Study On The Mechanism Of Cardiac Dopamine D5 Receptor Through Oxidative Stress And PKG-ERK1 / 2 / AKT In The Pathogenesis Of Dilated Cardiomyopathy

BackgroundDilated cardiomyopathy (DCM) is a progressive disease of heart muscle that is characterized by ventricular chamber enlargement and contractile dysfunction, but so far its molecular rmechanism is not clear. D5 dopamine receptor gene knockout (D5-/-A) mice exhibit high blood pressure, heavy heart and cardiac hypertrophy. In 3th month, compared with CMV-hD5WT transgenic mice, CMV-D5F173L transgenic mice became hypertensive, and in 4th month the transgenic mice became left ventricular hypertrophy, which indicate that D5 dopamine receptor is related to the progression of cardiac diseases. Therefore,we generated heart-specific hD5F173L(a-MHC-hD5F173L) and hDWT (a-MHC-hD5WT) transgenic mice, and hD5F173L and hDWT transfection H9C2 cell lines to investigate the role and mechanism of D5R-mediated regulation of cardiac function.MethodsThe NOX2 expression and ROS production were tested in the transgenic mice at three month of age. The α-MHC-hD5F173L mice were treated with either NADPH oxidase inhibitor Apocynin (1mmol/kg/day) or phosphate-buffered saline (PBS) as control by intraperitoneal injection for 4 weeks. After then, the indexes of heart function were measured. The hD5WT and hD5F173L were transfected respectively in rat H9C2 cells, in which ROS production and NOX2 expression were detected at basal level.And to determine if PKA or PKG is involved in the mechanism of the regulation of NADPH oxidase activity by dopamine D5 receptor, the H9C2 cells were treated by PKA inhibitor (H89) and PKG inhibitor (KT5823) respectively.ResultsWe found that a-MHC-hD5F173L mice presented dilated cardiomyopathy with high NADPH oxidase activity and NOX2, ERK1/2 and AKT expression compared with a-MHC-hD5WT mice, which were ameliorated or decreased by NADPH oxidase inhibitor Apocynin. NOX2, pro-ERK1/2 and pro-AKT expressions were higher in hD5F173L transfected H9C2 cells than hDWT cells. The increased expressions of NOX2, pro-AKT and pro-ERK1/2 in H9C2-D5F173L cells were prevented by PKG inhibitor KT5823 but not by PKA inhibitor H89.ConclusionDopamine D5 receptor may play an important role in the regulation of heart function by inhibiting ROS production via PKG induced ERK1/2 and/or AKT pathway.The role of platelet NHE-1 in the mechanism of salt-sensitive hypertensionHypertension is a complex and multifactorial disease resulting from the interaction of multiple genetic and environmental factors. The salt sensitive hypertension is one of the important types of hypertension and salt intake plays a pivotal role in the development of it. However, the mechanism is still unclear. It has been found that platelet or/and inflammation are important factors causing salt-sensitive hypertension. Human studies have reported that serum P-selectin levels were higher in salt-sensitive hypertensives than normaltensives. It was reported that the blood pressures were increased with inctreased introcellular calcium concentration of platelet and platelet dysfunction in salt-sensitive hypertensive rats fed withhigh-salt diet. We hypothesyze that high sodium intake causes an increase in intracellular calcium concentration and platelet activation via platelet Na+/H+exchanger (NHE-1),and then results in inflammation and high blood pressure. In this study, we will test the the role of platelet NHE-1 in the mechanism of salt-sensitive hypertension via In Vivo and In Vitro studies, so as to provide new information for treatment or prevention of hypertension.Methods1. In vivo StudyDahl salt-sensitive rats and SS13BN rats (control) were fed with high salt diet (8% Nacl) and low salt diet (0.12% Nacl) for four weeks. Systolic and diastolic blood pressures were measured by tail artery. The positive rates of NHE-1, p-selectin (platelet activation) and the introcellular concentration of Ca2+ of platelet were measured by flow cytometry (FCM). The expression of IL-6, IL-1, NFκB was analyzed by Western blotting.2. In vitro StudyPlatelets were isolated from blood of SD rat for in vitro study. After pre-incubated with NaCl saline (1.3%NaC1) for 30mins, the isolated platelets were then stimulated in medium with or without NHE-1 inhibitor HOE642(5μM) for 15 mins. After then, the extracellular membrance NHE-1 and P-selectin expression and introcellular calsium concentration of platelet were measured.Results1. In vivo StudyCompared with low salt diet (0.12% NaCl) or SS13BN groups, the Dahl SS rats presented significantly increased blood pressure after four weeks high-salt diet (8% NaCl), and accompanied with increased platelet extracelluar membrance NHE-1 expression, introcellular calcium concentration, platelet activation, and inflammation factors expression.2.1n vitro studyNHE-1 inhibitor HOE642 preventes the incease of platelet extracelluar membrance NHE-1 expression, introcellular calcium concentration and platelet activation, which were stimulated by high sodium.ConclusionHigh salt intake develops salt-sensitive hypertension via inflammation and increased platelet activation through stimulating platelet membrance NHE-1 activity.