The Effects And Mechanisms Of Prolyl Hydroxylase 3 In Diabetic Cardiomyopathy

BackgroundDiabetic cardiomyopathy (DCM) is defined as left ventricular (LV) dysfunction independent of hypertension and underlying coronary artery disease in the diabetic population. Many pathogenic mechanisms, including myocardial apoptosis and fibrosis, which are mediated by hyperglycemia, result in diabetic cardiomyopathy. However, the molecular mechanisms that regulate cardiomyocyte apoptosis and fibrosis in the setting of hyperglycemia remain incompletely understood.Prolyl hydroxylase 3 (PHD3) is a member of the prolyl hydroxylases (PHDs) family and is induced by hypoxia. It plays a critical role in regulating the abundance of hypoxia-inducible factor (HIF). Many other environments and intracellular factors regulate the expression of PHD3 in addition to hypoxia. In human endothelial cells interferon-gamma (IFNy) induced PHD3 expression in the absence of hypoxia. In nucleus pulposus cells the expression of PHD3 was induced by tumor necrosis factor a (TNFa) and interleukin 1β (IL-1 β). In addition to its role in response to hypoxia, PHD3 also has numerous additional functions that may not be directly related to the HIF pathway under normoxic conditions. PHD3 expression is upregulated in some cardiac diseases such as cardiomyopathy, ischemia/reperfusion injury and congestive heart failure. Recent studies have demonstrated that PHD3 expression is increased in rat hearts affected by DM; however, its role remains unclear.We hypothesized that upregulated PHD3 induced by hyperglycemia participates in the pathophysiological processes of DCM. Knockdown of PHD3 may exert protective effects in the setting of diabetes.Objectives1. To investigate the role and mechanism of cardiac PHD3 in myocardial dysfunction induced by diabetic cardiomyopathy.2. To study the effect of PHD3 on cardiac apoptosis and fibrosis in diabetic cardiomyopathy.Methods1. Induction of type2 diabetesin ratsSixty male Sprague-Dawley rats (100-120 g) were randomized into the following 4 groups (n = 15 per group):control, diabetes mellitus (DM), DM+shRNA-PHD3, and DM+shRNA-NC. The control group was fed a basal diet, and the other groups were fed a high-fat (HF) diet (16% fat and 0.25% cholesterol) for 4 weeks. The insulin resistance was determined by the intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPGTT) four weeks later. Diabetes was induced by a single intraperitoneal injection of streptozotocin (STZ; 35 mg/kg, Solarbio, China) dissolved in 0.1 ml of citrate buffer (pH 4.5). One week after STZ administration, blood glucose levels were measured (Roche, Germany) after the rats were fasted overnight. Only rats with blood glucose levels≥11.1 mmol/1 were considered for inclusion in the diabetic rat model. The results of echocardiography showed that diabetic rats exhibited cardiac dysfunction after 8 weeks of STZ injection. All experimental procedures were performed in accordance with animal protocols approved by the Shandong University Animal Care Committee.2. RNA interferenceThe target sequence for PHD3-shRNA was 5’-GGGCAAATACTATGTCAAG-3’, and the negative control sequence was 5’-TTCTCCGAACGTGTCACGT-3’. After 8 weeks of STZ injection, either 5 × 107 UT/50 μl of lentivector with PHD3 shRNA or the same volume of lenti-vehicle was injected into the jugular vein.3. Cardiac function measurementCardiac function was measured using the Vevo770 imaging system (VisualSonics, Toronto, Canada). The derived echocardiography parameters included the left ventricular ejection fraction (LVEF), fractional shortening (FS), peak E to peak A ratio (E/A), early (E’) to late (A’) diastolic velocity ratio (E’/A’), and left ventricular end-diastolic dimension (LVEDd).4. Histology and immunohistochemistryThe hearts of SD rats were dissected and fixed in 4%formalin. Tissue samples were embedded in paraffin and sectioned (4 μm) for subsequent analyses. Hematoxylin and eosin (HE) staining was used to measure cardiomyocyte width, determined as the shortest dimension per cardiomyocyte (μm). Masson’s trichrome and Sirius red staining were used to detect collagen. Immunostaining was used to detect the expression of PHD3、collagen Ⅰ、collagen III.5. TunelassayTUNEL assay was used to measure cell apoptosis rates.6. Gelatin zymographyThe activity of matrixmetalloproteinase (MMP-2) and MMP-9 in diabetic rat heart tissues was determined by zymography.7. Western blotThe protein expression of PHD3, cleaved caspase-3, collagen Ⅰ, collagen Ⅲ, MMP-2 and MMP-9 was analyzed in our experiment.8. Data analysisThe data are reported as the means ± standard deviation. All statistical analyses were performed using SPSS v18.0 (SPSS Inc., Chicago, IL). Significance was defined as p<0.05.Results1. HF diet induced insulin resistanceAll rats were performed by IPGTT and IPITT after 4 weeks of HF diet. The results of IPGTT and IPITT showed that the blood glucose and AUC in HF group were higher than that in control group at 0min、15min、30 min、60 min and 120 min.2. PHD3 was increased in diabetic rat heartsThe diabetic rats showed increased expression of PHD3 in the myocardial tissues compared with the control rats. In diabetic rats, shRNA-PHD3 treatment downregulated myocardial PHD3 protein levels significantly.3. PHD3 inhibition ameliorated diabetes-induced myocardial remodelingThe heart weight and ratio of heart weight to body weight in diabetic rats were higher compared with the control rats. PHD3 gene silencing attenuated the heart weight and ratio of heart weight to body weight as compared with vehicle treatment. Cardiomyocyte widths were increased in diabetic rats compared with control rats, and PHD3 gene silencing attenuated this enlargement compared with vehicle treatment.4. PHD3 inhibition ameliorated diabetes-induced cardiac dysfunction.LVEF, FS, the E/A ratio and the E’/A’ ratio were each lower in diabetic rats than in control rats. Compared with vehicle treatment, shRNA-PHD3 treatment enhanced the attenuation of LVEF, FS, the E/A ratio and the E’/A’ ratio in diabetic rats. LVEDd was significantly increased in diabetic rats, and shRNA-PHD3 treatment attenuated wall thickening.5. PHD3 inhibition limits diabetes-induced myocardial apoptosisThe proportion of TUNEL-positive apoptotic cells was significantly increased in diabetic hearts. Additionally, the level of myocardial cleaved caspase-3 increased significantly. PHD3 inhibition effectively decreased the proportion of TUNEL-positive cells and the protein expression of cleaved caspase-3 in diabetic hearts.6. PHD3 inhibition limits diabetes-induced myocardial fibrosisMasson’s trichrome and Picrosirius red staining demonstrated that collagen deposition was increased in diabetic hearts compared with the hearts of control rats, and shRNA-PHD3 treatment reduced collagen deposition compared with vehicle treatment. Diabetic animals exhibited enhanced expression of collagens Ⅰ and Ⅲ compared with control animals, whereas shRNA-PHD3 transfection significantly reduced the levels of each protein in diabetic rats compared with vehicle treatment.7. PHD3 inhibition reduced the MMP-2 and MMP-9 expression and MMP-2 activityDiabetes increased the protein expression of MMP2 and MMP9, and gelatin zymography demonstrated that diabetes also increased the activity of MMP-2. These effects were significantly downregulated by shRNA-PHD3 treatment.Conclusion1. PHD3 expression increased in diabetic rat hearts.2. PHD3 expression in heart was inhibited effectively using lentivirus-mediated short-hairpin RNA.3. PHD3 inhibition attenuated cardiac dysfunction in diabetic cardiomyopathy.4. The effects of PHD3 on cardiac dysfunction were involved in the decreased myocardial apoptosis and fibrosis.BackgroundThe prevalence of diabetes mellitus (DM) is increasing and becoming to a severe threat to human health. Hyperglycemia can induce damages of various organs, including kidney、heart、eye and nerve, et al. Cardiovascular complications are the most important causes of death in diabetic patients. Diabetic cardiomyopathy (DCM) is defined as left ventricular dysfunction independent of hypertension and underlying coronary artery disease in the diabetic population.Myocardial apoptosis plays an important role in cardiac remodeling and heart dysfunction in diabetic cardiomyopathy. Numerous studies have found that myocardial apoptosis increased in the process of diabetic cardiomyopathy. However, the mechanism of myocardial apoptosis induced by diabetes remains unclear.Prolyl hydroxylase 3 (PHD3) is a member of the prolyl hydroxylases (PHDs) family. PHD3 is induced by hypoxia and regulates the protein expression of hypoxia-inducible factor (HIF). Under normoxic conditions, IFNγ、 TNFα and IL-1β can induce the expression of PHD3. Our previous studies have found that PHD3 expression increased in diabetic cardiac tissues, however, the mechanism is still understood. The production of ROS increased in cardiomyocytes cultured with high glucose. Under hypoxia conditions, ROS can regulate the activity of PHDs. Whether ROS regulates the expression of PHD3 induced by high glucose in cardiomyocytes in the absence of hypoxia remains unclear. Additionally, the mechanisms of PHD3 promoting cardiac apoptosis in diabetic rat hearts need to be elucidated.In this study, we would investigate the mechanisms of PHD3 expression induced by high glucose and the role of PHD3 on apoptosis in cardiomyocytes induced by high glucose.Objectives1. To investigate the mechanism of expression of PHD3 induced by high glucose.2. To investigate the effect of PHD3 on apoptosis in cardiomyocytes induced by high glucose.3. To study the signaling pathway involved in PHD3-mediated cardiomyocyte apoptosis.Methods1. Cell cultureH9c2 cardiomyoblasts and primary cardiomyocytes isolated from neonatal rat ventricular tissues were cultured in normal glucose (5.5 mM, NG) and stimulated by high glucose (33.3 mM, HG) in our experiments.2. Interference of gene expressionTransient transfection with PHD3-siRNA was performed to inhibit PHD3 expression in H9c2 cardiomyoblasts.3. Laser scanning confocal microscopyThe expression level and disposition of PHD3 was evaluate by immunofluorescence analysis.4. Measurement of ROSROS production was assessed using the peroxide-sensitive fluorescent probe,2’,7’-dichlorofluorescein diacetate (DCFH-DA, Sigma, Shanghai, China). Fluorescence intensity was observed under a microscope.5. TUNEL assayTUNEL assay was used to determine the apoptotic rate of H9c2 cardiomyoblasts.6. Western blottingThe expression of PHD3, HIF-la, cleaved caspase-3, p-ERK, ERK, p-JNK, JNK, p-p38, p38 was analyzed by western blotting.7. Statistical analysisThe data are reported as the means ± standard deviation. All statistical analyses were performed using SPSS v18.0 (SPSS Inc., Chicago, IL).Results1. The expression of PHD3 was increased in H9c2 cardiomyoblasts and primary cardiomyocytes in high glucose environment.H9c2 cardiomyoblasts and primary cardiomyocytes were maintained in DMEM with 5.5 mM glucose (control group, NG) and DMEM with 33.3 mM glucose (high glucose group, HG) for 6,12, 24 and 48 h. The PHD3 protein levels began to increase significantly after H9c2 cardiomyoblasts and primary cardiomyocytes were maintained in an HG environment for 24 h. Confocalimmuno fluorescence microscopy revealed that PHD3 is expressed in both the nucleus and the cytoplasm.2. ROS mediates HG-induced PHD3 overexpressionHigh glucose induces ROS overproduction in H9c2 cardiomyoblasts. The addition of NAC inhibited the overexpression of ROS and PHD3.3. Inhibition of PHD3 reduced high glucose-induced H9c2 cardiomyoblasts apoptosisPHD3-siRNA was used to inhibit PHD3 expression. The results of western blot and Tunel assay showed that high glucose increased the apoptosis of H9c2 cardiomyoblasts while PHD3 inhibition significantly reduced the expression of cleaved caspase-3 and apoptotic cells induced by high glucose.4. The effect of PHD3 on H9c2 cardiomyoblasts apoptosis was independent of HIF-1α in high glucose environment.The HIF-la protein levels were not altered in response to an HG environment in primary cardiomyocytes. HIF-la expression was not altered after PHD3 inhibition in H9c2 cardiomyoblasts.5. Inhibition of PHD3 reduces HG-induced MAPKs activationPHD3 inhibition reduced ERK1/2 and JNK phosphorylation but had no effect on p38 phosphorylation induced by high glucose in H9c2 cardiomyoblasts.Conclusion1. ROS was involved in the overexpression of PHD3 induced by high glucose.2. Inhibition of PHD3 reduced high glucose-induced H9c2 cardiomyoblasts apoptosis.3. PHD3 induced apoptosis via MAPKs activation in high glucose-treated H9c2 cardiomyoblasts.