The Role Of Asymmetric Dimethylarginine In Myocardial Aging Of Aged Rats And Its Mechanisms

BACKGROUD With aging, cardiac function declines, accompanying with increased cardiovascular diseases. Myocardial aging plays an important role in the Abnormal Cardiac Function of old people and also is an potent risk factor for cardiovascular diseases. However, The mechanism for these have not been well elucidated. We found that serum concentrations of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NOS, were significantly elevated in aged rats, accompanying with impaired endothelium-dependent relaxation. Besides,ADMA significantly accelerated senescence associatedβ-galactosidase activity, accelerated the shortening of telomere length and reduced the telomerase activity in Human umbilical vein endothelial cells.All these suggest that ADMA is mostly associated with Endothelial Cell Senescence. However, the effect of ADMA on cardiocyte Senescence is not sure. Previous studies demonstrated that Oxidative stress, changed expression of aging-related genes and mitochondrial dysfunction are closely related to aging. Our and other researches indicated that ADMA can induce oxidative stress, and that the elevation of ADMA plays an important role in myocardial mitochondrial dysfunction of diabetic rats. So it is deserve to study the effect of ADMA on myocardial aging and to explore the possible mechanisms for ADMA leading to myocardial aging from several aspects, including Oxidative stress, changed expression of aging-related genes and mitochondrial dysfunction. This study is firstly to determine the important effect of ADMA in myocardial aging of aged rats and cultured rat primary cardiocytes Senescence. Furthermore, effects of calorie restriction, antioxidant N-acetylcysteine (NAC) and Resveratrol,a agonist of longevity gene Sirtl on ADMA induced cardiocytes Senescence were also investigated in the present study. This study provides new insight into the pathogenesis & prevention or treatment of cardiocytes Senescence.Methods①In animal experiment, the rats were divided into four groups at random, including six months ad lib-fed(AL) group, six months calorie restriction(CR) group, twenty-four months ad lib-fed group and twenty-four calorie restriction group. CR groups consume 40%fewer calories over time compared with animals fed AL. Oral glucose tolerance test (OGTT) was performed to assess insulin sensitivity. Parameters including blood levels of glucose, insulin, glycosylated hemoglobin (GHb), and lipid profiles were determined to assess metabolic control in the experiments of animal model. The mRNA level of P53, P21, Sirtl in rats myocardium were measured to reflect the degree of aging. The transcription of mitochondrial COX-Ⅰ, PGC-1αand UCP-2 gene and the mitochondrial ATP levels were measured to reflect mitochondrial function. The transcription of dimethylarginine dimethylaminohydrolase (DDAH), the major metabolic enzyme of ADMA, was detected. Serum ADMA was analyzed by high performance liquid chromatography; activities of DDAH & NOS and NO contents in myocardium of aged rats were analyzed by colorimetry to reflect changes in the pathway of DDAH/ADMA/NOS/NO. Myocardial superoxide dismutase (SOD) activity and contents of Malondialdehyde(MDA) were measured to reflect the antioxidant ability.②In cell experiments, rat primary cardiomyocytes were incubated with 30μmol/L ADMA for 72 hours in the absence or presence of Sirtl agonist resveratrol (30μmol/L) and antioxidant NAC(10μmol/L).β-gal staining was used to assess the degree of myocardial aging; the beating rate of primary cardiomyocytes was calculated to reflect the cardiac function. In addition, the transcription of aging related genesP21,P53,Sirt 1, the changes of DDAH/NOS/NO pathway and the degree of oxidative stress were assessed.RESULT Our studies have found that in comparison with the young control group, the old ad-lib rats have abnormal lipid metabolism and decreased plasma insulin, glucose and area under curve. More importantly, this study found that in the ad-lib fed older group, the transcription of myocardial DDAH, the activity of DDAH and NOS, the content of NO were largely decreased, with increased serum ADMA concentration. These changes of DDAH/ADMA/NOS/NO pathway are accompanied with increased transcription of aging genes P53, P21 and decreased transcription of longevity gene Sirtl. Besides, the transcription of COX-Ⅰ, PGC-1αand mitochondrial ATP levels in myocardial of aged rats were also greatly decreased. Further studies found that the activity of SOD was declined in myocardial of aged rats, accompanying with increased MDA content. It has long been held that calorie restriction is the most potent measure for extending the life span of rodents and other species. Our research supports that calorie restriction can mostly improve the ADMA-induced changes above. In cell experiments, after incubated with 30μmol/L ADMA for 72 hours, the beating rate of primary cardiomyocytes were mostly declined, accompanying with increased senescence-associated P-galactosidase activity. Agree with the animal experiment,the transcription of aging genes P53,P21 were increased,and which of Sirtl decreased. Further studies found that the antioxidant NAC (10μmol/L) can significantly improve the ADMA-induced Senescence of rat primary cardiomyocytes. Besides, as the most potent agonist of Sirtl, resveratrol (30μmol/L) also has a little therapeutic effect on ADMA-induced Senescence.CONCLUSIONS①These results indicate that the elevated endogenous ADMA play a very important role in myocardial aging of aged rats;②Calorie restriction can reduce the elevation of serum ADMA levels in aged rats, decline the transcription of aging genes, and increase the transcription of longevity genes.③Exogenous ADMA could directly induce senescence of rat primary cardiomyocytes; The mechanisms underlying which ADMA induced myocardial aging could be related to increased oxidative stress via uncoupling NOS and the decrease of NO content.