The Role And Mechanism Of Protein Kinase D In Diabetic Cardiomyopathy

BackgroundsDiabetic cardiomyopathy (DCM), characterized by left ventricular hypertrophy and reduced diastolic function, is a distinct myocardial disease leading to heart failure independently of hypertension and coronary artery disease in patients with diabetes. DCM is the major cause of mortality among diabetic patients, and the number of newly diagnosed cases of DCM is rising at epidemic rates throughout the world. Diverse pathogenic mechanisms contribute to diabetic cardiomyopathy, including myocardial cell death and fibrosis.The pathogenesis of diabetic cardiomyopathy is a chronic and complex process which is attributed to abnormal cellular metabolism and defects of many organelles, such as mitochondria, endoplasmic reticulum (ER) and sarcolemma. Endoplasmic reticulum is an organelle involved in the intrinsic pathway of apoptosis. Recently, apoptosis mediated by ER stress was demonstrated to contribute to the progression of diabetic cardiomyopathy.Protein kinase D (PKD) is a recent addition to the calcium/calmodulin-dependent protein kinase (CAMK) group of serine/threonine kinases, increasingly implicated in the regulation of multiple cell biological processes, including cell survival, migration, differentiation, and proliferation. Recent advances demonstrate an important role of PKD-mediated signaling pathways in the cardiovascular system, particularly in the regulation of myocardial contraction and hypertrophy. Previous studies also reported a significant increase of PKD expression and activity in the myocardium of spontaneously hypertensive heart failure rats, as well as in heart failure patients. However, little is known about the potential role of PKD in the development of DCM.Angiotensin Ⅱ receptor blocker (ARB) has been demonstrated to offer effective cardioprotection in diabetic patients. In the RENAAL and LIFE studies, losartan significantly lowered the hospitalization rate because of new onset heart failure in diabetic patients. In another clinical trial, irbesartan significantly lowered the incidence of congestive heart failure in patients with type 2 diabetes. Additionally, telmisartan was shown to have an efficacy in improving left ventricular diastolic properties in a diabetic population. Researchers also reported that candesartan induced an improvement in diastolic function parameters in asymptomatic diabetic patients by a decrease in collagen synthesis and an increase in collagen degradation. Previous studies indicate the cardioprotective effects of ARB, such as preventing myocardial fibrosis, reducing cardiomyocyte apoptosis, and improving in Ca2+ signaling parameters. However, the molecular mechanism of ARB in reversing cardiac remodeling and dysfunction is still incompletely understood. The present study was designed to investigate the role of PKD in type 2 DCM, and whether the cardioprotective effects of irbesartan (Irb) were mediated by PKD and ER stress.Objectives1. To establish the type 2 DCM rat model and explore the role of PKD and ERS in DCM.2. To investigate the cardioprotective effects and mechanisms of irbesartan in the pathogenesis DCM.Methods1. Animal model and drug treatmentSixty male Sprague-Dawley rats weighting 120g to140g were obtained from the experimental animal center of Shandong University of Traditional Chinese Medicine (Ji’nan, China). The animals were maintained under conditions of standard lighting (alternating 12 h light/dark cycle), temperature (20-22℃) and humidity (50-60%). After 1 week of acclimatization, the rats were then randomly divided into 5 groups (n=12 each):control, diabetes mellitus (DM), DM+Irb-15, DM+Irb-30, DM+Irb-45. The control group received normal chow and the other diabetic groups a high fat diet (34.5% fat,17.5% protein,48% carbohydrates; Beijing HFK Bio-Technology, China). After 4 weeks, intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were performed. The rats with insulin resistance in the diabetic groups received an intraperitoneal injection of a single dose of streptozotocin (STZ; 35 mg/kg, in 0.1 mol/L citrate buffer, pH 4.5; Solarbio, China). Rats of the control group received injections of citrate buffer vehicle of equivalent volume. One week later, tail vein fasting blood glucose (FBG) was measured (Roche, Germany) and rats with blood glucose levels>11.1 mmol/1 were considered diabetic. After induction of diabetes for 8 weeks, rats in the DM+Irb-15, DM+Irb-30, and DM+Irb-45 group were treated with Irbesartan at dosages of 15 mg/kg/day,30 mg/kg/day, and 45 mg/kg/day respectively. The control and DM group received vehicle. Irbesartan (Sanofi-Aventis Pharmaceuticals Company, Hangzhou, China) and vehicle were administered orally by gavage once a day. After 8 weeks of treatment, all rats were killed.2. Serum MeasurementsAfter rats fasted overnight, we collected jugular blood samples. Serum triglycerides (TG) and total cholesterol (TC) were determined using an automatic analyzer (Roche, Basel, Switzerland). Serum level of fasting insulin (FINS) was measured by enzyme-linked immunosorbent assay (Jiancheng, Nanjing, China). And the insulin sensitivity index (ISI) was calculated [ISI= In (FBGxFINS)-’].3. Blood pressure measurementsHeart rate (HR), systolic blood pressure (SBP), and diastolic blood pressure (DBP) were measured with a noninvasive tail-cuff system.4. Echocardiographic evaluationTransthoracic echocardiography was performed using the Vevo 770 imaging system (VisualSonics, Toronto, Canada). Images were obtained from two-dimensional, M-mode, pulsed-wave Doppler and tissue Doppler imaging.5. HistopathologyImmunohistochemistry were performed for detecting the expressions of Glucose regulated protein 78 kDa (GRP78), collagen I and collagen III. Slides were incubated overnight at room temperature with the primary antibodies rabbit anti-GRP78, anti-collagen I and III. Goat anti-rabbit antibody was the secondary antibody.6. TUNEL assayApoptotic cells in myocardium were detected by the terminal transferase-mediated DNA nick end labeling (TUNEL) assay according to the manufacturer’s instructions (ApopTag Plus Peroxidase In Situ Apoptosis Detection Kit, Millipore, USA).7. Western Blot analysisTotal protein from rat myocardium tissues were extracted and separated with a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred to PVDF membranes. The membranes were blocked with 5% defatting milk for 2 h at room temperature, then incubated overnight at 4℃ with primary antibodies specific against PKD, p-PKD, GRP78, collagen I, collagen III, cleaved caspase-3, Bax, Bcl-2, and P-actin. After being washed, the membranes were incubated with an appropriate secondary antibody for 1 h at room temperature. The membranes were subsequently detected with a chemiluminescence kit (Millipore, Billerica, MA, USA).8. Statistical analysisSPSS (version 18.0, SPSS, Chicago, IL, USA) for Windows was used for statistical analysis. Data are expressed as mean±standard deviation (Mean±SD). P＜0.05 was considered statistically significant.Results1. Effect of irbesartan on metabolic indexes and blood pressureAt the end of the experiment, diabetic rats showed significantly increased FBG, TG, TC and FINS levels compared with the control rats (p<0.05). Rats in DM group also showed severe impaired insulin sensitivity (p<0.05). There were no significant differences in SBP and DBP between diabetic and control rats.Both 30 and 45mg/kg irbesartan improved insulin imbalance and impaired insulin sensitivity (p<0.05), while 15mg/kg irbesartan failed to improve these metabolic abnormities. No significant differences were found in blood glucose, serum lipids and blood pressure between rats treated with and without irbesartan.2. Irbesartan alleviated diabetes-induced cardiac dysfunctionBoth systolic and diastolic indexes were evaluated in our study. According to the results, LVEF, FS, and E/A were significantly decreased in the diabetic group compared with the control (all p<0.05). LVEDd and E/E’of DM rats was higher than the control (p<0.05). Irbesartan treatment at 30 and 45mg/kg improved LVEF, FS, E/A significantly (p<0.05) in diabetic rats, but had little effect on LVEDd.15mg/kg irbesartan treatment only increased E/A in diabetic rats (p<0.05).3. Irbesartan ameliorated diabetes-induced cardiac remodelingHeart weight and the ratio of heart weight to body weight were significantly higher in diabetic than control rats (p<0.05). Masson’s trichrome and Sirius red staining of heart sections revealed severe myocardial fibrosis in diabetic rats. Quantitative analysis of Masson’s trichrome staining indicated a significant increase of myocardial interstitial collagen deposition in diabetic rats compared with controls (p<0.05). The immunohistochemistry analysis showed increased level of collagen Ⅰ and Ⅲ in DM rats compared with control group. Western blotting analysis also showed increased expression level of collagen Ⅰ and Ⅲ, and collagen Ⅰ-to-Ⅲ ratio in DM rats compared with control (p<0.05). TUNEL assay revealed that the percentage of myocardial apoptotic cells in DM rats was much higher than the controls (p<0.05).30 and 45mg/kg irbesartan treatment reduced heart weight and the ratio of heart weight to body weight significantly in diabetic rats (all p＜0.05). Meanwhile, myocardial fibrosis, collagen deposition and myocardial apoptosis were ameliorated by irbesartan treatment (all p<0.05).4. Irbesartan attenuated ER stress-mediated cardiomyocyte apoptosis in DM ratsCompared with control rats, diabetic rats showed a significant up-regulation of GRP78 expression level (p<0.05), which was dose-dependently reduced by irbesartan (all p<0.05). To further examine the myocardial apoptosis, expression levels of cleaved caspase-3, Bax and Bcl-2 were evaluated by Western Blot analysis. Our data showed that caspase-3 activity (p<0.05) and Bax/Bcl-2 ratio (p<0.05) were significantly increased in diabetic rats. Both of these increases were reduced by irbesartan treatment in a dose-dependent manner (all p<0.05).5. Irbesartan inhibited the activation of PKD in DM ratsCompared with control rats, diabetic rats showed significantly increased phosphorylation of PKD (p<0.05). The increased expression level of p-PKD was dose-dependently down-regulated by irbesartan treatment (p<0.05).6. Diabetes-induced cardiac remodeling and dysfunction were correlated with PKD activationTo clarify the role of PKD activation in the progression of DCM, correlation analysis was performed in our study. Data showed that both the percentage of TUNEL positive cells (r=0.883, p<0.01) and myocardial fibrosis area (r=0.946, p<0.01) were positively correlated with the p-PKD protein expression, demonstrating that PKD activation may be involved in diabetes-induced myocardial apoptosis and fibrosis. E/A ratio was negatively correlated with the p-PKD protein expression (r=-0.867, p<0.01), suggesting that PKD activation might be implicated in diabetes-induced diastolic dysfunction.Conclusion1. PKD was activated in the myocardium of rats with type 2 diabetes, and was correlated with diabetes-induced cardiac remodeling and dysfunction.2. Irbesartan treatment alleviated the diabetes-induced cardiac remodeling and dysfunction.3. The beneficial effect of irbesartan in DCM was associated with its ability to suppress the activation of PKD and ER stress.BackgroundsDiabetic cardiomyopathy (DCM) is a distinct myocardial disease characterized by structural and functional cardiac changes in patients with diabetes, which develops independently of other confounding factors such as hypertension and coronary artery disease. The pathological features of DCM include cardiomyocyte hypertrophy, apoptosis, necrosis, interstitial fibrosis and microangiopathy. In recent years, accumulating evidence indicates that cardiomyocyte apoptosis, which initiates cardiac remodeling and results in cardiac dysfunction finally, is a key element in the pathogenesis and progression of DCM. 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 underlying mechanisms for increased cardiac apoptosis are still incompletely understood.More recently, endoplasmic reticulum (ER) stress mediated apoptosis was suggested to contribute to the onset and progression of diabetic cardiomyopathy. At the early stage of ER stress, unfolded protein response (UPR) is initiated aiming to restore the ER homeostasis by decreasing the load of proteins in the ER via translational attenuation, increasing the transcription of chaperones and other proteins involved in the folding and maturation of proteins, and inducing the degradation of misfolded proteins via the ER-associated degradation (ERAD). But prolonged ER stress may initiate the apoptosis signaling pathways, which are mediated by C/EBP homologous protein (CHOP), c-Jun NH2-terminal kinase (JNK) and caspase-12. The ER stress initially activates intracellular signaling pathways mediated by three ER-resident proteins in mammalian cells, and among them inositol-requiring kinase-la (IREla), which manifests both kinase and endoribonuclease activity, was considered to be the dominant pathway.Protein kinase D (PKD) is a newly discovered serine/threonine kinase belonging to the calcium/calmodulin-dependent protein kinase (CAMK) group. Studies have shown that PKD participates in the regulation of apoptosis. Nevertheless, the role and mechanism of PKD in apoptosis regulation is very complex, depending on different cell types and the way of its activation. PKC is the chief up-streaming kinase of PKD, and it seems that multiple biological responses attributed originally to PKC are in fact executed by PKD. Previous studies have confirmed that PKC is involved in the regulation of ER stress mediated apoptosis. However, the potential role of PKD in regulating ER stress have not yet clarified at present.Objectives1. To explored the effect of high glucose on the expression and activation of PKD in cardiomyocytes.2. To investigate the role of IRE1α/CHOP signaling pathway in high glucose-induced cardiomyocyte apoptosis.3. To explored the effect and underlying mechanism of PKD in high glucose-induced cardiomyocyte apoptosis.Methods1. Cell cultureH9c2 cardiomyocytes were cultured with DMEM containing 5.5 mmol/L glucose (normal glucose, Control),5.5 mmol/L glucose plus 27.5 mmol/L mannose (osmotic control, OC), or 33 mmol/L glucose (high glucose, HG).2. Transfection of small Interfering RNA (siRNA)PKD-siRNA and IREla-siRNA were transfected into H9c2 cardiomyocytes respectively to explore the role of PKD and IREla in high glucose-induced cardiomyocyte apoptosis. H9c2 cardiomyocytes were divided into the following groups according to different interventions:control group, HG group, HG+PKD-siRNA group, HG+IRE1α-siRNA group and HG+N.C-siRNA group.3.TUNELassayCardiomyocyte apoptosis in different groups was determined by TUNEL assay.4. Western blottingProtein were extracted from cardiomyocytes underwent different stimulation. The protein expression of PKD、p-PKD、GRP78、IRE1α、CHOP、Cleaved caspase-3、 Bax、Bcl-2 and P-actin was analyzed in our experiment.5. Statistical analysisSPSS (version 18.0, SPSS, Chicago, IL, USA) for Windows was used for statistical analysis. Data are expressed as mean±standard deviation (Mean±SD). P＜0.05 was considered statistically significant.Results1. High glucose could induce the apoptosis of H9c2 cardiomyocytesH9c2 cardiomyocytes were cultured in DMEM with 5.5mmol/L glucose (Control), 5.5mmol/L glucose plus 27.5mmol/L mannose (OC), or 33mmol/L glucose (HG) for Oh,12h,24h and 48h. Compared with the control, the expression levels of cleaved caspase-3 and Bax/Bcl-2 ratio were significantly evaluated in the HG group at 24h and 48h (all p<0.05). HG also significantly increased the rate of apoptotic cells according to TUNEL analysis (p<0.05). No significant difference of the TUNEL-positive cells was found between OC group and control group at all of the time points (all p>0.05).2. High glucose could induce the activation of PKD in H9c2 cardiomyocytesNo significant difference of the Drotein expression level of total PKD was found between OC group and control group at all time points (all p>0.05). After HG treatment for 12h, the level of p-PKD protein expression was slightly increased, but had no significant difference compared with the control group (p>0.05). The p-PKD protein expression was significantly elevated at 24h and lasted to 48h (both p<0.05). There was no significant difference of p-PKD between OC group and control group at all time points (p>0.05).3. Inhibition of PKD reduced high glucose-induced cardiomyocytes apoptosisTo confirm whether PKD activation was involved in HG-induced cardiomyocyte apoptosis, we used PKD-siRNA to knock down its protein expression. The expression level of PKD protein decreased significantly after cells were transfected with PKD-siRNA (p<0.05). Inhibition of PKD significantly reduced the HG-induced activation of caspase-3 protein, Bax/Bcl-2 ratio and TUNEL-positive cells as compared with the negative control group which transfected with nonsense-siRNA (all p＜0.05).4. The IRE1α/CHOP pathway of ERS was involved in high glucose-induced cardiomyocytes apoptosis4.1 High glucose could induce ER stress in H9c2 cardiomyocytesCompared with the control group, stimulation of HG for 24h significantly increased the expression levels of GRP78, IREla and CHOP protein in H9c2 cardiomyocytes (all p<0.05). No significant difference of these three protein was found between OC group and control group (all p>0.05).4.2 Inhibition of IRE1α reduced high glucose-induced cardiomyocytes apoptosisTo explore the role of IREla in HG-induced cardiomyocytes apoptosis, we inhibited the expression of IREla by siRNA transfection. The expression level of IRE1α protein decreased significantly after cells were transfected with IRE1α-siRNA (p<0.05). And inhibition of IRE1α significantly reduced the HG-induced activation of caspase-3 protein, Bax/Bcl-2 ratio and apoptotic cell rate as compared with the negative control group which transfected with nonsense-siRNA (all p<0.05).5. Inhibition of PKD down regulated the IRE1α/CHOP signaling pathwayTo investigate the mechanism of PKD in mediating HG-induced cardiomyocytes apoptosis, we further examined the effect of PKD inhibition on the IREla/CHOP pathway. Western blot analysis demonstrated that PKD-siRNA transfection significantly reduced the HG-induced increase of IRE1α and CHOP protein levels.Conclusion1. High glucose could elevate the protein expression of p-PKD in H9c2 cardiomyocytes.2. IREla/CHOP signaling pathway was involved in high glucose-induced H9c2 cardiomyocytes apoptosis.3. Inhibition of PKD reduced high glucose-induced cardiomyocytes apoptosis through down regulation of IRE1α/CHOP signaling pathway.