The Role Of Endogenous Inhibitor Of Nitric Oxide Synthase In Cardiac Dysfunction And Myocardial Mitochondrial Dysfunction Of Type1Diabetic Rats

BACKGROUDDiabetes is a common disease that is serverely harmful to the human’s health. Cardiovascular disease is the main causes of death and disability in patients with diabetes mellitus, especially diabetic cardiomyopathy. However, its mechansim is still unclear. Diabetic cardiomyopathy is a classic non-ischemic cardiomyopathy which does not depend on atherosclerosis or hypertension. The earliest clinical appears features are left ventricular diastolic dysfunction, decreased left ventricular compliance and left ventricular hypertrophy. Then it develops to contractive function dysfunction, Left ventricular expanding and showing all score of cardiac arrhythmia, finally leading to the heart failure. The main pathological changes are damage to myocardial microvascular endothelial cells, the fiber hyperplasia beneath the endothelium, film of blood capillary base, interstitial inflammation, myocardial fibrosis, myocardial cell apoptosis, etc. Diabetes clinically divide into type1and type2diabetes. Although90%of the clinical diabetes is type2diabetes, but type1diabetes frequently occurring early in the young with the longer disease courses, the treatment is depended on insulin, therefore there are more riskes to develop cardiovascular complications. There is accumulating evidence that diabetic cardiomyopathy is closely related to mitochondrial dysfunction. In recent years, our group has found that in the blood of type2diabetic rats induced by high-fat diet and a single small dose intraperitoneal injection of streptozotocin (STZ), endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) significantly increased, which has a relationship with reduced mitochondrial biogenesis and myocardial mitochondrial dysfunction, but the changes in cardiac function of diabetic rats were not detected. Therefore, this study intends to further explore the relation among endogenous ADMA, diabetic cardiac dysfunction and myocardial mitochondrial dysfunction in STZ-induced diabetic rats. This study provides new experimental data for the mechanism of the pathogenesis of diabetic cardiomyopathy.METHODS1. In animal experiments, the two-month weight200～220g male were given a single injection of streptozotocin(65mg/kg,0.1mol/L citrate buffer, pH4.2, i.p.). Type1diabetic rats underwent left ventricular catheterization through the left carotid artery to detect the changes in hemodynamics and cardiac function. The ATP contents of myocardial tissue were assessed. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the mRNA levels of myocardial tissue uncoupling protein (UCP2). RT-PCR was used to detect the mRNA levels of PGC-1a, and polymerase chain reaction (PCR) was used to detect the copy numbers of mitochondrial genes such as COX I and nuclear genes such as β-actin, these indexes were used to reflect mitochondrial biogenesis. Serum ADMA concentration was measured by high performance liquid chromatography. Western blotting were used to assess the expression of protein N-arginine methyltransferase (PRMT1), ADMA main metabolizing enzymes dimethylarginine dimethylamine hydrolase (DDAH), eNOS and iNOS. NOS activity and NO concent were measured by colorimetry to reflect the changes of PRMT1/DDAH/NOS/NO pathyway, Malondialdehyde (MDA) contents and superoxide dismutase activity were also measured by colorimetry to reflect oxidative stress.2. In cell experiments, rat cardiocytes (H9C2) were incubated with30μmol/L ADMA or L-NNA or L-NAME for48hours, the changes of protein expression of PRMT1/DDAH/NOS/NO pathyway were detected by western blottingRESULTS1. In animal experiment, STZ-induced diabetic rats’blood glucose was significantly elevated, and the urine glucose was strongly positive. T1DM rat weight significantly decreased, the heart weight declined, then the relative heart mass index (HMI) increased. Diabetic rats’ heart rate (BPM) was much slower than normal rats with prolonged cardiac cycle and systole. Left ventricle isovolumic relaxation period (IRP)average dp/dt was significantly lower, maximum rate of change of left ventricular diastolic pressure decreased (Min dp/dt) was significantly slowed down, these index reflect obvious damage in diabetic rats diastolic function; In addition, left ventricular systolic pressure (Max pressure) decreased, diastolic blood pressure (Min pressure) increased, left ventricular end diastolic pressure (EDP) increased,although these changes are not statistically significant, but the left ventricular developed pressure (Max-Min pressure) decreased significantly, suggesting that systolic function was mild damage.Compared with normal control rats, mitochondrial DNA content significantly decreased in the heart of diabetic rat, the key regulator of mitochondrial biogenesis PGC-la transcription was down-regulated, indicating that diabetic rats heart mitochondrial biogenesis reduced; uncoupling protein UCP2transcription levels of myocardium increased, while ATP content increased. Serum ADMA levels were markedly elevated in type1diabetic rats compared to control rats. Myocardium DDAH activity decreased, while NOS activity did not change markly, iNOS activity increased, NO’s metabolites increased. Diabetic myocardium ADMA produce enzymes PRMT1protein expression increased, ADMA metabolism enzymes in the cardiovascular system DDAH2protein expression was reduced, DDAH1showed no significant change. iNOS expression was regulated, while eNOS expression decreased, suggesting that ADMA high concentration of diabetes was due to the upregulation its generating enzyme PRMTl and downregulation of its metabolic enzymes DDAH2. Correlation analysis revealed that the serum concentrations of endogenous ADMA was negatively related to left ventricular developed pressure (Max-min pressure), and negatively correlated with left ventricular diastolic pressure decreased the maximum rate (Min dp/dt). In addition, endogenous ADMA concentration was negatively correlated with the content of mitochondrial DNA or mitochondrial biogenesis, and positively correlated with the level of UCP2transcription.2. In cellular experiment, incubation of H9C2cells with30μmol/L ADMA or L-NNA or L-NAME for48h obviously increased PRMT1expression, reduced DDAH1and DDAH2expression.Meanwhile,the expression of iNOS was upregulated,and the expression of iNOS was downregulated.CONCLUSIONS1.The elevation of endogenous inhibitor of nitric oxide synthase ADMA in serum may be as a result of upregulation of PRMT1and dowregulation of DDAH2.2. Inhibition of mitochondrial biogenesis in diabetic rats myocardium inhibition of may be related to downregulation of PGC-1α transcription3.Obvious damage was observed in diabetic rats diastolic function, and systolic function was mild damaged.4.The elevation of endogenous inhibitor of nitric oxide synthase ADMA may have a relationship with diastolic dysfunction systolic dysfunction. Endogenous ADMA concentration was negatively correlated with the content of mitochondrial DNA or mitochondrial biogenesis, and positively correlated with the level of UCP2transcription.