Translated Abstract
Objective and Significance:
Diabetic cardiomyopathy (DCM) is a common cardiovascular complication in diabetic patients. With the increasing incidence of diabetic mellitus, DCM has become a serious social and family burden. However, the pathogenesis of DCM remains to be elucidated in detail. And the prognosis is poor because of the lack of specific treatment. Cardiomyocyte mitochondrial injury resulted from diabetic mellitus plays an important role in the pathogenesis of DCM, and is one of the pathogenesis of DCM. Improving mitochondrial function can be used as the central link of the effective prevention and treatment of DCM. Mitochondria are the main places where intracellular reactive oxygen species (ROS) are generated, and also vulnerable to ROS attack, damaged mitochondria generate ROS much further. Eventually vicious spiral appears between ROS generation and mitochondrial damage. Mitochondrial uncoupling protein 2 (UCP2) is a proton transporters, which is located in the mitochondrial inner membrane. UCP2 can cause H+ leakage from mitochondrial membrane spaces into the matrix, and reduce the generation of intracellular ROS, thereby regulate mitochondrial function, including regulating the level of oxidative phosphorylation, maintenance of mitochondrial membrane potential, inhibition of apoptosis and keeping calcium homeostasis et al. The studies above strongly suggest that UCP2 may ultimately improve cardiac function by reducing ROS production and inhibiting mitochondrial injury caused by diabetes. Protein kinase B (AKT) can regulate mitochondrial pathway apoptosis and mitochondrial biogenesis, and UCP2 activated AKT phosphorylation in rats brain tissue, suggesting that AKT may involved in the regulation effect of UCP2 on mitochondrial function. Therefore, in this study, we will analyze the regulation effects of UCP2 on mitochondrial and myocardial function in DCM, and reveal the underlying mechanism.
In addition, previous studies suggest that resveratrol (RES), as a mitochondria protective botanical agent, can increase the expression of UCP2 in liver and skeletal muscle of diabetic mouse. Therefore, in this study, we will also investigate whether RES improve mitochondrial and myocardial function in DCM mediated by UCP2. This study will make a preliminary exploration of the regulation effects of UCP2 on mitochondrial and cardiac function in DCM，shed a light on the UCP2 as a potential therapeutic target of DCM, and also provide a theoretical basis on clinical application of RES in the treatment of DCM.
Methods:
1) Changes of myocardial mitochondrial function and UCP2 expression in DCM rats
The SD rats were used as the research object. DCM rat models were constructed by high-fat diet and intraperitoneal injection of streptozotocin (STZ). The rats were divided into two groups: control group and DCM group. Venous blood glucose, lipid level andplasma brain natriuretic peptide (BNP) were analyzed by biochemical analyzer; Echocardiography, electrocardiogram and histopathology methods were used to detect the changes of cardiac structure and function; Transmission electron microscopy was used for observing cardiac ultrastructure; The expressions of UCP2 in myocardium was examined by Real-time quantitative PCR, Western-blot and immunohistochemical method; Dihydroethidium (DHE) labeling was used to evaluate the level of ROS; The level of mitochondrial membrane potential (MMP) was determined by fluorescent enzyme labeling; The activity of mitochondrial respiratory chain complex was examined by spectrophotometry.
2) Effects of UCP2 on mitochondrial function and cell apoptosis in cardiomyocytes incubated with high-glucose
H9C2 cardiomyocytes were used as the research object. Cardiomyocytes were incubated with high-glucose. To construct a cell model of differential expressions of UCP2, UCP2siRNA and UCP2 agonist TTNPB were used to inhibit and increase the expression of UCP2, respectively. Cardiomyocytes were divided into four groups: control, high-glucose group, high-glucose+UCP2siRNA group, and high-glucose+TTNPB group. ROS level was detected by DCFH-DA labeling; The activation of mitochondrial permeability transition pore (MPTP) was examined by Calcein-AM labeling; Spectrophotometry was used to detect the activity of mitochondrial respiratory enzyme complexes; MMP level and apoptotic rate were analyzed by flow cytometry; The expressions of UCP2 and its downstream molecule AKT, and the activity of apoptotic pathway were examined by Western-Blot.
At the same time, to reveal the molecular mechanism of UCP2 in modulating mitochondrial function, we used AKT inhibitors LY294002 to intervene AKT expression in H9C2 cells. Cardiomyocytes were divided into four groups: control group, high-glucose group, high-glucose+TTNPB group, and high-glucose+TTNPB+LY294002 group, the activation of MPTP and cell viability were detected.
3) The role of resveratrol in regulating UCP2 and myocardial mitochondrial function in DCM rats
The SD rats were used as the research object. DCM rats were intervened with resveratrol by gavage. The rats were divided into three groups: control group, DCM group, DCM+RES group. Echocardiography, electrocardiogram and histopathology methods were used to detect the change of cardiac structure and function; Transmission electron microscopy was used for observing cardiac ultrastructure; TUNEL staining was used to detect myocardial apoptotic level; Western-blot was used to detect the expression of UCP2 and its downstream molecule AKT in myocardium; DHE labeling was used to detect the level of ROS; MMP level was analyzed with fluorescence enzyme labeling; The activity of mitochondrial respiratory chain complex was determined by spectrophotometry.
On the basis of identifying the effects of RES on mitochondrial and myocardial function in DCM rats, we further analyzed whether UCP2 was the target of the effect of RES. First, we examined the effect of different concentrations of RES on expression of UCP2 by Western-blot. Then, H9C2 cardiomyocytes were divided into four groups: control group, high-glucose group, high-glucose+RES group and high-glucose+RES+UCP2siRNA group. The levels of ROS, the activation of MPTP, and cell viability were detected in each group.
Results:
1) Changes of myocardial mitochondrial function and UCP2 expression in DCM rats
A DCM model of type 2 diabetes mellitus was successfully established by high-fat diet combined with intraperitoneal injection of STZ. Compared with the control group, the levels of blood glucose,lipid and BNP in the DCM group were significantly increased (P＜0.05). Echocardiography showed that DCM rats had enlarged hearts with left ventricular systolic and diastolic dysfunction (P＜0.05). Electrocardiogram showed that the intraventricular conduction time was prolonged in DCM group (P＜0.05). HE and Masson staining showed that in DCM group, the myocardial fibers was derangement, partially broken, accompanied by interstitial edema, and collagen deposition was obvious. Transmission electron microscopy showed that the myocardial mitochondria in DCM group were edematous and the cristae of mitochondria were disordered. Mitochondrial function tests showed that the level of ROS in myocardium was elevated, mitochondrial respiratory enzymes activities and the level of MMP were reduced in DCM group compared with the control group (P＜0.05). The mRNA and protein expression of UCP2 in DCM group were both elevated measured by R-T PCR, Western-blot and immunohistochemistry (P＜0.05).
2) Effects of UCP2 on mitochondrial function and cell apoptosis in cardiomyocytes incubated with high-glucose
High-glucose incubation increased the level of ROS, enhanced the opening of MPTP, inactive the mitochondrial respiratory enzyme complexes, decreased the level of MMP, and increased apoptotic rate in cardiomyocytes. The release of cytochrome C from mitochondria to cytoplasm and the activation of caspase-9 were both increased, suggesting that high-glucose activated mitochondrial apoptotic pathway in cardiomyocytes. While, up-regulation of UCP2 prevented mitochondrial damage and cardiomyocytes apoptosis from high-glucose incubation. Whereas, inhibition of UCP2 expression aggravated mitochondrial damage and cardiomyocytes apoptosis induced by high-glucose. Together, UCP2 has protective effects on mitochondrial injury and cardiomyocytes apoptosis induced by high-glucose.
We also found that up-regulation of UCP2 could increase the phosphorylation of AKT in cardiomyocytes under high-glucose condition, inhibition of UCP2 expression decreased the phosphorylation level of AKT. These results suggested that AKT phosphorylation may be involved in the protective effects of UCP2 on the mitochondrial function. LY294002 was applicated to inhibit the phosphorylation of AKT. Compared with HG+TTNPB group, the opening of MPTP in HG+TTNPB+ LY294002 group was significantly enhanced, cell viability was decreased, indicating that AKT phosphorylation mediated the protective effects of UCP2 on the mitochondrial function and cell viability under high-glucose condition.
3) The role of resveratrol in regulating UCP2 and myocardial mitochondrial function in DCM rats
Compared with the DCM group, echocardiography showed that RES intervention ameliorated the cardiac systolic and diastolic function. Histological examination showed that RES significantly decreased the destruction of myocardial fibers, collagen deposition and mitochondrial destruction in DCM rats. TUENL staining showed that RES inhibited myocardial apoptosis in DCM rats. The results of mitochondrial function tests showed that RES reduced the ROS level, increased MMP and respiratory chain complexes activities in DCM rats. Meanwhile, RES up-regulated the expression of UCP2 and pAKT in the myocardium of DCM.
In addition, results in vitro showed that RES up-regulated the expression of UCP2 in H9C2 cardiomyocytes incubated with high-glucose in a dose-dependent manner. Moreover, compared with the HG+RES group, the ROS level was increased, MPTP opening was enhanced, and cell viability was reduced in HG+RES+UCP2siRNA group, indicating that UCP2 mediates the protective effects of RES on mitochondrial function in DCM.
Conclusions：
1） In the course of DCM pathogenesis, ROS were accumulated in the myocardium, the structure and function of myocardial mitochondria were impaired, accompanied by elevated expression of UCP2. Up-regulation of UCP2 may be a protective feedback reaction of mitochondrial to oxidative stress in DCM;
2） Up-regulation of UCP2 suppressed high glucose-induced ROS generation in cardiomyocytes, thereby improved mitochondrial function and inhibited cardiomyocytes apoptosis;
3） Phosphorylation of AKT was an important molecular target of UCP2, and involved in the UCP2 protective effects on cardiomyocytes under high-glucose condition;
4） RES can prevent ROS generation, mitochondrial damage, and excessive myocardial apoptosis in DCM, subsequently improve the cardiac function; UCP2 mediated the protective effects of RES on myocardial mitochondria.
Translated Keyword
[Apoptosis, Diabetic cardiomyopathy, Mitochondria, Reactive oxygen species, Uncoupling protein2]
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