| Background and Objectives With the rapid development of economy, the aggravation of society competes, the improvement of living standard as well as alterd work, life style and traditional diet, the incidence of diabetes mellitus (DM) rises steadily and it has been become a serious public health issue. DM is easily complicated by cardiovascular disease which contributes to most of the excess handicap and mortality associated with DM. Especially diabetic cardiomyopathy (DCM) remain a major concern recently. It is has known that DCM is idiopathic complications of DM patients and a distinct entity characterized by the absence of epicardial coronary artery disease, hypertension, and significant valvular disease. The main performance of DCM is that left ventricular diastolic dysfunction comes out at first followed by left chamber compliance decrease and left ventricular hypertrophy, then contractile dysfunction, left ventricular dilation, arrhythmia and heart failure development. The main pathological changes of DCM are microvascular endothelial cells impairment, collagen fibers in endocardium proliferation, vascular basement membrane thickening, myocardial interstitial inflammation and fibrosis as well as cardiomyocytes hypertrophy and apoptosis. However, the mechanisms involved in pathogenesis of DCM are not fully understood. It is reported recently that mitochondrial dysfunction is associated with DCM. Our previous studies found that endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) significant increase in the blood of diabetic rats was closely related to vascular endothelial dysfunction and major vessel complications induced by DM, furthermore, in recent years ADMA accumulation was associated with mitochondrial biosynthesis inhibition and dysfunction in myocardium. However, we has not been explored the relationship between endogenous ADMA accumulation and cardiac insufficiency and the direct effects of NOS inhibitor on the functions of mitochondria and heart have not been reported as yet. The present study was therefore designed that control SD rats were given intraperitoneally of exogenous NOS inhibitor NG-nitro-L-arginine (L-NNA), then the effects of cardiac function, mitochondrial function and biosynthesis in myocardium were observed, and further the effects as above of L-NNA were explored from the two aspects of nitric oxide (NO) precursor L-Arigine,(L-Arg) compensation and mitochondrial biosynthesis increase via metformin (Met). This study is to elucidate the inhibition of endogenous and exogenous NOS inhibitor on mitochondrial function and biosynthesis and reveal the crucial roles and mechanisms of ADMA in DCM. Our study will provide the new experimental data for the pathogenesis elucidation of DCM and provide the new insight into the clinical prevention and treatment of DCM and drug development.Methods (1) Animal experiments:Adult male Sprague-Dawley (SD) rats weighing150～180g were randomized into six groups.①Control,②L-NNA group (30mg/Kg/d),③L-Arg+L-NNA group (100mg/Kg/d+30mg/Kg/d),④Met+L-NNA group (125μg/Kg/d+30mg/Kg/d),⑤L-Arg group (100mg/Kg/d),⑥Met group (125μg/Kg/d). Among them, the②,⑤and⑥groups were treated corresponding medicine respectively intraperitoneally according to suggested doses, the③and④groups were given L-Arg (100mg/Kg) or Met (125μg/Kg), then given L-NNA (30mg/Kg) after1hour intraperitoneally every day. The Control group were daily treated PBS of the same volume intraperitoneally. After8weeks of the animal model preparation, myocardial performance and structure changes of the rats were detected M-Mode Echocardiography; After10weeks, hemodynamic index of carotid artery and left ventricle in groups were measure by carotid artery intubation, then the hearts were rapidly harvested and weighed, papillary muscles of left ventricle and thoracic aorta were separated and papillary muscles contraction of elicited by electrical stimulation and endothelium-dependent relaxation of aortic rings in response to acetylcholine were determined, respectively. The copies ratio of mitochondrial gene cytochrome oxidase I (COX I) and nuclear gene β-actin, the gene transcription and protein expression levels of the key gene of mitochondrial biosynthesis regulation peroxisome proliferation co-activated factor receptor y (PGC-1α), uncoupling protein2(UCP2) mRNA in mitochondria and ATP contents in myocardium were detected to reflect the changes of myocardial mitochondria function. RT-PCR and western blotting were used to measure the gene transcription and protein expression levels of the main generating enzymes protein arginine methyltransferasel (PRMT1) and the main metabolism enzymes dimethylarginie dimethylaminohydrolase (DDAH) of endogenous ADMA as well as endothelial nos (eNOS). The activities of NOS in myocardium and metabolic products contents of NO were used to reflect the changes of PRMT1/DDAH/NOS/NO pathway in myocardium by colorimetry. In addition, the level of lipid peroxidation product malondialdehyde (MDA) and the activity of super oxygen dehydrogenises (SOD) in myocardium were measured to reflect oxidative stress status.(2) Cell experiments:primary cardiomyocytes of neonatal SD rat were incubated by glucose (Glu,30mmol/L), oleinic acid (OA,100μmol/L), ADMA (30μmol/L) and L-NNA (30μmol/L) for48h, respectively. Mitochondrial transmembrane potential was detected by specific fluorescent probe JC-1; the copies ratio of mitochondrial gene cytochrome oxidase I (COX I) and nuclear gene β-actin and the transcription levels of UCP2gene were determined by PCR and RT-PCR; the protein expression changes of PRMT1/DDAH/ NOS pathway and PGC-1α were detected by Western blotting. Addition-ally, the effects of L-Arg (1mmol/L) and Met (300μmmol/L) pretreatment on mitochondrial transmembrane potential in primary cardiomyocytes and the protein expression of PRMT1/DDAH/NOS pathway.Results (1) Results of animal experiment:①After8weeks intraperitoneal injection of exogenous NOS inhibitor L-NNA to SD rats, the M-type ultrasonic testing revealed that the structure of the heart had changed, such as the augment of Left ventricular posterior wall end-diastolic thickness (LVPWd) and interventricular septum end-diastolic thickness (IVSd), as well as the increase of Left ventricular end-diastolic diameter (LVIDd,) and Left ventricular end-systolic diameter (LVIDs), which suggests that the left ventricular wall hypertrophy and cardiac structure reconstruction. In addition, IVSd%that reflecting the magnitude of cardiac interventricular septum and LVPW%that revealing the contraction amplitude of the Left ventricular posterior wall were both significantly reduced. The left ventricular end diastolic volume (EDV), left ventricular ejection fraction (EF%) and fractional shortening (FS%) were also significantly lower than the normal control rats, which indicate that the decrease of left ventricular systolic function.②After intraperitoneal injection of exogenous NOS inhibitor L-NNA in model rats fed for10weeks, rats cardiac index increased significantly; carotid arterial blood pressure tested by the carotid artery catheterization of the left ventricular displayed that the systolic and diastolic blood pressure of rat carotid artery, mean arterial pressure, mean diastolic blood pressure and dicrotic notch pressure were significantly higher than normal control group; in vitro vascular rings experiment to acetylcholine (ACh) induced endothelium-dependent relaxation response decreased, suggesting that exogenous injection of L-NNA can cause a strong contraction of blood vessels and inhibition of endothelium-dependent function.③Compared with normal control rats, the copy number ratio of mitochondrial genes such as COX I and nuclear genes like β-actin, mRNA level and expression of PGC-1α decreased in the myocardium of L-NNA rats. It showed inhibited mitochondrial biogenesis. In addition, reduced ATP contents and increased UCP2mRNA level indicated mitochondrial dysfunction.④The PRMT1/DDAH/NOS/NO pathway changed in the myocardium of L-NNA rats, such as increased PRMT1expression but mRNA level,decreased DDAH expression and mRNA level, eNOS expression and NO contents.⑤MDA contents were elevated significantly while SOD activity was reduced markedly in L-NNA model rats heart tissue. It indicated that exogenous intraperitoneal injection NOS inhibitor L-NNA increased oxidatie stress.⑥Whether to give NO synthetic precursor L-Arg or Met which could increase mitochondrial biosynthesis, can both improve, to some different extent, heart structure and function, endothelial function, mitochondrial function, mitochondrial biosynthesis and the changed of PRMT1/DDAH/NOS/NO in L-NNA to rat.(2)Results of cell experiments:it is the use of either high glucose, high fatty acids, or the endogenous and exogenous NOS inhibitor ADMA and L-NNA in the incubation of the primary cultured cardiac myocytes from neonatal SD rat after48h, that can reduce the mitochondrial transemembrane potential, increase the uncoupling protein UCP2gene transcription level, reduce mitochondrial DNA content, and down regulate the expression of PGC-la protein. All this results suggest that the endogenous and exogenous NOS inhibitors are the same as high glucose and fatty acids that could also inhibit mitochondrial function and mitochondria biosynthetic. In addition, high glucose and high fatty acids as well as endogenous and exogenous NOS inhibitors may also increase the expression of PRMT1, DDAH and eNOS protein in primary cultured cardiac myocytes, and cause the abnormalities of PRMT1/DDAH/NOS pathway. NO precursor L-Arg and Met treatment which could increase mitochondrial biosynthesis, can both improve, to some different extent, the endogenous and exogenous NOS inhibitor caused changes above.CONCLUTIONS:①Normal SD rats treated with chronic intraperitoneal injection of exogenous NOS inhibitors can significantly increased the peripheral arterial pressure. This may be due to inhibition of vascular endothelial function;②Chronic injection of exogenous NOS inhibitor can cause SD rats left ventricular wall thickening and reduced left ventricular pump function. This may be related with high peripheral arterial blood pressure, overload cardiac pressure and myocardial mitochondrial dysfunction;③The chronic injection of exogenous NOS inhibitor led to inhibition of cardiac mitochondrial dysfunction and mitochondrial biosythesis, which may be associated with increased oxidative stress, up-regulation of the UCP2gene transcription and down-regulation of PGC-la protein expression;④Chronic injection of exogenous NOS inhibitor can cause the disorder of myocardial PRMT1/DDAH/NOS/NO pathway... |
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