| BackgroudDiabetic cardiomyopathy (DCM) which develops independent of coronary artery disease and hypertension is a primary cause of death in patients with diabetes. DCM is characterized by early-onset diastolic dysfunction and late-onset systolic dysfunction. Many pathological changes are involved in the occurrence of DCM, including cardiomyocyte hypertrophy, apoptosis, degeneration and necrosis, and interstitial fibrosis. Cardiomyocyte apoptosis plays an important role in the development of cardiac dysfunction. It causes progressive loss of effective myocardial contractile unit, initiates cardiac remodeling and finally results in both systolic and diastolic dysfunction of the heart. However, the mechanisims underlying cardiomyocyte apoptosis in DCM remain incompletely understood.Smad2and Smad3play a crucial role in transducting the signal of TGF-β family from the receptor on the surface of membrane into the nucleus. They form a heteromeric complex with the co-Smad (Smad4) after phosphorylated by activin type I receptors and then translocate into the nucleus to regulate target gene transcription. Smad2/3are important apoptosis regulatory proteins. Previous studies have proved that nitric oxide, angiotensin II and activator protein-1could induce increased phosphorylation of Smad2/3which subsequently stimulated apoptosis-related genes such as Caspase-3/6/9. However, it is not clear whether Smad2/3participate in high glucose-induced cardiomyocyte apoptosis or not.ALK7, a new member of type Ⅰ TGF-β receptors, is firstly isolated from rat brain as an orphan receptor and consists of a transmembrane domain, a serine/threonine kinases domain and a GS domain between them. Nodal, activin AB, activin B and GDF are ligands specific for ALK7. During the signal transduction processes, the ligands firstly bound to activin type Ⅱ receptors (ActRⅡ), and once bound, ALK7is recruited and phosphorylated by ActRⅡ. The activation of ALK7then brings about a series of transformation of downstream substrates, such as Smad2/3, and participates in the regulation of cell proliferation, differentiation and apoptosis. Studies have shown the existence of ALK7in pancreas, brain tissue, adipose tissue, human liver and intestine. ALK7also participates in the proliferation and apoptosis of pancreatic beta-cell, human ovarian epithelial cells and hepatoma cell line, however, the role of ALK7in cardiomyocyte apoptosis remains unclear. In this study we would explore whether ALK7-Smad2/3signaling pathway is involved in high glucose-induced cardiomyocyte apoptosis.Objectives1. To explored the effect of high glucose on cardiomyocyte apoptosis.2. To explore the role of Smad2/3in high glucose-induced cardiomyocyte apoptosis.3. To explore the effect of high glucose on the expression level of ALK7.4. To explore whether ALK7is involved in high glucose-induced cardiomyocyte apoptosis or not.Materials and MethodsH9c2cardiomyoblasts and neonatal rat primary cardiomyocytes were cultured, and RT-PCR, western blotting, flow cytometry and transfection of siRNA were performed in this study.1. H9c2cardiomyoblasts were cultured with DMEM containing5.5mmol/L glucose (normal glucose),5.5mmol/L glucose plus27.5mmol/L mannose (osmotic control), or33mmol/L glucose (high glucose). The protein levels of cleaved Caspase3and Bcl2were measured by western blotting, and apoptosis rate was measured by flow cytometry.2. H9c2cardiomyoblasts were cultured with DMEM containing normal glucose, mannose or high glucose, and the mRNA level of ALK7was measured by RT-PCR while protein level of ALK7and phosphorylated Smad2/3were measured by western blotting.3. Smad2-siRNA and Smad3-siRNA were transfected into H9c2cardiomyoblasts respectively to explore the role of Smad2/3in high glucose-induced cardiomyocyte apoptosis. The protein level of Bcl2and cleaved Caspase3were measured by western blotting while apoptosis rate was measured by flow cytometry.4. ALK7-siRNA plasmid was transfected into H9c2cardiomyoblasts to explore the role of ALK7in high glucose-induced cardiomyocyte apoptosis. The the protein level of phosphorylated Smad2/3, Bc12and cleaved Caspase3were measured by western blotting while apoptosis rate was measured by flow cytometry.Results1. High ambient glucose at a concentration of33mmol/L elevated the expression level of ALK7in H9c2cardiomyoblasts. The results of RT-PCR and western blotting showed the expression of ALK7in both H9c2cardiomyoblasts and primary cardiomyocytes. H9c2cardiomyoblasts were maintained in DMEM with5.5mmol/L,11mmol/L,22mmol/L,33mmol/L and38.5mmol/L glucose for24h. The protein expression of ALK7increased significantly (P<0.05) after H9c2cardiomyoblasts were maintained in DMEM with33mmol/L glucose for24h, so DMEM with33mmol/L glucose was used as high glucose stimulation in subsequent experiments.2. High ambient glucose at a concentration of33mmol/L could induce the apoptosis of H9c2cardiomyoblasts. H9c2cardiomyoblasts were maintained in DMEM with5.5mmol/L glucose (Control),5.5mmol/L glucose plus27.5mmol/L mannose (OC), or33mmol/L glucose (HG) for Oh,4h,8h,12h,24h and48h. The protein expression level of cleaved Caspase3in HG group was84%higher than that in LG group (P<0.001) while the level of Bcl2in HG group was49%lower than that in LG group (P<0.001) at the time point of24h. The protein expression level of cleaved Caspase3in HG group was141%higher than that in LG group (P<0.001) while the level of Bcl2in HG group was57%lower than that in LG group (P<0.001) at the time point of48h. No significant difference of the expression levels of cleaved Caspase3and Bcl2were found between OC group and LG group at all time points.The apoptosis rate detected by Annexin V through flow cytometer showed that H9c2cardiomyoblasts underwent early stage of apoptosis after maintained in high ambient glucose condition for24h and experienced late stage of apoptosis after exposed to high glucose for48h.3. Smad2/3were involved in high glucose-induced H9c2cardiomyoblasts apoptosis.The expression levels of phosphorylated Smad2/3began increased significantly after H9c2cardiomyoblasts were maintained in DMEM with high glucose for12h and lasted to48h. The results were also confirmed in primary cardiomyocytes. The levels of phosphorylated Smad2/3began increased significantly after primary cardiomyocytes were maintained in DMEM with high glucose for12h and lasted to72h.The expressions level of phosphorylated Smad2/3decreased significantly after cells were transfected with Smad2-siRNA and Smad3-siRNA respectively for48h (P<0.001, P<0.001). The expression level of cleaved Caspase3was42%and48%lower (P<0.05, P<0.05) while the level of Bcl2was104%and101%higher (P<0.01, P<0.001) in cells transfected with Smad2-siRNA or Smad3-siRNA than those in cells transfected with nonsense-siRNA.The apoptosis rate was42%lower (P<0.01) in cells transfected with Smad2-siRNA than that in cells transfected with nonsense-siRNA while55%lower (P<0.01) in cells transfected with Smad3-siRNA than that in cells transfected with nonsense-siRNA. 4. High ambient glucose elevated the expression level of ALK7in both H9c2cardiomyoblasts and primary cardiomyocytes.The mRNA expression of ALK7began to increase after H9c2cardiomyoblasts were exposed to high glucose for1h (P<0.05) and reached the peak after cultured for8h (P<0.01). The protein level of ALK7elevated significantly after H9c2cells were exposed to high ambient glucose for12h (P<0.001) and lasted to48h (P<0.001).The results were also confirmed in primary cardiomyocytes. The protein level of ALK7began increased significantly after primary cardiomyocytes were maintained in DMEM with25mmol/L glucose for8h (P<0.01) and lasted to72h (P<0.01).5. ALK7participated in high glucose-induced H9c2cardiomyoblasts apoptosis through activating Smad2/3signaling pathway.The expression level of cleaved Caspase3was24%lower (P<0.01), apoptosis rate was38%lower (P<0.01) while the level of Bcl2was242%higher (P<0.05) in cells transfected with ALK7-siRNA plasmid than those in cells transfected with nonsense-siRNA plasmid.Both the expression levels of phosphorylated Smad2and phosphorylated Smad3decreased significantly after the expression of ALK7was inhibited (P<0.01,P<0.05).Conclusion1. High ambient glucose at a concentration of33mmol/L could induce the apoptosis of H9c2cardiomyoblasts.2. Smad2/3participate in high ambient glucose-induced H9c2cardiomyoblasts apoptosis.3. High glucose could elevate the expression level of ALK7in both H9c2cardiomyoblasts and rat cardiomyocytes.4. ALK7is involved in high glucose-induced H9c2cardiomyoblasts apoptosis through activating Smad2/3signaling pathway. BackgroudDiabetic cardiomyopathy (DCM), an independent diabetes-associated cardiovascular complication, is one of the leading causes of increased morbidity and mortality in diabetic patients. Previou studies showed that multifaceted stimuli such as hyperglycemia, insulin resistance, oxidative stress, inflammation and lipid accumulation were involved in the onset and progression of DCM. Both cardiomyocytes apoptosis and interstitial fibrosis are important pathological mechanisms for the development of cardiac dysfunction in DCM. The apoptosis and fibrosis could cause persistent loss of effective contractile tissue and increased myocardial stiffness both of which inevitably result in early-onset diastolic dysfunction and late-onset systolic dysfunction. However, the underlying mechanisms by which diabetes leads to apoptosis and fibrosis remain incompletely elucidated.Activin receptor-like kinase7(ALK7), a new member of type I TGF-β receptors, has been demonstrated to be associated with cell proliferation, differentiation and apoptosis. Recent studies in rodents showed that the nonsense mutation of ALK7ameliorates fat accumulation and obesity-induced glucose intolerance and insulin resistance. The observations suggested that the activation of ALK7contributed to the abnormalities of lipometabolism and insulin sensitivity which are important triggers of DCM. Our previous study in H9c2cardiomyoblasts has demonstrated that the inhibition of ALK7attenuated high ambient glucose-induced cardiomyocytes apoptosis. However, its pathophysiological significance in diabetic heart is incompletely understood.Both Smad2/3and Akt are crucial downstream mediators of ALK7signaling pathway. Previous findings showed that Smad2/3were critical for the fibrosis induced by profibrotic factors, such as angiotensin II and advanced glycation end products. Akt plays a key role in the maintenance of normal glucose homeostasis and selective insulin resistance, and depressed activity of Akt is considered to be a potential inducer of insulin resistance. Besides, both ALK7-Smad2/3and ALK7-Akt signaling pathway are involved in the regulation of cell apoptosis.On the basis of the above considerations, we hypothesized that ALK7plays a critical role in myocardial remodeling during the development of DCM, and this action of ALK7may be associated with cardiomyocyte apoptosis and cardiac fibrosis in diabetic hearts. In the present study, we established the rat model of type2DCM and used ALK7gene silencing in vivo to elucidate the role of ALK7in the pathogenesis of DCM. We try to look forward for a new potential target for the treatment of human DCM.Objectives1. To investigate the effect of ALK7gene silencing on glucose and lipid metabolism and insulin sensitivity in type2diabetic rats.2. To investigate the effect of ALK7gene silencing on cardiac structure and cardiac function in type2diabetic rats.3. To investigate the effect of ALK7gene silencing on cardiomyocyte apoptosis and interstitial fibrosis in type2diabetic rats.4. To investigate the effect of ALK7gene silencing on the expression level of phosphorylated Smad2/3and Akt in type2diabetic hearts.Materials and Methods1. Induction of type2diabetes in rats. Sixty male Sprague-Dawley rats (120-140g) were randomized into four groups:control, DM, DM+ALK7-siRNA, and DM+vehicle after intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were performed. The control group received normal chow and the other three groups were fed a high-fat (HF) diet. Four weeks later, IPGTT and IPITT were repeated. Rats with insulin resistance were intraperitoneally injected with streptozotocin (STZ) at the dose of30mg/kg for only one time. After12weeks of diabetes, rats in DCM+ALK7-siRNA group and DCM+vehicle group received adenovious injection. After16weeks of diabetes, the rats were killed.2. Intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT).IPGTT was performed after rats fasted for12h. The rats were intraperitoneally injected with the20%glucose solution (1g/kg) and blood samples were collected sequentially from the tail vein at the time point of0,15,30,60, and120min. IPITT was performed after rats fasted for4h. The rats were intraperitoneally injected with insulin solution (1unit/kg), and the blood was sampled and serum glucose was measured as described above.3. Blood serum analysis.Serum levels of total cholesterol (TC), triglyceride levels (TG), and fasting blood glucose (FBG) were determined using the Bayer1650blood chemistry analyzer and fasting insulin level (FINS) was analyzed using enzyme-linked immunosorbent assay.4. Measurement of blood pressure and heart rate.The heart rate, systolic blood pressure, diastolic blood pressure, and mean arterial pressure were measured by a noninvasive tail-cuff method.5. Evaluation of left ventricular function by cardiac catheterization.Left ventricular (LV) function was assessed by invasive hemodynamic measurement at the end of the experiment. A pressure tip-catheter filled by fluid was advanced from the right carotid artery into the left ventricle, and LV end-diastolic pressure (LVEDP) and LV end-systolic pressure (LVSP) were measured.6. Gene silencing of ALK7.After12weeks of diabetes, rats in DCM+ALK7-siRNA group and DCM+vehicle group received an adenovirus harboring ALK7gene with green fluorescent protein (GFP) or a control empty virus with GFP at a dose of2.5×1010plaque-forming units via the jugular vein. Adenovirus transfer was repeated in two weeks.7. Tissue preparation.Histological paraffin-embedded sections (5μm) were prepared for histology staining. The remaining portion of the left ventricular tissue was stored at-80℃for RT-PCR and western blotting analysis.8. Histology and morphometric analysis.The LV paraffin-embedded tissue sections were stained with hematoxylin and eosin (H&E), masson’s trichrome and0.5%sirius red to assess LV architecture and perivascular and interstitial fibrosis.9. In situ detection of apoptotic cells.In situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was performed to detect apoptotic cells. Percent TUNEL stained positive nuclei=number of TUNEL positive nuclei/total nuclei×100.10. Immunohistochemical staining.Tissue sections were incubated with polyclonal rabbit anti-rat collagen I and collagen III primary antibodies at4℃overnight, and then incubated with biotinylated goat anti-rabbit secondary antibody. The reaction was visualized with3,3’-diaminobenzidine (DAB) solution and stained sections were then counterstained with hematoxylin. The positive staining was analyzed with Image-Pro Plus5.0analysis software.11. Real-time quantitative RT-PCR.Total RNA was preparing with trizol agent. The reverse transcription and PCR reactions were performed using standard methods. The relative expression of the genes was analyzed with the2(-Delta Delta C (T)) method with β-actin as endogenous reference gene.12. Western blotting analysis.A total of15μg protein from each sample was separated by10%SDS—polyacrylamide gels and electrophoretically transferred to polyvinylidene fluoride membranes. The membranes were incubated with different primary antibodies overnight at4℃, and then treated with horseradish peroxidase-conjugated secondary antibodies for2h. β-actin was used as a loading control. The ECL kit was used to visualize the blots, and Photoshop CS4software was used to quantify the bands.13. Statistical analysis.Values are presented as mean mean±SD. Intergroup comparisons involved one-way ANOVA, followed by Tukey’s post hoc test and independent samples t-test. SPSS17.0was used for statistical analysis. A value of P<0.05was considered statistically significant.Results1. ALK7gene silencing ameliorated insulin resistance in type2diabetic rats.The4-week HF diet induced insulin resisitance in rat. Both the results of IPGTT and IPITT showed that levels of blood glucose in rats fed with HF diet were significantly higher at week4than at baseline at all of the time points tested. The area under the curve (AUC) of IPGTT and IPITT across the time for glucose level was also significantly higher at week4than at baseline.Both IPGTT and IPITT were repeated at the end of the experiment, and the results showed significantly higher glucose levels in DM group than control group (P<0.001, P<0.001). With ALK7gene silencing, the results of IPGTT and IPITT showed significant decreases of glucose levels in ALK7-siRNA group compared with vehicle group (P<0.01, P<0.01). The AUC for IPGTT and IPITT was also lower in ALK7-siRNA group than vehicle group (P<0.001, P<0.001). Besides, the decreased ISI in DCM group was restored after ALK7silencing (P<0.001). 2. ALK7gene silencing ameliorated metabolism abnormalities in type2diabetic rats. The diabetic rats got increased serum levels of TC, TG and FBG compared with control group. The elevated TC and TG appeared at week4while the FBG increased markedly after the onset of diabetes (at week5). With ALK7silencing, the elevated serum levels of TC, TG and FBG decreased significantly in ALK7-siRNA group compared with vehicle group (P<0.001, P<0.01, P<0.001).3. ALK7gene silencing improved cardiac function in DCM. Rats in DM group exhibited increased LVEDP (7.14±0.90vs.23.43±1.81mmHg, respectively; P<0.001) and decreased LVSP (109.29±9.78vs.88±10.6mmHg, respectively; P<0.01) compared with control group. This impaired cardiac function was partially restored by ALK7-siRNA treatment. Rats in ALK7-siRNA group showed significantly lower LVEDP compared with the vehicle group (12.43±1.62vs.22.85±2.91mmHg, respectively; P<0.001).4. ALK7gene silencing ameliorated cardiac remodeling in DCM. Rats of DM group showed a significantly larger heart compared with the control group, and the heart weight to body weight ratio (HW/BW) was22%higher in DM group than the control group. With ALK7silencing, HW/BW was significantly decreased in DM group compared with vehicle group (2.82±0.33vs.3.19±0.27mg/g, respectively; P<0.05).The cross-section staining of H&E at papillary muscle level showed that rats of DM group had dilated ventricle and thickened ventricular wall compared with control group. The cardiomyocytes were compact and well-organized in control group while hypertrophic, disorganized and twisty in DM group. With ALK7-siRNA treatment, the myocyte hypertrophy, rupture, disorganization as well as LV dilation and wall thickening were ameliorated. The myocyte size were significantly decreased in ALK7-siRNA group compared with vehicle group (0.35±0.03vs.0.50±0.04mm2, respectively; P<0.001).5. ALK7gene silencing prevented cardiomyocyte apoptosis in DCM.Rats in DM group showed significantly increased cardiomyocyte apoptosis rate (P<0.001) and elevated levels of cleaved Caspase3and Bax/Bcl2(P<0.001, P<0.001, respectively) compared with rats in control group. After a4-week ALK7-siRNA treatment, the cardiomyocyte apoptsis rate as well as protein level of cleaved Caspase3and Bax/Bcl2was significantly decreased in ALK7-siRNA group compared with vehicle group (P<0.001, P<0.001, P<0.001, respectively).6. ALK7gene silencing attenuated diabetes-induced cardiac fibrosis.Rats of DM group showed a diffuse, reticular, pockety and disorganized collagen network structure in the interstitial and perivascular areas. The collagen volume fraction (CVF) and perivascular collagen area/luminal area (PVCA/LA) were higher in the DM group than control group (P<0.001; P<0.001). With ALK7-siRNA treatment, the collagen deposition was significantly ameliorated in both the interstitial and perivascular areas, and the CVF and PVCA/LA were reduced significantly (P<0.01; P<0.001) compared with vehicle group.The immunohistochemistry analysis showed decreased level of collagen Ⅰ-to-Ⅲ ratio in ALK7-siRNA group compared with vehicle group (P<0.001). Western blotting analysis also showed decreased protein level of collagen Ⅰ and Ⅲ, and collagen Ⅰ-to-Ⅲ ratio in ALK7-siRNA group compared with vehicle group (P<0.001; P<0.01; P<0.01).7. ALK7regulated Akt and Smad2/3signaling pathways in DCM.The expression of phosphorylated Akt decreased markedly (P<0.001) while phosphorylated Smad2/3increased significantly in DM group (P<0.001, P<0.001). With ALK7silencing, the depressed phosphorylation of Akt was restored, and the phosphorylation of Smad2and Smad3was abolished by62%and37%, respectively.ConclusionThe inhibition of ALK7significantly attenuates cardiomyocyte apoptosis, cardiac fibrosis as well as insulin resistance in type2diabetic rats. The protective effects of ALK7gene silencing may be mediated through the suppression of Smad2/3signaling pathway and restoration of Akt signaling pathway. The cardioprotective effects with ALK7gene silencing suggest a potential therapeutic approach for the treatment of DCM in type2diabetes. |
PDF Full Text Request