Effects Of Benefiting Qi,Activating Blood And Promoting Diuresis For Treatment Of Heart Failure Mediated By Mtln Regulation On Calcium Homeostasis During Sarcoplasmic Reticulum-mitochondrial Crosstalk

Objective:By exploring the role of Mtln in regulating calcium homeostasis under the mitochondrial-associated endoplasmic reticulum membrane in chronic heart failure,and exploring the role of Nuanxinkang in regulating Mtln-mediated calcium homeostasis under the mitochondrial-associated endoplasmic reticulum membrane in chronic heart failure,To explore the effect and mechanism of Fang Nuanxinkang,the representative of the method of benefiting qi,activating blood and diverting water,in the prevention and treatment of chronic heart failure.Methods:1.Animal experiment:A mouse model of chronic heart failure induced by ischemia-reperfusion was established.In the first part of the experiment,mice were randomly divided into 4 groups:① control group（CTRL）,② Mtln knockout mice（MtlnKO）,③control+model group（CTRL+I/R）,④ Mtln knockout+model group（MtlnKO）+I/R),30 mice in each group.The Mtln knockout animal model was established by tail vein injection of adeno-associated virus,and the model was established four weeks later.In the second part of the experiment,mice were randomly divided into 3 groups:sham operation group（SHAM）,model group（I/R）and Nuanxinkang group（NXK+I/R）,with 50 mice in each group.After I/R operation,the mice in the Nuanxinkang group were given Nuanxinkang suspension 1.65 g/kg/d by gavage,and the sham-operated group and the model group were given the same amount of normal saline by gavage.2.Cell experiment:In the first part of the experiment,HL-1 cardiomyocytes were used to establish stable Mtln knockout/overexpressing cell lines.The grouping settings were:NC,KO,LCK,OE,and cultured under normoxia and hypoxia/reoxygenation cell.The second part of the experiment cultured the H9C2 cardiomyocyte cell line.The cells were cultured in MEM medium containing 10%fetal bovine serum,100U/ml penicillin and/100mg/ml streptomycin,and the incubator environment was set to 37℃,5%carbon dioxide,95%air.3.In this study,experimental methods such as cardiac ultrasound,Masson staining,Sirius red staining,HE staining,TUNEL staining,MitoSOX fluorescence staining,Seahorse energy metabolism detection,calcium flow detection,transmission electron microscopy,fluorescence co-localization analysis and Western Blot detection were used.Detection of cardiac function,pathological changes of cardiac tissue,myocardial fibrosis level,myocardial cell apoptosis,myocardial mitochondrial reactive oxygen species level,myocardial cell mitochondrial ATP generation rate and aerobic oxidation level in mice with chronic heart failure induced by ischemia-reperfusion.Cytoplasmic and mitochondrial calcium concentration changes,MAM structure,number and spacing,mitochondria and sarcoplasmic reticulum coupling,MAM calcium transporter expression and Mtln and Mfn2 protein expression changes.To explore the role and mechanism of calcium homeostasis in cardiac energy metabolism remodeling in heart failure under Mtln regulation of sarcoplasmic reticulum-mitochondrial interaction at the overall and cellular levels of animals,and the regulation of Mtln-mediated muscle remodeling by Yiqi Huoxue Lishui method Calcium homeostasis under plasma-reticulum-mitochondria interaction improves energy metabolism remodeling and prevents heart failure.In order to clarify whether Mtln regulates calcium homeostasis under the interaction of sarcoplasmic reticulum-mitochondria plays an important role in heart failure,and whether Fang Nuanxinkang,a representative of Yiqi Huoxue Lishui method,can regulate Mtln-mediated sarcoplasmic reticulum-mitochondria Interaction of calcium homeostasis in the prevention and treatment of heart failure.Results:1.Mtln and Nuanxinkang have protective effects on heart failure.After myocardial ischemia-reperfusion,compared with the CTRL+I/R group,the cardiac LVEF and LVFS values of the mice in the MtlnKO+I/R group were significantly decreased（P<0.01,P<0.05）,and the LVID;s value was significantly increased（P<0.05）.Compared with SHAM group,the values of LVEF and LVFS of mice in I/R group decreased significantly at 1,2,and 4 weeks after operation,and the values of LVID;d and LVID;s increased significantly.At the 4th week,the results of M-mode echocardiography showed that the cardiac diastolic and systolic functions of the mice in the I/R group were significantly reduced.The LVEF and LVFS values of the NXK+I/R group were significantly higher than those of the I/R group at the 2nd and 4th week（P<0.0001,P<0.0001）,and the LVID;d and LVID;s values were significantly decreased（P<0.0001,P<0.0001）.2.Mtln and Nuanxinkang inhibit ventricular remodeling in failing hearts.Compared with the CTRL+I/R group,the myocardial fibrosis in the MtlnKO+I/R group was significantly increased（P<0.0001）,the pathological changes were aggravated,and the number of myocardial cell apoptosis was significantly increased（P<0.0001）.Compared with the I/R group,the deposition of collagen fibers in the heart of the mice in the NXK+I/R group was significantly reduced（P<0.0001）,the cardiac pathological changes were alleviated,and the number of myocardial cell apoptosis was significantly reduced（P<0.0001）.3.Mtln and Nuanxinkang can improve the energy metabolism of failing heart.The content of ROS in the heart mitochondria of MtlnKO+I/R group was significantly higher than that of CTRL+I/R group（P<0.0001）.At the same time,compared with the I/R group,the mitochondrial respiration ATP and total ATP generation rates in the Nuanxinkang group increased,and the basal respiration and maximum mitochondrial respiration rates increased（P<0.01,P<0.05）.4.Mtln and Nuanxinkang alleviate cytoplasmic and mitochondrial calcium overload in failing cardiac myocytes.Compared with normoxia,we found that the Ca²⁺ retention capacity of cardiomyocytes decreased in the hypoxia/reoxygenation state,but the Ca²⁺retention of cells in the NXK+H/R group was significantly increased compared with the control group（P<0.0001）.At the same time,the mCa²⁺retention capacity of cells in NXK+H/R group was also significantly higher than that in control group.（P<0.0001）FCCP stimulation was used to observe the change of free calcium ion concentration.The results showed that the free calcium ion in the NXK+H/R group was significantly lower than that in the CTRL+H group（P<0.0001）.mCa²⁺ was also significantly lower than the control group.（P<0.0001）.5.Mtln and Nuanxinkang regulate MAM conformation and reduce the coupling between sarcoplasmic reticulum and mitochondrial structure.After I/R,the co-localization of mitochondria and sarcoplasmic reticulum in myocardial cells of mice in CTRL+I/R group and MtlnKO+I/R group was significantly increased（P<0.001,P<0.0001）,among which,MtlnKO+I/The MAM coupling part in the R group was significantly more than that in the CTRL+I/R group（P<0.01）.Compared with the I/R group,the number of MAM couplings in the NXK+I/R group was reduced,and the distance between the sarcoplasmic reticulum and mitochondria was narrowed（P<0.001）,and Nuanxinkang could reduce the myocardial cell sarcoplasmic reticulum after heart failure,stable MAM coupling structure（P<0.0001）.6.Mtln and Nuanxinkang reduce the expression of calcium transporter on MAM.Western Blot was used to detect the expression levels of cardiac calcium transporters IP3R,GRP75 and VDAC in mice in each group.The results showed that the expression of IP3R and VDAC proteins in MtlnKO+I/R group was significantly higher than that in CTRL+I/R group（P<0.0001,P<0.001）,indicating that Mtln may mediate the role of MAM in regulating calcium transporter.Meanwhile,Nuanxinkang could significantly reduce the expression of GRP75 and VDAC in failing hearts（P<0.001,P<0.0001）.7.Nuanxinkang can increase the expression of mtln in heart failure and inhibit the expression of Mfn2 protein.Western Blot detected the expression levels of Mtln and Mfn2 proteins in the heart tissue of mice in each group.The results showed that Nuanxinkang significantly increased the expression of Mtln（P<0.0001）and inhibited the expression of Mfn2 protein（P<0.0001）when heart failure occurred.Conclusion:Mtln can reduce calcium overload in failing cardiac myocytes by regulating calcium homeostasis under the interaction of sarcoplasmic reticulum-mitochondria,thereby improving energy metabolism in chronic heart failure and delaying ventricular remodeling.Fang Nuanxinkang,representative of benefiting qi,activating blood and promoting diuresis method,may reduce the distance and number of coupling chains between endoplasmic reticulum and mitochondria by regulating Mtln,and reduce myocardial cell cytoplasmic and mitochondrial calcium overload in chronic heart failure caused by ischemia-reperfusion,thereby effectively improving Ventricular remodeling in chronic heart failure to protect cardiac function.