the hypothesis that ER stress in DCM can up-regulate the expression of CHOP through the activation of XBP1-s, and then induce the transcription of Puma, contributing to cardiocyte apoptosis. Focusing on this subject, we induce the occurrence of ER stress in rat primary cardiocytes by the use of high D-glucose stimulation. The role and relationship among XBP1, CHOP and Puma in the process of cardiocyte apoptosis mediated by ER stress will be investigated, and thus providing a new theoretical support on elucidating the relative mechanism of cardiocyte apoptosis mediated by ER stress.Objectives 1. To demonstrate the influence of high D-glucose on the occurrence of ER stress and apoptosis in the rat primary cardiocytes.2. To investigate the role of XBP1, CHOP and Puma in the process of cardiocyte apoptosis mediated by ER stress.3. To elucidate the relationship among XBP1, CHOP and Puma expression in the process of cardiocyte apoptosis mediated by ER stress.Methods1. Neonatal rat primary cardiocytes cultureNeonatal rat primary cardiocytes were obtained by the following procedures:the hearts from2-3-day-old Wistar rats were minced finely and placed in trypsin. The pooled cell suspensions were centrifuged and resuspended in DMEM supplemented with10%fetal bovine serum and then plated onto culture flasks, which could allow for selective adhesion of cardiocytes to the bottom of the culture flask. The majority of cultured cardiocytes (>90%) began to contract spontaneously within24-48h of adhesion (130-150beats/min).2. Identification of neonatal primary cardiocytesThe antibody of cardiac troponin T was used to identify the neonatal primary cardiocytes through immunofluorescence.3. Experiment designNeonatal rat cardiocytes were divided into different groups:control group, high D-glucose with different concentration gradients (5.5,16.7and33.3mmol/L) groups, mannitol (27.8mmol/L) group, high D-glucose (33.3mmol/L) plus CHOP siRNA group, high D-glucose (33.3mmol/L) plus XBP1siRNA.4. Small interfering RNA (siRNA)The siRNA of XBP1and CHOP were used to observe the influence of XBP1and CHOP activation in the ER stress mediated cardiocyte apoptosis. Meanwhile, the relationship of XBP1, CHOP and Puma was also investigated by using the siRNA technique.5. Detection of the cardiocyte apoptosis The rate of the cardiocyte apoptosis were detected by the use of flow cytometer.6. Real time RT-PCR and semi-quantitative RT-PCR analysisTotal RNA was extracted from neonatal rat cardiocytes by the use of Trizol, and then reverse transcribed. Real time RT-PCR was used to detect the mRNA levels of GRP78, CHOP and Puma. And semi-quantitative RT-PCR was used to detect the expression of XBP1-smRNA.7. Western blotAfter being stimulated respectively, neonatal rat cardiocytes were harvested for protein extraction. Western blot was used to detect the protein expression levels of GRP78, XBP1-s, CHOP, Puma, cleaved caspase3and cytochrome c.Results1. High D-glucose stimulation induces cardiocyte apoptosis in a dose-dependent manner.Neonatal rat cardiocytes were treated with different concentration gradients (5.5,16.7and33.3mmol/L) of D-glucose and mannitol (27.8mmol/L) for48hours. The rate of the cardiocyte apoptosis were detected by the use of flow cytometer. Compared with the group treated with5.5mmol/L D-glucose, the apoptosis rate of high D-glucose group (16.7and33.3mmol/L) were increased significantly (P<0.05), while the group treated by mannitol (27.8mmol/L) had no uncommon difference (P>0.05). These results indicate that the induction of cardiocyte apoptosis by high D-glucose is in a dose-dependent.2. High D-glucose stimulation triggers ER stress in neonatal rat cardiocytes Neonatal rat cardiocytes were treated with different concentration gradients (5.5and33.3mmol/L) of D-glucose and mannitol (27.8mmol/L) for48hours. By using real time RT-PCR and Western blot analysis, our data showed that the expression of GRP78were up-regulated in the high D-glucose group (33.3mmol/L) at both mRNA and protein levels compared with the control group (5.5mmol/L D-glucose) which suggested the activation of ER stress in this process (P<0.05). Meanwhile, the expression of GRP78in the group treated by mannitol (27.8mmol/L) had no uncommon difference (P>0.05).3. High D-glucose stimulation up-regulates the expression of XBP1-s, CHOP and Puma in the progression of cardiocytes apoptosis Neonatal rat cardiocytes were treated with different concentration gradients (5.5and33.3mmol/L) of D-glucose and mannitol (27.8mmol/L) for48h. By using semi-quantitative RT-PCR analysis, real time RT-PCR and Western blot analysis, the result showed that the expression of XBP1-s, CHOP and Puma were up-regulated in the high D-glucose group (33.3mmol/L) at both mRNA and protein levels compared with the control group (5.5mmol/L D-glucose)(P<0.05). Meanwhile, the expression of XBP1-s, CHOP and Puma in the group treated by mannitol (27.8mmol/L) had no uncommon difference (P>0.05).Since the levels of cleaved caspase3and cytochrome c could reflect the apoptosis of cells, the expression of cleaved caspase3and cytochrome c in neonatal rat cardiocytes was also investigated. Compared with the control group (5.5mmol/L D-glucose), both cleaved caspase3and cytochrome c were up-regulated in the high D-glucose group (33.3mmol/L)(P<0.05). Meanwhile, the expression of them in the group treated by mannitol (27.8mmol/L) had no uncommon difference (P>0.05). Thus, our data demonstrated that cardiocyte apoptosis mediated by ER stress is closely related to the up-regulations of XBPl-s, CHOP and Puma.4. XBP1siRNA and CHOP siRNA can inhibit the cardiocyte apoptosis induced by high D-glucose stimulationAfter being transfect by XBP1siRNA for24h, semi-quantitative RT-PCR and Western blot analysis showed that the expression levels of XBP1-s was down-regulated significantly at both mRNA and protein levels (P<0.05) compared with the control group. Our result demonstrated that the siRNA technique could down-regulate the expression of XBP1-s effectively.After being transfect by CHOP siRNA respectively for24h, real time RT-PCR and Western blot analysis showed that the expression levels of CHOP was down-regulated significantly at both mRNA and protein levels (P<0.05) compared with the respective control group. Our result demonstrated that the siRNA technique could down-regulate the expression of CHOP effectively.After being treated with different concentration gradients (5.5and33.3mmol/L) of D-glucose and the siRNA of XBP1and CHOP respectively, the rate of the neonatal rat cardiocyte apoptosis were detected by the use of flow cytometer. Compared with the group treated with5.5mmol/L D-glucose, the apoptosis rate of high D-glucose group (16.7and33.3mmol/L) were increased significantly (P<0.05). Meanwhile, compared with the group treated with33.3mmol/L D-glucose, the group treated by the siRNA of XBP1and CHOP were reduced significantly (P<0.05). These results indicate that both XBP1siRNA and CHOP siRNA can inhibit the cardiocyte apoptosis induced by high D-glucose stimulation. The activation of XBP1and CHOP may play a key role in the progression of the cardiocyte apoptosis mediated by ER stress.5. High D-glucose stimulation induce the cardiocyte apoptosis through the XBP1/CHOP/Puma signaling pathwayAfter being transfect by XBP1siRNA for24h, neonatal rat cardiocytes were treated with D-glucose (33.3mmol/L) for another48h. Through using semi-quantitative RT-PCR, real time RT-PCR and Western blot analysis, our results showed that the expression of XBP1-s was down-regulated by XBP1siRNA effectively (P<0.05). At the same time, with reduced XBP1-s level, the expression of CHOP and Puma were also down-regulated significantly (P<0.05), suggesting that the activation of CHOP and Puma could be regulated by XBP1-s and both of them could be the downstream factors of XBP1.After being transfect CHOP siRNA for24h, neonatal rat cardiocytes were treated with D-glucose (33.3mmol/L) for another48h. By the use of semi-quantitative RT-PCR, real time RT-PCR and Western blot analysis, our data showed that the expression of CHOP was down-regulated by CHOP siRNA effectively (P<0.05). Meanwhile, the expression of XBP1-s and Puma were also been observed. Our data showed that the expression of XBP1-s was not influenced by the reduced level of CHOP (P>0.05), suggesting that XBP1could be the upstream factor of CHOP. But accompany with the reduced level of CHOP, the expression of Puma was down-regulated significantly (P<0.05), suggesting that the activation of CHOP was necessary for the Puma transcription and Puma could be the downstream factors of CHOP. Based on the results mentioned above, we come to a conclusion that there must be a signaling transduction relationship among the XBP1, CHOP and Puma. And the XBP1/CHOP/Puma signaling pathway play a crucial role in the the cardiocyte apoptosis induced by high D-glucose stimulation.Conclusions1. In the present study, high D-glucose stimulation can induce cardiocyte apoptosis through activating ER stress.2. XBP1plays a crucial role in cardiocyte apoptosis mediated by ER stress.3. Cardiocyte apoptosis mediated by ER stress is closed related to XBP1/CHOP/Puma signaling pathway.
|
PDF Full Text Request