| Pathological cardiac hypertrophy is one of adaptive responses of the heart to a variety of pathological stimuli, and always one of the complications of some cardiovascular diseases, such as hypertension, myocardial infarction, congenital heart diseases, cardiovalvular diseases, and etc. Currently pathological cardiac hypertrophy is believed to be an independent risk factor influencing incidence and mortality of cardiovascular diseases, since the incidence of cardiovascular events in population with cardiac hypertrophy is 2~4-fold of that in normal population. Cardiac hypertrophy will cause severe arrhythmia and progress pathologically to heart failure at the final stage, thus worsening prognosis of clinical patients and accordingly justifying research on formation mechanisms of cardiac hypertrophy to facilitate invention of more efficient treatments. Recently, researchers have gained an insight into alterations of physiological function and gene expression profile in the heart during formation of hypertrophy, however, detailed formation mechanisms of cardiac hypertrophy remains uncertain. It is now well accepted that cardiac hypertrophy belongs to adaptive responses of the heart to various stimuli and is closely related to pressure overload and abnormal neuroendocrine. Several studies have demonstrated that various neuroendocrine cytokines, such as angiotensin, norepinephrine, endothelin, platelet-derived growth factor, and etc., could bind to their corresponding receptors to activate multiple signaling cascade, including JAK-STAT signaling, MAPK signaling, calcineurin signaling, and etc. As a result, transcription of proto-oncogenes is then activated to upregulate expression levels of cell behavior-specific genes, causing cell growth, proliferation, differentiation and other long-term effects. To maintain homeostasis, known as negative feedback, some other cytokines such as bradykinin, nitric oxide (NO), and etc., are simultaneously released to negatively regulate the above cell behavior, thus making cells and tissues live with complicated regulation network. Morphological investigation has suggested that cardiac hypertrophy is characterized not only by hypertrophy of cardiomyocytes, but also by proliferation of interstitial cells and accumulation of collagen. Consequently, studies on signal transduction molecules relative to these cellular effects are requisite for better understanding of cardiac hypertrophy, and even indispensable for invention of novel anti-hypertrophy agents. Recently L-arginine was found to generate NO under catalytic action of NO synthase and this process was known as L-arginine-NO pathway. NO, in the heart, could dilate coronary arteries, increase blood influx in coronary arteries and reduce inotropy of myocardium. Judged by these bioactivity, NO may play an important role in regulation of cardiac hypertrophy. To further authenticate the roles of NO in cardiac hypertrophy, this study was designed to investigate expression levels of extracellular inducer production and intracellular signaling molecules, and changes of gene expression profiles during formation of cardiac hypertrophy or under the influence of L-arginine, with the intention to interpret relationship between cardiac hypertrophy and multiple signaling molecules, and clarify effects of L-arginine-NO pathway on cardiac hypertrophy at both cellular and molecular levels.1 Study of establishment and evaluation of rat models with abdominal aortic coarctationThis experiment is aimed to improve the traditional surgical procedure used for the establishment of rat models with abdominal aortic coarctation, and to clarify influences of different degrees of abdominal aortic coarctation on the survival rate and the level of cardiac hypertrophy and hypertension.(1) The survival rate following the revised surgical procedure was compared with that derived from the traditional procedure; (2) The revised surgical procedure was utilized to operate on abdominal aortas of additional four rat groups. The abdominal aortas'internal diameters of these four groups were respectively coarctated to 0.5mm, 0.6mm, 0.7mm and 0.8mm (correspondingly called 0.5mm group, 0.6mm group, 0.7mm group and 0.8mm group). Four weeks after the operation, the survival rates were compared with one another; (3) According to parameters such as Left Ventricular Mass Index (LVMI), Left Ventricular Wall Thickness (LVWT), Diameter of Cardiac Myocyte (DCM) and Mean Arterial Blood Pressure (MABP), we compare the level of cardiac hypertrophy and hypertension in 0.8mm group with that in 0.7mm group and sham group four weeks after the operation.As indicated by the results: (1) Four weeks after the operation, the survival rate following the revised surgical procedure was 90.00% (27/30), significantly higher than 66.67% (20/30) derived from the traditional surgical procedure; (2) The survival rate 86.67% (26/30) in 0.8mm group were respectively higher than 63.33% (19/30) in 0.7mm group, 43.33% (17/30) in 0.6mm group and 3.33% (1/30) in 0.5mm group; (3) As for the level of cardiac hypertrophy and hypertension, there was no significant difference between 0.8mm group and 0.7mm group, according to the detection of LVMI, LVWT, DCM and MABP.The revised surgical procedure is more suitable than the traditional surgical procedure due to increase in the survival rate of rat models. Moreover, coarctating the internal diameter of abdominal aorta to 0.8mm can increase the survival rate without influence on the level of cardiac hypertrophy and hypertension, compared with coarctating its internal diameter respectively to 0.7mm, 0.6mm or 0.5mm.2 L-arginine-NO pathway negatively regulates expression level of angiotensin II and its receptor type 1We evaluate the effects of L-arginine (L-Arg) on cardiac hypertrophy in vivo and in vitro, with the intention to elucidate the role of L-Arg-NO pathway in the heart and relevant mechanisms involved.Grouping was respectively conducted in rat models (by utilizing rat models with abdominal aortic coarctation) and in angiotensin II (ATII)-induced cardiomyocytes cultured in vitro, according to the presence/absence of L-Arg and N-nitro-L-arginine methyl ester (L-NAME). In vivo, the heart rate, blood pressure level, left ventricular mass index (LVMI) were measured; Total protein level in the heart, cardiac nitric oxide (NO) content were detected by colorimetric methods and cardiac ATII content was determined by radioimmunological methods; The expression level of inducible NO synthase (iNOS) mRNA, angiotensin receptor type 1 (ATR1) mRNA, angiotensin receptor type 2 (ATR2) mRNA,angiotensin converting enzyme (ACE) mRNA were examined using RT-PCR analysis; Besides, the protein expression level of iNOS was detected by Western-Blotting.As indicated by the results: (1) In vivo, L-Arg could lower the LVMI and reduce cardiac ATII content, ACE mRNA expression level and total protein level in the heart, as the same time it raised cardiac NO content; (2) In vitro, L-Arg could increase the expression level of iNOS and NO concentration in the medium, whereas down-regulate ATR1 expression. The expression level of ATR2 was not affected by L-Arg, L-NAME or ATII. The above effects of L-Arg could be abolished by NOS inhibitor L-NAME in vivo and in vitro; (3) As revealed by multiple stepwise regression analysis, no relationship of regression was found between total protein level in the heart and blood pressure level or the heart rate. However, cardiac NO and ATII content could act as predictors of total protein level in the heart; (4) Correlation analysis displayed that, the relationship of negative correlation was significant between cardiac NO content and each of following factors, cardiac ATII content, ACE expression level and ATR1 expression level.Summaries are as follows: (1) The development of cardiac hypertrophy is closely related to cardiac NO and ATII level; (2) By enhancing myocardial iNOS expression level, L-Arg could increase cardiac NO content. NO, derived from L-Arg-NO pathway, could inhibit the development of pathological cardiac hypertrophy by reducing the production of ATII and down-regulating the expression level of ACE and ATR1 in the heart; (3) ATR2 seems to be independent from the development and regression of cardiac hypertrophy. 3 L-arginine-NO pathway inhibits activation of MAPK signaling by dephosphorylationThis study is designed to analyze the influence of L-arginine (L-Arg) on expression level of angiotensin II receptors (ATR) and the mitogen-activated protein kinase (MAPK) in cultured cardiomyocytes, with the intention to clarify the mechanisms relative to L-Arg's inhibitory effects on formation of cardiac hypertrophy.Five groups of cardiomyocytes were established: (1) Control group; (2) Angiotensin II (ATII) group; (3) ATII + Saralasin group; (4) ATII + L-Arg group; (5) ATII + L-Arg + L-NAME (N-nitro-L-arginine methyl ester) group. After 48 hours in supplemented culture, synthetic velocity of protein, NO production, expression level of ATR and p38 MAPK in cardiomyocytes were detected through the [3H]-leucine incorporation method, colorimetry, RT-PCR and western blotting, respectively.As indicated by the results: (1) L-Arg could decrease the expression level of ATR1 and phosphorylated p38 MAPK, enhance NO production and reduce the synthetic velocity of protein in cultured cardiomyocytes stimulated by ATII. (2) Correlation analysis revealed that the relationship of negative correlation was significant between NO production and each of following factors: ATR1 expression level and phosphorylated p38MAPK expression level; As indicated by multiple stepwise regression analysis, not ATR2 expression level, but ATR1 expression level acted as the regression predictor of expression level of phosphorylated p38 MAPK.Summaries are as follows: (1) Phosphorylation of p38 MAPK is mediated by ATR1. (2) By enhancing myocardial NO production, L-Arg-NO pathway inhibits the p38MAPK activation mediated by ATR1, leading to inhibition of the hypertrophic response of cardiomyocytes. (3) ATR2 seems to be independent from the activation process of p38 MAPK. 4 L-arginine-NO pathway inhibits activation of calcium-mediated calcineurin signalingIn acutely isolated cardiomyocytes, effects of L-arginine (L-Arg) on intracellular calcium concentration ([Ca2+]i) and expression level of caicineurin (CaN) are analyzed, with the aim to interpret the role of CaN during formation of cadiac hypertrophy, and clarify relationship between L-Arg-NO pathway and CaN signaling.Grouping was conducted first: (1) Sham-operation group (without constriction operation on the exposed abdominal aorta); (2) Operation group (The internal diameter of abdominal aorta was constricted to 0.8mm at the ligation site); (3) L-Arg group ( L-Arg at 600mg/kg?day-1 was administered to aorta-constricted rats intragastrically immediately after operation); (4) N-nitro-L-arginine methyl ester (L-NAME) group (L-Arg at 600mg/kg?day-1, and L-NAME at 50 mg/kg?day-1 were sequentially administered to aorta-constricted rats intragastrically after operation). As control, water without drugs was used for intragastric administration to rats in Sham-operation group and Operation group. Six weeks later, total protein content and NO production in left ventricular myocardium were respectively detected by means of Bradford methods and colorimetry. In acutely isolated cardiomyocytes, [Ca2+]i is tested by fluorescent colorimetry and expression levels of CaN and embryonic stage-specific atrial natriuretic factor (ANF) in the myocardium are detected by means of RT-PCR. Significant up-regulation of ANF is regarded as a marker of cardiac hypertrophy formation.As indicated by the results: (1) In cardiomyocytes of rats in Operation group, [Ca2+]i is significantly elevated and expression levels of CaN and ANF are obviously up-regulated, in comparison to those in Sham-operation group; (2) As compared to Operation group, L-Arg group exhibits lower level of [Ca2+]i , as well as CaN and ANF expression; (3) In L-NAME group, L-NAME could abolish L-Arg's above effects, making detection values of above indices similar with those in Operation group; (4) Myocardial NO production is found to negatively correlate with [Ca2+]i , CaN and ANF expression level, respectively; On the contrary, myocardial total protein content positively correlates with [Ca2+]i and CaN expression level respectively.Summaries are as follows: (1) Cardiac hypertrophy is closely related to intracellular calcium concentration and CaN expression level in cardiomyocytes; (2) L-Arg-NO pathway could significantly reduce intracellular calcium concentration and down-regulate CaN expression level, resulting in inactivation of CaN signaling.Conclusion1 The revised surgical procedure is more suitable than the traditional surgical procedure, due to increase in the survival rate of rat models.2 Coarctating the internal diameter of abdominal aorta at the ligation site to 0.8mm can increase the survival rate without influence on the level of cardiac hypertrophy and hypertension, in marked comparison to coarctating its internal diameter respectively to 0.7mm, 0.6mm or 0.5mm.3 The development of cardiac hypertrophy is closely related to cardiac NO and ATII level; By enhancing myocardial iNOS expression level, L-Arg could increase cardiac NO content, which could inhibit the development of cardiac hypertrophy by reducing the production of ATII and down-regulating expression level of ACE and ATR1 in the heart.4 ATR2 appears to be independent from the development and regression of cardiac hypertrophy.5 Phosphorylation of p38 MAPK is mediated by ATR1.6 By enhancing myocardial NO production, L-Arg-NO pathway inhibits the p38 MAPK activation mediated by ATR1, leading to inhibition of the hypertrophic response of cardiomyocytes.7 Cardiac hypertrophy is closely related to intracellular calcium concentration and CaN expression level in cardiomyocytes.8 L-Arg-NO pathway could significantly reduce intracellular calcium concentration and down-regulate CaN expression level, resulting in inactivation of CaN signaling. |
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