Study On Neuroendocrine Mechanisms Of Diastolic Heart Failure

Objective:To establish the rat model of hypertensive diastolic heart failure through different methods.We evaluated the characteristics of DHF model through echocardiography,hemodynamics,histomorphology and level of NT-proBNE We studied the effects of kidney relative protein renalase and sympathetic nervous system on DHF and renal hypertension through DHF and renal hypertension animal models of two kidney-one clip.We studied the molecular mechanisms of diastolic heart failure through DHF animal models and evaluated roles and mechanisms of diltiazem on DHF.Methods:We used 6-week-old Wistar rats to establish the animal model through coarctation of renal artery and abdominal aorta in different positions.The rats sacrificed after twelve weeks of feeding.We measured the changes of cardiac structure and function using echocardiography after operation.The hemodynamic parameters were determined before sacrifice.Serum NT-proBNP levels were determined before operation and before sacrifice.The left ventricular weight index was determined after sacrifice.The histomorphology of myocardium was estimated by HE staining.CVF and PVCA were determined through Masson staining.We established renal hypertension model by two kidney-one clip method and established DHF model by both infrarenal abdominal aorta and left renal artery coarctation.After feeding 12 weeks,serumal renalase and catecholamine levels were determined by ELISA.Myocardial catecholamine levels were determined by ELISA.And myocardial renalase levels were determined by Western blot technique and they were corrected byβ-actin.Plasmatic AngⅡand ET levels were determined by radio-immunity method and serumal renin and aldosterone levels were determined by ELISA.Myocardial apoptosis was determined by TUNEL staining and apoptosis index(N)was calculated.We established DHF model through both infrarenal abdominal aorta and left renal artery coarctation method.Some rats were given diltiazem through intragastric administration everyday for four weeks from the eighth week after operation. Then all the rats sacrificed.Heart function parameters of rats were determined by echocardiography and hemodynamics before sacrifice.Masson staining was adopted.Myocardial apoptosis was determined by TUNEL staining.The serumal levels ofⅠtype collagen,Ⅲtype collagen,MMP-2,MMP-9 and TIMP-1 were determined by ELISA.The SERCA2,RYR and PCNA levels ofmyocardium were determined by Western blot technique.Results:IVSD,LVPWD and LVmass of rats in suprarenal abdominal aorta coarctation group and both infrarenal abdominal aorta and left renal artery coarctation group increased(P＜0.05)four weeks after operation.The rats in the two groups showed cough,dyspnea,decrease of appetite and insensitivity of reaction.And the rats in the two groups showed diastolic dysfunction in echocardiography and hemodynamics.There were hypertrophy,derangement of cardiac cells,hyperplasia of cardial interstitium and increase of collagen content in pathology.The levels of serumal and myocardial NT-proBNP of rats in the two groups increased.All the results showed that we could estabolish DHF animal model successfully through the two operation methods.Serumal catecholamine levels of rats in renal hypertension group and DHF group were higher and serumal renalase levels were lower than those in the control group.And there were negative correlations between the levels of serumal renalase and serumal dopamine,epinephrine,norepinephrine(P＜0.05).The levels of myocardial dopamine and norepinephrine of rats in DHF model group were higher than those of control group and renal hypertension group(P＜0.05)and there were no difference between the two groups.The myocardial renalase levels of rats in renal hypertension group and DHF group were lower than those of control group(P＜0.05,P＜0.01 respectively).The levels of myocardial renalase of DHF group were lower than those of renal hypertension(P＜0.01).There were no relationships between the myocardial renalase and myocardial dopamine, epinephrine,norepinephrine.There were positive correlation between serumal renalase level and myocardial renalase level(r=0.613,P＜0.05).The diastolic function of DHF rats improved after four weeks of diltiazem therapy.CVF and PVCA of DHF rats decreased and apoptosis cells reduced after diltiazem therapy. Compared with sham group,Ⅰtype collagen,Ⅲtype collagen,and TIMP-1 levels of rats in DHF group and diltiazem therapy group increased obviously(P＜0.01)and MMP-2,MMP-9 levels decreased(P＜0.05).Ⅰtype/Ⅲtype collagen ratios of DHF rats decreased after diltiazem therapy of four weeks (P＜0.05).Compared with those of sham group,myocardial levels of SERCA2 and RYR in DHF model group decreased obviously and levels of PCNA increased. And levels of the above proteins of rats in diltiazem therapy group were between those of sham group and DHF model group.Conclusion:(1)Diastolic heart failure animal model can be established through both infrarenal abdominal aorta and renal artery coarctation for twelve weeks. (2)Serumal NT-proBNP had good correlations with diastolic functional parameters.So serumal NT-proBNP will be a important means appreciating DHF. (3)Myocardial cell apoptosis and myocardial interstitial fibrosis may participate the formation of diastolic heart failure.(4)Dysequilibrium of calcium homeostasis may participate the formation of diastolic heart failure and SERCA2 and RYR may play important roles in it.(5)There were activation of sympathetic nervous system and decrease of kidney relative protein renalase in renal hypertension and diastolic heart failure.This indicates that the activation of sympathetic nervous system may be the result of insufficiency of renalase secretion.(6)Serumal dopamine,epinephrine and norepinephrine of rat were negatively correlated with serurnal renalase.This indicates that as same as the vitro result,catecholamine in vivo may also mainly be metabolized by renalase.(7)Calcium channel blocker diltiazem can effectually improve cardiac diastolic function and can be an elementary medication of diastolic heart failure.