Experimental Study On Regulating Myocardial Energy Metabolism And Calcium Circulation Homeostasis In Heart Failure By Qishen Granule

Objective:To dynamically evaluate syndrome property from micro and macro signs in heart failure(HF)rats induced by high-fat diet(HFD)and left anterior descending(LAD)ligation.Based on those HF models,to evaluate the cardio-protective effects of Qishen Granule(QSG)and explore the pharmacological mechanism in regulating cardiac energy metabolism and sarcoplasmic reticulum calcium cycle.Methods:1.Male SD rats were underwent ligation surgery of LAD and were fed with high-fat diet from one week before and four weeks after the surgery.Three groups,HFD group(HFD+sham operation),Sham group(normal diet+sham operation)and LAD group(normal diet+LAD ligation),were set as controls.Blood and heart tissues were harvested in 7,14,28 days after surgery.The successful rate of HF rats and their syndrome property were determined based on results of echocardiography,blood lipid testing,electrocardiography,histopathology of heart and liver,open-field test,respiratory rate under stress,RGB value of planta pedis,body weight,daily-intake,BMI and body weight growth.2.The biomarkers in blood,including markers of myocardial injury(AST,cTnI,CK-MB and LDH),inflammatory factors(TNF-α,IL-1β,IL-6 and hsCRP),neural hormone(AngⅡ,ALD,BNP and NTpro-BNP),markers of oxidative stress(MDA,ox-LDL and NO)and cardiac fibrosis(ICTP,PINP and PⅢNP),were detected.A correlation analysis was conducted between the blood biomarkers and syndrome indexes.3.The HF rats were divided into 5 groups and received gavage administration from the day after LAD ligation:QSG group(2.2g/kg daily),trimetazidine group(6.3mg/Kg,daily),metoprolol group(10.5mg/kg daily),atorvastatin group(1.05 mg/Kg,daily)and model group(same volume of distilled water,daily).A sham group received distilled water gavage was set as control.All rats were treated for 28 days.Syndrome indexes were collected once a week after LAD or sham surgery.Those indexes consisted of endurance(staying time hanging in the air),respiratory rate under stress,RGB value of tongue,body weight daily growth and skin temperature.At the end of treatment,all rats underwent echocardiography and then euthanasia to have their hearts and blood harvested for hematoxylin and eosin(HE)staining and lipid testing.The heart weight and lung weight were measured.4.Study on cardiac energy metabolism was performed between the sham,model,QSG and trimetazidine group.ATP concentrations in cardiac tissues were measured by phosphomolybdic acid colorimetry.Ultrastructures of myocardium were observed by transmission electron microscopy.Apoptotic cells of hearts were detected by TUNEL method.Levels of free fatty acid and lactate both in heart and serum were measured by chemical method.Expressions of protein and mRNA involved in energy metabolic pathways were detected by means of Western Blot and RT-qPCR.5.Study on regulation of SR calcium cycle was performed between the sham,model,QSG and metoprolol group.Concentrations of Ca2+in cytoplasm and activity of Ca2+-ATPase were measured according to the manufacturer’s instructions.Expressions of protein and mRNA involved in RyR2 leak and SERCA2a uptake were detected by means of Western Blot and RT-qPCR.Results:1.HF rat models were successful,evidenced by significant decreased EF value(P<0.01),elevated blood lipid(TC,TG and LDL-C)levels(P<0.05),disordered myocardial structure and abundant fatty deposited liver.At the 7th,14th and 28th day after surgery,the decreased movement distance in 3min(P<0.01),increased respiratory rate under stress(P<0.05)and decreased RGB and R value of Planta pedis(P<0.05)indicated a qi-deficiency and blood stasis syndrome.At the 14th and 28th day after surgery,body weight growth and BMI increased remarkably(P<0.05),indicating syndrome of turbid phlegm appeared.2.The biological characteristics of HF models were detected.At the 14th day after surgery,it is LDH increased significantly(P<0.01)while at the 28th day it evolves to elevated CK-MB and cTn-I(P<0.05).Inflammatory response mainly occurred at the 7th and 14th days after surgery,characterized by elevated TNF-α and IL-1β and decreased IL-6(P<0.05).Ang Ⅱ and ALD both increased significantly(P<0.05),indicating a persistent activation of RAAS system.Oxidative stress was the noticeable characteristic of the HFD-LAD induced HF models,manifesting in significant increased levels of MDA,NO and ox-LDL(P<0.05).The degree of cardiac fibrosis grow progressively,with the ways of main collagen metabolism changing from Col I into Col III.There were 17 syndrome related indicators were selected through correlation analysis,of those 8,9,14 indicators participated in the development of qi-deficiency,blood stasis and turbid phlegm syndrome,respectively.These indicators were not specific for one syndrome but cross one another.3.QSG treatment increased EF and FS values(P<0.01),inhibited ventricular dilatation,decreased lung weight/body weight(P<0.01)and serum TC levels(P<0.01).QSG improved qi-deficiency syndrome by strengthening endurance(P<0.05)and decreasing respiratory rate under stress(P<0.05).QSG treatment improved blood syndrome by increasing RGB value of tongue(P<0.05).QSG treatment also improved turbid phlegm syndrome by inhibiting body weight growing too fast and prevented yin-deficiency syndrome by decreasing the higher skin temperature(P<0.05).4.HF rats showed reduced cardiac ATP concentrations(P<0.01),abnormal mitochondrial morphology,up-regulated UCP2(P<0.01)and increased TUNEL positive cells accompanied by elevated fatty acid oxidation and glycolysis proportion.QSG treatment increased cardiac ATP concentrations significantly(P<0.01),maintained mitochondrial structure,promoted mitochondrial biosynthesis by up-regulating PGC-1a/mtTFA(P<0.05),down-regulated UCP2(P<0.05)and reduced apoptotic cells.QSG treatment down-regulated CPT1 and up-regulated GLUT4,decreased cardiac and serum FFA and LA levels.Those data indicated that QSG increased cardiac energy supply mainly through inhibiting FFA oxidation and glycolysis and promoting glucose oxidation.5.HF model of rats developed calcium overload and defective Ca2+ release-uptake cycling in cardiomyocytes.Treatment with QSG reduced the basal intracellular Ca2+ level(P<0.01).QSG prevented defective Ca2+leak by attenuating hyperphosphorylation of RyR2,inhibiting expression of PKA and up-regulated transcriptional expression of PP1(P<0.05).QSG also restored Ca2+ uptake by up-regulating expression and activity of SERCA2a and promoting phosphorylation of PLB(P<0.05).Conclusion:1.HF rat models could be established by HFD combined with LAD ligation.This model belongs to syndrome of qi-deficiency and blood stasis during the 7-14th days after operation with a transition to syndrome combined with turbid phlegm during the 14-28th days2.HDF rat models showed dynamic evolution in biomarkers of myocardial injury and inflammatory factors,persistent activation of RAAS system,enhanced oxidative stress,progressive aggravated cardiac fibrosis.8,9,14 indicators participated in the development of qi-deficiency,blood stasis and turbid phlegm syndrome,respectively.3.QSG improved contractive function,attenuated ventricular remodeling,increased cardiac energy supply through inhibiting FFA oxidation and glycolysis and promoting glucose oxidation and restored SR calcium cycle by inhibiting hyperphosphorylation of RyR2 and up-regulating expression and activity of SERCA2a.Significance:In this study,HF rat models were built by HFD combined with LAD ligation and their syndrome property and biological characteristics were detected roundly and dynamically.We verified the effects of QSG on treating HF and declared its pharmacological mechanism on improving cardiac energy metabolism and SR calcium regulation.