| Heart failure is one of the most common cardiovascular epidemics of the 21st century.Nearly 50%of heart failure patients are diastolic heart failure,manifested as normal or retained echocardiographic heart failure(heart failure with preserved ejection fraction,HFpEF).Today,the incidence of HFpEF has been further increased,accounting for 38%to 54%of all heart failure events,and systolic heart failure prevention and treatment has made more progress compared to that of diastolic heart failure.HFpEF has become the difficulty and hot spot of heart failure research.Calcium homeostasis in cardiomyocytes is a dynamic process of calcium ion distribution and phase change,which directly affects the systolic and diastolic function of cardiomyocytes through the excitatory-contractile coupling mechanism.Myocardial cell calcium abnormalities throughout the pathological state of myocardial compensatory hypertrophy to diastolic dysfunction,and then to the whole process of diastolic dysfunction.Protein kinase CaMKII-mediated sarcoplasmic systolic calcium release and diastolic calcium recovery is a key link in the regulation of cardiomyocyte systolic and diastolic function,and also a potential important intervention target.However,currently,no effectively targeted drug is available,traditional Chinese medicine may be a breakthrough in solving this problem.Chinese medicine believes that Qi deficiency and blood stasis is the basic pathogenesis of heart failure,throughout the development of heart failure.Both HFpEF and systolic heart failure can occur palpitations,shortness of breath,fatigue and chest tightness(severer after exertation),which are typical signs of the pattern of Qi dificency;and may be accompanied with dark purple lip,dark purple tongue with purple speckles,which aer typical signs of the pattern of Blood stasis.It can be concluded that the Qi deficiency and Blood stasis syndrome is certainly the foundamental pattern of HFpEF.Based on above understanding,a number of clinical studies have conducted and confirmed that Yiqihuoxue drugs can resulted in diastolic function alleviation of HFpEF patients and,meanwhile,Qi deficiency and Bloos stasis syndrome improvement at the same time.Furthermore,our previous research also found that Yiqihuoxue drugs can improve both left ventricular diastolic and systolic functions of HFpEF rat induced by pressure overload and the hypertrophy cardiomyocyte model induced by Ang Ⅱ injection,and calcium homeostasis got improvement at the same time.However,whether or not different effectiveness of Yiqi drug,Huoxue drug or integration of these two drugs on HFpEF and the underlined mechanisms need to be explored futher.In this research,we will copy the HFpEF rat model induced by pressure overload,and use animal ultrasound,left ventricular suspension catheter,laser confocal and immunofluorescence technique,Western blot technology,etc.,to testify the hypothesis of "Yiqihuoxue drugs can prevent and cure HFpEF through regulating calcium homeostasis and there are difference among Yiqi drug,Huoxue drug and their integrations(Yiqi vs Huoxue=1:1 and Yiqi vs Huoxue=2:1),from the systematic,histological,cellular and molecular level.Methods:1.Animal modeling:HFpEF rat model induced by pressure overload constructed by constricting the abdominal aorta.2.Grouping and administration:In addition to sham operation group,the model rats were randomly divided into model group(given saline),Yiqi group(given Astragalus + Codonopsis),Huoxue group(given salvia miltiorrhiza +pseudo-ginseng),Yiqihuoxue group(Yiqi vs Huoxue=1:1 and Yiqi vs Huoxue=2:1)and metoprolol group.3.Observational methods:1)Observed general conditions of model rats every week after surgery,including body weight,symptoms,signs and death including;Evaluated left ventricular function by animal ultrasound at the 4th week and 12th week after sugery,including diastolic wall thickness(LVAWd,LVPWd),diastolic diameter(LVIDd),left ventricular ejection fraction(LVEF),shortening ratio(FS),mitral valve forward blood flow velocity E and mitral annular motion velocity e;measured exertion tolerance by animal treadmill and left ventricular function by invasive suspension catheter;collected vein blood to measure platelet aggregation rate(PAR)and fibrinogen,then the heart and lung were removed to measure standardized heart weight(SHW)and standardized lung weight(SLW);2)Twelve weeks after surgery,acute left ventricular myocytes were isolated and the cardiomyocyte systolic and diastolic function and calcium release were measured by laser confocal and immunofluorescence;After 12 weeks,the expression of CaMK,PKA,NCX1,PLB(S16),PLB(T17),SERCA2a and RyR2 were detected by Western blot.Results:(1)Model evaluation:① At 12 weeks after operation,interstitial fibrosis and perivascular fibrosis in the myocardium of the model rats were significantly detected(P<0.05),LVAWd and LVPWd were significantly thickened(P<0.05),the exertion tolerance was significantly decreased,the E/e ratio,SHW,SLW,left ventricular end diastolic pressurewere(LVEDP)(about 12mmHg at rest and 16mmHg nder load condition),carotid pressure were significantly higher(P<0.05),which have proved the success of HFpEF rat model;when compared with sham group,the LVEF and FS of model rats decreased significantly(P<0.05)but with normal LVEF(>50%),suggesting that model rat suffered a slight but normal systolic disfunction at 12th week after sugery;② when compared with sham group,diastoling velocity of cardiomyocyte(-dl/dt)decreased significantly(P<0.05),suggesting diastolic dysfunction in cardiomyocyte of model rats;meanwhile,the systoling velocity(+dl/dt)decreased significantly(P<0.05)and 50%time consumed by systole of model rats(T50s)prolonged significantly(P<0.05),which suggested that cardomyocyte of model rats also suffered a slight but normal systolic disfunction at 12th week after sugery;In addition,50%time to peak of calcium transient(T50s-c),50%time to baseline calcium(T50d-c);diastolic calcium elimination time constant(Tau)prolonged significantly(P<0.05),while calcium leak increased signigicantly(P<0.05)and calcium content decreased significantly(P<0.05)when compared with sham group;on molecular level,calcium homeostasis-related protein expression of rats in model group were significantly different from that of the sham group,manifented as higher expression of CaMKII、PKA、NCX1、N/S(NCX1/SERCA2a)and lower expression of PLB(S16)、PLB(T17)、SERCA2a(P<0.05).What’s more,model rats showed exertion intolerance and higher level of fibrinogen,which are representive symptoms of Qi deficienc and Bloos stasis syndrome(QDBSS).(2)Effects of Yiqihuoxue drugs on SHW,SLW,myocardial fibrosis and representive symptoms of QDBSS of HFpEF rats on 12th week after sugery.① Compared with the model group,both SHW,SLW of all the intervention groups were lower than that of the model group(P<0.05);within the intervention groups,1:1 group and 2:1 group had better effectiveness in SHW than Huoxue group(P<0.05),2:1 group took the most priority on SLW when compared with Yiqi group,Huoxue group and moteroplol group(P<0.02);Compared with the model group,only 2:1 group could significantly alleviate myocardium fibrosis(P<0.05);③Compared with the model group,Yiqi group,1:1 group and 2:1 group could significantly improve the exertion distance and exertion time(P<0.05),but Huoxue group and metoprolol group failed to show effectiveness on exertion tolerance(P>0.05);within these groups,2:1 group showed better effectiveness than Yiqi group and 1:1 group(P<0.05);The PARmax and fibrinogen counted in Huoxue group were significantly lower than those in the other interventional group,while all interventional groups showd a tendacy of PARmax and fibrinogen reduction(P<0.05).(3)Effect of Yiqihuoxue drugs on left ventricular systolic and diastolic function of HFpEF rats on 12th week after sugery.① a.Systolic function evaluated by animal ultrosound-2:1 group could significantly alleviate LVEF of HFpEF rats when compared with model group(P<0.05);b.Diastolic function-when compared with model group,Yiqi group and metoprolol group could significantly decrease LVAWd and LVPWd(P<0.05),1:1 group and 2:1 group can significantly reduce not only LVAWd and LVPWd but also E/e(P<0.05),Huoxue group had no significant difference on all indexes(P>0.05);and within these groups,no evident difference was discovered(P>0.05);② a.Left ventricular diastolic function measured by left ventricular suspension catheter-when compared with model group,in the resting state,both 1:1 group and 2:1.group had a lower LVEDP(P<0.05);only 2:1 group had a higher-dp/dt(P<0.05),while Huoxue group and metoprolol group had no significant difference on all indexes(P>0.05);within these groups,2:1 group had a better effectiveness on LVEDP than 1:1 group(P<0.05);under load condition,2:1 group and 1:1 group showed lower LVEDP and higher-dp/dt when compared with model group(P<0.05),while Huoxue group and metoprolol group had no significant effect on LVEDP and-dp/dt(P>0.05);within these groups,2:1 group showed better effect on-dp/dt than 1:1 group(P<0.05).(4)Effect of Yiqihuoxue drugs on systole and diastole of cardiomyocyte,calcium homepstasis and calcium transient-related protein expression of HFpEF rats on the 12th week after sugery.① a.Cardiomyocyte systolic function-compared with model group,Yiqi group,1:1 group and 2:1 group could shorten T50s(P<0.05),but had no significant difference on other indexes;within these groups,1:1 group,2:1 group had better effects on T50s than Yiqi group(P<0.05),while no significant difference existed between 2:1 group and 1:1 group(P>0.05);b.Diastolic function-compared with model group,all interventional groups could alleviate T50d(P<0.05),but had no significant difference on other indexes;within these groups,2:1 group was discovered a better effect regarding T50d than Yiqi group(P<0.05),and 2:1 group,1:1 group and metoprolol group showed better effects than Huoxue group(P<0.05);② a.Calcium transient of cardiomyocytes-compared with the sham group,the Huoxue and metoprolol group was discovered to reduce the calcium transient(ph/bl)(P<0.05),but Yiqi group,1:1 group and 2:1 group were discovered to shoten T50s-c(P<0.05),and no evident difference found among these groups(P>0.05);b.calcium recovery of cardiomyocyte-compared with model group,Yiqi group,1:1 group and 2:1 group could significantly alleviate both T50d-c and Tau(P<0.05),but no evident difference found among them(P>0.05);however,no significant effectiveness was found in Huoxue group and metoprolol group(P>0.05);c.Calcium leak and content-only 2:1 group could statistically signigicantly reduce calcium leak of HFpEF rats(P<0.05);however,no difference of calcium content was detected in all interventional groups(P>0.05);③Calcium homeostasis-related key protein expression.Compared with the model group,all interventional groups could significantly reduce the expression of CaMK Ⅱ,PKA and N/S ratio(P<0.05);only 2:1 could result in a lower expression of RyR2 and a higher expression of SERCA2a(P<0.05);Yiqi group,1:1 group and 2:1 group could significantly reduce NCX1 expression(P<0.05);in addition,Yiqi group and 2:1 group also showed effect on.increasing the expression of PLB(S16)、PLB(T17)(P<0.05);meanwhile,metoprolol groups also had the function of increasing PLB(T17)expression(P<0.05);within these groups,2:1 group was more effectively on reducing CaMKII,PKA than Yiqi group(P<0.05)and on reducing N/S than Huoxue group and metoprolol group(P<0.05).Conclusions:(1)Yiqi group had better effects on activity intolerance,left ventricular remodeling,diastolic function of left ventricular,systolic and diastolic function of cardiomyocyte,calcium retaking ability and PLB expression than that of Huoxue group.(2)Overall,Yiqihuoxue group had better effect on prevention and treatment of HFpEF than Yiqi group,Huoxue group and Metopolol group.(3)Yiqihuoxue group with more tonishing Qi drugs had better effect on prevention and treatment of HFpEF than that with equal quantity of tonishing Qi drugs and activating Blood drugs. |
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