The Role And Mechanism Of High Mobility Group Box1Protein In Diabetic Cardiomyopathy

BackgroundDiabetes mellitus is associated with increased risk of heart failure, independent of hypertension and underlying coronary artery disease, called diabetic cardiomyopathy (DCM). DCM is characterized by functional and structural cardiac changes, including myocardial cell death and accumulation of extracellular matrix (ECM) protein. In particular, myocardial fibrosis is the most frequently proposed mechanism responsible for the cardiac changes in DCM. Although chronic hyperglycemia plays an important role in the pathogenesis of diabetic complications, the molecular mechanisms underlying cardiac fibrosis are not clear, and factors contributing to the cardiac dysfunction remain to be elucidated.High-mobility group box1(HMGB1) is a nuclear protein existing widely in almost all eukaryotic cells. In addition to its nuclear role, HMGB1is also released from activated macrophages, monocytes and necrotic cells but not apoptotic cells, which results in an inflammatory reaction and signal transduction pathway. In addition to its secretion by macrophages and monocytes, HMGB1can be secreted by other viable non-immune cells, including cancer cells, pituicytes, enterocytes, and cardiomyocytes. Therefore, it may participate in other pathological processes such as oncogenesis, proliferation and differentiation, which challenges the limited role of HMGB1as an important mediator in inflammatory cellular processes. Increasing evidence has demonstrated the multiple functions of HMGB1in various heart diseases.Serum HMGB1levels in patients with type1diabetes are significantly higher than in healthy controls so HMGB1may be a newly identified cytokine associated with DCM.Objectives1. To investigate whether high glucose could induce HMGB1translocation and secretion in mice heart.2. To investigate the role and mechanism of cardiac HMGB1in diabetic cardiomyopathy.3. To study the effect of recombinant HMGB1on cardiac fibroblasts collagen synthesis, proliferation and migration.Methods1. Lentivirus vector RNA interferenceDesigning and synthesizing3pieces of short-hairpin RNAs (shRNAs) against HMGB1according to RNAi principle. We used a lentivirus vector containing a green fluorescent protein (GFP) reporter and a U6promoter upstream of the cloning site for the most effective shRNA. The target sequence for HMGB1was5’-GGCTCGTTATGAAAGAGAAAT-3’ and negative control sequence5’-GTTCTCCGAACGTGTCACGT-3’.2. Animal model and RNA interferenceC57BL/6J wild-type (WT) mice8weeks-old (25～30g) were divided into2groups:control (n=60) and diabetes (n=15). Diabetes was induced by injecting streptozotocin dissolved in0.1ml citrate buffer (pH4.5) intraperitoneally (i.p.) at50mg/kg per mouse for5consecutive days. Control mice were injected with citrate buffer only. After7days, whole blood was obtained from the tail vein, and random glucose levels were measured. Diabetes was determined as blood glucose at least16.7mmol/1. After the induction of diabetes (12weeks), mice were divided into3groups for treatment:control, shRNA-HMGBl and shRNA-N.C for the following experiments. An amount of1×107UT/30μl of lentivector with HMGB1shRNA or the same volume of lenti-vehicle was injected into various sites of the left ventricle.3. Cardiac function measurementCardiac diameter and function was measured by use of the Vevo770imaging system.2D echocardiography, M-mode echocardiography, pulsed-wave Doppler echocardiography and tissue Doppler imaging were used to evaluate cardiac function.4. Histology and immunohistochemistryTissue was paraffin-embedded and sectioned (4μm) for staining with hematoxylin and eosin (H&E), Masson’s trichome and Picrosirius red to examine heart size and extracellular matrix (ECM) deposition. Immunohistochemistry was used to determine the levels of HMGB1, collagen Ⅰ, collagen Ⅲ and transforming growth factor-β1(TGF-β1) in myocardial tissues.5. Cell cultureCardiomyocytes and cardiac fibroblasts (CFs) were isolated from neonatal rat ventricular tissues. When cell populations reached60%confluence, cultures were exposed to high glucose (HG;15-25mM). Some cell cultures were exposed to normal glucose (NG;5.5mM) as controls.6. Laser scanning confocal microscopyImmunofluorescence analysis was used to evaluate HMGB1and Ki67expression levels and disposition.7. Real-time RT-PCRTotal RNA was extracted from cardiomyocytes and CFs. In the experiment, the mRNA expression of HMGB1in cardiomyocytes and CFs was analyzed.8. Western blottingProteins were extracted from cardiomyocytes and CFs. The protein expression of HMGB1, lactate dehydrogenase (LDH), collagen I, collagen Ⅲ, TGF-β1, matrix metalloproteinase (MMP-2), MMP-9, ERK, p-ERK, JNK, p-JNK, p38and p-p38was analyzed in our experiment.9. Gelatin zymographyThe activity of MMP-2and MMP-9in CFs was determined by zymography.10. Enzyme-linked immuno sorbent assay (ELISA)HMGB1released into cell culture supernatants was evaluated by Elisa in CFs in vitro.11. Assessment of cell proliferation and migration assay:CFs proliferation assays were determined by use of the Cell Counting Kit-8(CCK-8). CFs migration assays were performed in Transwell chambers.12. Propidium iodide staining for necrosis:Necrotic cells were stained positive for propidium iodide and negative for Annexin-V.13. Statistical analysis:All statistical analyses involved use of SPSS18.0. Data are reported as mean±standard deviation.Results1. Hyperglycemia increased the level of HMGB1and induced HMGB1translocation and secretion in mice heartDiabetic mice showed significantly increased myocardial HMGB1mRNA level and protein level. Immunohistochemistry analysis revealed HMGB1expression in nuclei of normal heart, while hyperglycemia induced HMGB1to diffuse from the nucleus to the myocardial interstitium in diabetic heart. 2. HMGB1inhibition prevented diabetes-induced myocardial remodeling and cardiac dysfunctionAfter shRNA-HMGB1treatment for4weeks, we measured cardiac structure and function. Inhibition of HMGB1mitigated heart structural alterations in type1diabetic mice. Heart weight and ratio of heart weight to body weight were lower with shRNA-HMGB1than vehicle treatment. HMGB1gene silencing attenuated the enlarged cardiomyocytes as compared with vehicle treatment. shRNA-HMGB1and vehicle-transfected diabetic mice did not differ in body weight.After16weeks of diabetes, cardiac function features were mainly diastolic dysfunction, which showed deceased E/A and E’/A’as well as thickened LVPWd. Cardiac systolic function lightly lower in diabetic than control mice, showed that deceased EF and FS. ShRNA-HMGB1treatment attenuated diabetes-induced cardiac dysfunction.3. HMGB1inhibition limited diabetes-induced myocardial fibrosisMasson’s trichome and Picrosirius red staining of heart sections revealed greater ECM in the interstitial regions of the diabetic than control myocardium. Diabetes enhanced the expression of the fibrotic markers collagen Ⅰ, Ⅲ and fibrotic TGF-β1as compared with the control. While ShRNA-HMGB1treatment reduced collagen deposition, the expression of collagen Ⅰ, Ⅲ and TGF-β1as compared with vehicle treatment4. HG induced HMGB1translocation and secretion in viable primary cardiomyocytes and CFsCardiomyocytes and CFs were exposed to various concentrations (15mM,20mM,25mM) of high glucose for48h. High glucose significantly increased levels of extracellular HMGB1in both cardiomyocytes and CFs and these effects were not due to cell necrosis. Immunofluorescence analysis revealed HMGB1expression in nuclei of both cardiomyocytes and CFs cultured under NG. As compared with NG, HG induced HMGB1to diffuse from the nucleus to the cytoplasm. HMGB1mRNA activity began to increase in cardiomyocytes after8h, peaking at24h with HG.5. HMGB1increased cardiac fibroblasts collagen systhesis, proliferation and migrationWith physiological glucose concentration, rHMGB1dose-dependent increased the protein level of collagen I, III and TGF-β1in as compared with NG with maximum effect at200ng/ml. CFs showed fast growth rate with rHMGB1treatment, which was simliar to CFs with HG. We used Transwell migration assay to evaluate CFs migration. After24h, we found prominent dose-dependent HMGB1chemotaxis of CFs. Meanwhile, pharmacological or genetic inhibition of HMGB1inhibited the HG-induced increase in cell collagen systhesis, proliferation.6. HMGB1inhibition reduced the MMP-2and MMP-9expression and activity in CFsHG increased the expression of MMP-2and MMP-9. These effects were significantly downregulated by Ab-HMGB1or shRNA-HMGB1treatment. HG had a similar effect of HMGB1as compared with NG. Gelatin zymography showed that HG increased the activity of MMP-2, and inhibition of HMGB1downregulated this effect. However, MMP-9activity was not detected. rHMGB1treatment had a similar effect of HG on MMP-2and MMP-9.7. HMGB1increased activation of ERK, JNK and Akt in CFsrHMGBl treatment increased ERK, JNK and Akt phosphorylation in CFs as compared with NG alone. While HMGB1inhibition reduced the HG-induced phosphorylation of ERK, JNK and Akt. HMGB1had no effect on the expression of p-p38. In vivo, HMGB1inhibition reduced diabetes-induced myocardium phosphorylation of ERK and JNK.Conclusion1. Hyperglycemia induced HMGB1translocation and secretion in type1diabetes mice heart.2. Silencing HMGB1gene could attenuate myocardial remodeling, fibrosis and cardiac dysfunction.3. Inhibition of HMGB1improved diabetic cardiomyopathy via ERK and JNK signal pathway. BackgroundDiabetes mellitus is an increasing worldwide systemic metabolic disease. Hyperglycemia is the major feature of diabetes mellitus and can induce organ damage such as cardiovascular disease, the most frequent cause of death in the diabetic population. The sharp rise in the incidence of diabetes in the world has become one of the major threats to human health. The rate of heart failure in diabetic patients is2-3times as large as it in non-diabetic patient. Heart failure in diabetes, which occurs independent of changes in blood pressure and coronary artery disease, is called diabetic cardiomyopathy.The process of diabetic cardiomyopathy consists of a series of sequential and interrelated steps, including myocardial apoptosis, hypertrophy and fibrosis. Apoptosis is programmed cell death. Cardiomyocyte apoptosis is the keystone in the process. The loss of cardiomyocytes has been implicated in the development of myocardial remodeling and heart dysfunction. Increased cardiomyocyte apoptosis has been detected in hearts of diabetic patient. However, little is known about the molecular mechanisms that regulate cardiomyocyte apoptosis under hyperglycemia. High mobility group box1protein (HMGB1) is a non-chromosomal nuclear protein that regulates gene transcription and maintains the nucleosome structure; it can be released from necrotic or activated immune cells. Released HMGB1together with its receptor TLRs or RAGE activates mitogen-activated protein kinases (MAPKs), which could mediate inflammatory response and apoptosis. Previous studies have found that inhibition of HMGB1could reduce cardiomyocyte apoptosis induced by chemotherapeutic drugs. Our previous study found that high glucose stimulated cardiomyocyte actively secreted HMGB1, which participated in diabetes-induced myocardial fibrosis and heart dysfunction. We suggested that HMGB1might involve in hyperglycemia-induced cardiomyocyte apoptosis.Objectives1. To investigate the effect of HMGB1in high glucose-induced cardiomyocyte apoptosis.2. To study the signaling pathway involved in HMGB1-mediated cardiomyocyte apoptosis.Methods1. Cell culture and cell model:Cardiomyocytes were isolated from neonatal rat ventricular tissues. Cardiomyocytes were cultured in normal glucose (5.5mM, NG) DMEM and stimulated by high glucose (33mM, HG) in our experiments. High mannose (33mM, OC) was served as osmolarity controls.2. Interference of gene expression:In vitro, lentivector with HMGB1shRNA transfection was performed to inhibit HMGB1expression; transient transfection with Ets-1siRNA was performed to inhibit Ets-1expression.3. Laser scanning confocal microscopy:Immunofluorescence analysis was used to evaluate p-Ets-1expression levels and disposition. 4. TUNEL assay:Cardiomyocyte apoptosis is determined by TUNEL assay.5. Western blotting:Proteins were extracted from cardiomyocytes. The protein expression of HMGB1, cleaved caspase-3, Bax, Bcl-2, Ets-1, p-Ets-1, ERK and p-ERK was analyzed in our experiment.6. Real-time RT-PCR:Total RNA was extracted from cardiomyocytes and CFs. In the experiment, the mRNA expression of HMGB1in cardiomyocytes and CFs was analyzed.7. Animal model:Type1diabetes was induced by injecting streptozotocin. Control mice were injected with citrate buffer only. After7days, random glucose＞16.7mmol/1was determined as diabetes. After the induction of diabetes (8weeks), mice were divided into3groups for treatment:control (n=8), shRNA-HMGBl (n=8) and shRNA-N.C (n=8) for the following experiments. An amount of1×107UT/30μl of lentivector with HMGB1shRNA or the same volume of lenti-vehicle was injected into various sites of the left ventricle.8. Immunohistochemistry:Immunohistochemistry was used to determine the levels of p-Ets-1in myocardial tissues.9. Statistical analysis:All statistical analyses involved use of SPSS18.0. Data are reported as mean±standard deviation.Results1. Inhibition of HMGB1reduced high glucose-induced cardiomyocytes apoptosisHMGB1shRNA was used to inhibit HMGB1expression. The cell apoptosis was determined by western blot and TUNEL assay. Results showed that HG increased the apoptosis of cardiomyocyte compared with the control; while HMGB1inhibition could significantly reduce the cleaved caspase-3, Bax/Bcl-2and apoptotic cells induced by HG.3. HMGB1mediated high glucose-induced activation of Ets-1in neonatal cardiomyocytesTo investigate the underlying mechanism of HG-induced apoptosis, we cultured cardiomyocytes in HG medium for6h,8h,12h,24h. After HG treatment for6h, the protein level of total Ets-1protein expression was slightly increased and p-Ets-1was significantly elevated at6h up to24h. Our results demonstrated that HG but not high media osmolarity enhanced the activation of Ets-1and accumulation of p-Ets-1in the nucleus; inhibition of HMGB1effectively reversed HG-induced increase in the level of p-Ets-1and its accumulation in the nucleus HMGB1.3. Inhibition of Ets-1reduced high glucose-induced cardiomyocytes apoptosisTo confirm whether Ets-1activation was involved in HG-induced cardiomyocyte apoptosis, we used Ets-1siRNA to knock down its protein level. Ets-1siRNA significantly reduced the activation of caspase-3protein, Bax/Bcl-2ratio and TUNEL-positive cells as compared with HG alone.4. ERK pathway was involved in high glucose-induced activation of Ets-1Stimulation of HG increased the phospho-ERK1/2level with a peak at60min. HG but not high osmolarity increased the level of p-ERK1/2in cardiomyocytes as compared with NG. Inhibition of HMGB1with HMGB1-specific shRNA significantly attenuated HG-induced ERK1/2activation in cardiomyocytes. Furthermore, we examined whether HG enhanced Ets-1activation via an ERK pathway. U0126was applied to inhibit p-ERK1/2. HG significantly increased the nuclear level of p-Ets-1, which was reduced by U0126. These observations supported that HMGB1mediated Ets-1activation via an ERK1/2pathway under HG treatment.5. Inhibition of HMGB1reduced diabetes-induced myocardial apoptosis in vivoHyperglycemia significantly increased myocardial caspase-3activity, the ratio of Bax/Bcl-2and apoptotic cells was significantly increased in diabetic hearts. HMGB1inhibition effectively ameliorated hyperglycemia-activated caspase-3and decreased Bax/Bcl-2ratio. Additionally, HMGB1inhibition decreased the proportion of TUNEL-positive cells in the diabetic mouse.6. Inhibition of HMGB1gene prevented ERK and Ets-1activation in the diabetic mouse heartWestern blot analysis demonstrated that inhibition of HMGBl by shRNA significantly reduced hyperglycemia-induced ERK phosphorylation. Immunohistochemistry showed that silencing HMGB1gene significantly decreased the level of p-Ets-1in the diabetic myocardium.Conclusion1. Inhibition of HMGB1reduced high glucose-induced cardiomyocyte apoptosis.2. HMGB1induced apoptosis via the ERK-dependent activation of Ets-1in HG-treated cardiomyocytes.