The Effect And Mechanism Of Dantrolene In Improving Post-Infarction Heart Failure By Regulating Calcium Homeostasis

BackgroundWith the continuous advancement of coronary vascular reperfusion therapy,the survival rate of patients with acute myocardial infarction（MI）has improved.However,heart failure（HF）has become a leading cause of increased mortality in the later stages of acute MI.This shift underscores the importance of research focused on the prevention and treatment of HF post-MI.The pathogenesis of ischemic HF is complex,involving abnormalities in myocardial calcium cycling and homeostasis,apoptosis,necrosis,remodeling of non-infarcted myocardium,and neurohumoral imbalance.These factors contribute to myocardial systolic dysfunction,ventricular enlargement,malignant arrhythmias,and even sudden cardiac death.Thus,inhibiting myocardial remodeling and electrical disturbances is crucial for improving patient prognosis.Studies have found that increased diastolic calcium leakage,caused by dysfunction of the cardiac ryanodine receptor type 2（Ry R₂）located in the myocardial endoplasmic reticulum（ER）,is linked to impaired cardiac function.This leakage leads to both systolic and diastolic dysfunction.Calcium leakage from the ER during diastole can trigger abnormal action potential changes,increasing the risk of arrhythmias.Disrupted calcium homeostasis also promotes the development of HF by activating ER stress and apoptosis pathways.Calcium/calmodulin-dependent protein kinase II（Ca MKII）has been extensively studied for its detrimental effects in experimental animal models.Ca MKII catalyzes the phosphorylation of the Ry R₂ serine 2814（S2814）site,causing calcium leakage,promoting arrhythmias,and adversely impacting heart disease models.Dantrolene has been established as a specific treatment for malignant hyperthermia due to its stabilization of skeletal muscle Ry R₁.Recently,studies have shown that dantrolene can inhibit myocardial Ry R₂ structural allosteric changes,reduce calcium leakage,and exert anti-arrhythmic effects.By inhibiting ER calcium leakage through its action on Ry R₂,dantrolene can restore ER calcium homeostasis and improve the activation of apoptotic pathways mediated by protein misfolding.This presents significant potential for improving cardiac function.In this study,we established a rat model of MI by ligating the left anterior descending coronary artery（LAD）and an H₂O₂-induced apoptosis model in H9c2 cells.We evaluated dantrolene’s protective and therapeutic effects on cardiac function post-MI through different administration timelines（one day and four weeks after MI）.We aimed to investigate dantrolene’s impact on myocardial calcium homeostasis,apoptosis,and cardiac function in rats after MI,exploring its potential protective mechanisms.This study seeks to identify new targets for HF treatment and explore new therapeutic uses for established drugs.MethodMale Wistar rats（12 weeks old）were randomly divided into the operation group（n=60）and the sham operation group（n=20）.Ligation of the LAD in rats to establish a rat model of MI.The sham operation group of rats underwent thoracotomy without LAD ligation.Elevation of electrocardiogram was used as the criteria for the immediate success of the model.The survived rats after MI were randomly divided into the MI group（n=16）;MI-Dan-early group（n=12）;MI-Dan-late group（n=12）.In order to explore whether dantrolene can prevent and improve cardiac function after MI,the drug was administered at different time nodes in early（postoperative day 1）and late periods（four weeks after surgery）.The MI group and Sham group were given DMSO（vehicle）after surgery.MI-Dan-early group rats were gavage dantrolene（15mg/kg/day）for eight weeks;the same dose of dantrolene began to gavage for four weeks after the MI as soon as the cardiac function of rats started to decline.We used echocardiography and hemodynamics to detect the changes in cardiac function at four and eight weeks after MI.The effect of dantrolene on the ventricular electrical stability of rats after MI was observed by the Langendorff perfusion system in ex-vivo.Elucidate the role of Ca²⁺/calmodulin-dependent protein kinase（Ca MKII）in the ER stress-apoptosis pathway by upregulating or inhibiting the expression of Ca MKII by plasmid transfection or KN93.Subsequently,we used hydrogen peroxide（H₂O₂）-induced cardiomyocyte H9c2 apoptosis.The apoptosis conditions of H9c2 cells induced by H₂O₂ were determined by cell viability under different H₂O₂ concentrations and time.The cells were divided into control group,H₂O₂ group,H₂O₂+dantrolene group and H₂O₂+dantrolene+Ca MKII overex group.The effect of dantrolene on cardiomyocyte apoptosis in vitro and in vivo was evaluated by TUNEL and flow cytometry,Caspase8enzyme activity,and molecular biology.At the same time,immunofluorescence was used to detect the change of calcium concentration in the cytoplasm and ER of cardiomyocytes.The objective of this study is to investigate the potential improvement effect of dantrolene on the calcium homeostasis of cardiomyocytes.The effects of dantrolene on the expression and activation of calcium pathway regulatory proteins Ca MKII and Ry R₂ were detected by Western blot and q PCR.Dantrolene was used as the ligand and Ca MKII as the receptor,and the molecular docking was carried out by Auto Dock Tools-1.5.7 software,and the binding energy and efficiency of the reference ligand and the receptor were used to test the binding energy and binding efficiency of dantrolene and Ca MKII-Thr287.Results1.Improvement effect of dantrolene on cardiac function in rats with HF.（1）Effect of dantrolene on hemodynamics in rats after MIThe hemodynamic test of rats in each group showed that the systolic blood pressure（SBP）of rats in the MI group significantly decreased compared with the sham group.（112.28±5.69mm Hg vs.98.1±10.66 mm Hg,P<0.05）;SBP increased in rats given Dantrolene for 4 or 8 weeks.At the end of the 8 weeks,the maximum change rate of left ventricular pressure rise and fall(±dp/dt_max)was significantly lower than those of the sham group(+dp/dt_max:3321.56±327.27mm Hg/s vs.4810.72±390.00mm Hg/s,P<0.001);(-dp/dt_max:-2766.29±200.11 mm Hg/s vs.-4067.82±490.4mm Hg/s,P<0.001).Dantrolene intervention can improve the+dp/dt_max and the-dp/dt_max.（2）Effect of dantrolene on ischemia-related myocardial remodeling.Eight weeks after the LAD ligation,the heart surface of the rats in the MI group was not smooth,the pericardial adhesion was severe,the appearance was large,the left ventricle part could be seen obvious pale infarct scar area,the myocardium in the infarct area was thinned and collapsed,and a few rats could see ventricular aneurysm.Histopathological staining such as HE and Masson showed that myocardial fibers were disordered,irregular morphology,fibrous tissue hyperplasia,vasodilation and congestion in fibrotic areas,and a small number of lymphocytes or plasma cell infiltration were found in rats in the MI group.The use of dantrolene can alleviate the above-mentioned myocardial damage and improve morphological changes.Compared with the sham group,both myocardial to body weight ratio（HW/BW）and left ventricular to body weight ratio（LVW/BW）were significantly elevated in the MI group（P<0.01）.In comparison to MI group,after dantrolene intervention,both HW/BW,LVW/BW are significantly decreased.Compared with the sham group,the left ventricular shortening rate（FS%）and ejection fraction（EF%）in the MI group were significantly decreased（FS%:34.25±7.59 vs.15.5±2.88,P<0.001）（EF%:68.25±11.12vs.36.83±5.88,P<0.001）.We found that dantrolene administration either on the one day or four weeks after MI resulted in improved EF%and reduced FS%（EF%:MI-Dan-early group:66.17±8.75,P<0.001;MI-Dan-late group:50.50±11.61,P<0.05）;（FS%:MI-Dan-early group:32.33±6.02,P<0.001;MI-Dan-late group:22.83±6.80,P<0.05）.Suggesting that dantrolene administration had a substantial effect on improving myocardial remodeling.（3）Effect of dantrolene on myocardial electrical stability after MI.We examined the impact of dantrolene intervention at four weeks post-MI on the electrical stability.When comparing the MI group to the sham group,we observed a significant prolongation of action potential duration APD₉₀ in the MI group（40.58±3.30ms vs.62.23±2.17 ms at 10 Hz pacing,P<0.001）.However,administration of dantrolene early resulted in a significant reduction in APD₉₀（46.23±4.04 ms vs.62.23±2.17 ms,P<0.01）.In comparison to the sham group,the MI group exhibited more pronounced electrical alternations of action potential（2.98±1.69 ms vs.17.82±3.98ms at 11 Hz pacing,P<0.001）.However,dantrolene intervention significantly inhibited these action potential alternations（MI-Dan-early group:3.10±1.79 ms,P<0.001）（MI-Dan-late group:5.12±2.32ms,P<0.001）.Calcium transient testing revealed significant calcium alternations ratio in the MI group compared to the sham group（11 Hz pacing,0.97±0.04 vs.0.72±0.12,P<0.001）.Dantrolene administration reduced these calcium alternations（MI-Dan-early group:0.94±0.04,P<0.001;MI-Dan-late group:0.88±0.08,P<0.05）.Additionally,we observed more pronounced spontaneous calcium release in the MI group（P<0.001）.Dantrolene significantly inhibits spontaneous calcium release（P<0.01）.When measuring the relative concentration of calcium（d F/F0）during the maximum calcium transient,the MI group was significantly lower than the sham group（P<0.001）.Early and late intervention with dantrolene can increase the amplitude of calcium transients and the relative concentration of calcium.Increased compared with the MI group（MI-Dan-early group,P<0.001）（MI-Dan-late group,P<0.05）.We used programmed ventricular stimulation to induce ventricular fibrillation,the induction rate of ventricular fibrillation（VF）was significantly higher in the MI group compared to the sham group（8.00±4.89%vs.92.00±4.89%,P<0.01）.Dantrolene intervention reduced the rate of VF induction（20.00±8.94%vs.92.00±4.89%,P<0.05）.3.The mechanism of dantrolene in improving cardiac function in rats with HF after MI.（1）Effect of dantrolene on calcium handing proteins and calcium homeostasis.To explore the effect of dantrolene on calcium handing protein in the myocardium of rats with HF after MI.Western blot assay was performed in vitro and in vivo to detect the expressions of Ry R₂,it is an upstream regulatory protein Ca MKII and its phosphorylated protein.We observed that total Ca MKII and phosphorylated Ca MKII significantly increased in HF rats’cardiac tissue and in vitro H₂O₂-induced apoptotic cardiomyocytes.Dantrolene intervention inhibited the expression of Ca MKII and its active form.Furthermore,in vitro and vivo detection of ER calcium regulatory protein Ry R₂ expression,we found the phosphorylation level at Serine 2814 was significantly increased in rats with HF and apoptotic cardiomyocytes after MI,and the expression of serine 2814 sites was significantly changed.Dantrolene intervention inhibited the expression of phosphorylated protein at the Ry R₂-S2814 site.Enhanced phosphorylation at Ry R₂-S2814 increased calcium leakage from ER and disrupted cellular calcium homeostasis.Therefore,we further used Fluo-3 AM and Mag-Fluo-4-AM,fluorescent Ca²⁺indicators of membrane permeability,to detect changes in intracellular Ca²⁺levels.Confocal microscopy and flow cytometry observed the fluorescence intensity of Ca²⁺in cytoplasm and ER.We found that apoptotic cardiomyocytes exposed to H₂O₂ significantly increased intracellular Ca²⁺levels and decreased Ca²⁺fluorescence intensity in the ER.At the same time,dantrolene intervention could partially reverse Ca²⁺concentration changes in the ER and cytoplasm,reducing cytoplasmic Ca²⁺overload and abnormal Ca²⁺leakage in the ER and restoring calcium homeostasis in cells by improving Ca²⁺storage in ER.Ca MKII overexpression plasmid up-regulated the expression and activation of Ca MKII,and the regulatory effect of dantrolene on Ry R₂-S2814 and intracellular Ca²⁺homeostasis was weakened.（2）Effects of dantrolene on ER stress and apoptosis pathways.Disturbance of calcium homeostasis is often accompanied by increased ER stress.We detected m RNA expression of ER stress-related molecular markers in H₂O₂-induced apoptotic cardiomyocytes.The results showed that the expression levels of ATF-6,CHOP,and GRP78 in apoptotic cardiomyocytes were significantly increased compared with those in the control group.Quantitative protein detection by Western blot showed that Caspase12 activation was significantly increased in myocardial cells of rats with HF after MI and those exposed to the H₂O₂ group.Dantrolene intervention could inhibit the expression of ATF-6,CHOP,GRP78,and Caspase12 activation.When ER stress occurs,ER stress-related apoptotic marker Caspase12 dissociates from the ER and activates,causing cell apoptosis.Eight weeks post-MI,immunohistochemical staining of the left ventricle using the TUNEL assay revealed a significant increase in apoptotic myocardial cells in the MI group compared to the sham group.Additionally,the expression levels of Bax and the Bax/Bcl2 ratio were upregulated.Both early and late dantrolene intervention inhibited these adverse changes in Bax and Bax/Bcl2 levels.In vitro experiments involving H9c2 cells were conducted to elucidate Ca MKII’s role further.Plasmid transfection and adding the inhibitor KN93 were used to upregulate and down-regulate Ca MKII,thereby clarifying its upstream regulatory position in the ER stress and apoptosis pathways.Cells were treated with 200μM H₂O₂for 24 hours.Flow cytometry,ELISA,immunofluorescence detection,Western blot,and q-PCR were utilized to assess the expression levels of critical apoptotic markers,including Bax,Bax/Bcl2 ratio,and Caspase3.The results revealed a significant induction of cell apoptosis in the H₂O₂-treated group.Dantrolene effectively mitigated the elevated expression of these apoptotic markers induced by H₂O₂.The overexpression of Ca MKII via plasmid transfection upregulated its expression and activation,thereby weakening dantrolene’s effect on inhibiting ER stress and apoptosis.This indicates that Ca MKII is a target of dantrolene and is located upstream in the pathway.Molecular docking studies further verified the potential site of action of dantrolene,revealing that it forms a hydrogen bond with the THR-287residue of Ca MKII.Combined with Western blot analysis,these findings suggest that dantrolene binding may alter the conformation of the THR-287 residue,inhibiting Ca MKII activation.Conclusion1.Both early and late administration of dantrolene can enhance cardiac function in rats with heart failure following myocardial infarction.Early intervention helps prevent the decline of cardiac function,while late administration can partially reverse myocardial remodeling.2.Overactivity of the Ca MKII/Ry R₂-S2814 signaling pathway disrupts calcium homeostasis,leading to ER stress and cardiomyocyte apoptosis.This pathological process is evident in the ventricular and electrical remodeling observed in rats with HF after MI.3.Dantrolene regulates calcium homeostasis in cardiomyocytes post-MI by inhibiting the Ca MKII/Ry R₂-S2814 signaling pathway.This inhibition reduces the activation of the ER stress pathway,decreases cardiomyocyte apoptosis,and consequently improves cardiac function.