The Effects And Mechanism Of Dantrolene On Cardiac Hypertrophy

RyR2,which is an important Ca²⁺release channel,distributed in the sarcoplasmic reticulum of cardiomyocytes and plays a role in excitation-contraction coupling.RyR channel blocker dantrolene was used for malignant hyperthermia,a kind of potentially fatal metabolic disorder caused by anesthesia.Recent studies indicated dantrolene a protective regulator in cardiovascular disorders,possibly though correcting the defective inter-domain interaction in RyR,enhancing the binding affinity of calcium and inhibiting calcium leak.However,current research of dantrolene is mainly limited in heart failure and arrythmia.Cardiac hypertrophy is a pathophysiological foundation for many cardiovascular diseases and an important stage of cardiac remodeling.Calcium imbalance is the core link of cardiac hypertrophy.Therefore,we speculate that dantrolene,an inhibitor of calcium channel RyR2,may have great potential in the treatment of cardiac hypertrophy.Here,we have investigated the effects and mechanisms of dantrolene on cardiac hypertrophy in vivo and in vitro,so as to provide a new idea and more evidences for the research of cardiac hypertrophy and the exploration of precise therapeutic targets.This study is divided into three groups.Firstly,we explore the protective effects of dantrolene on cardiac hypertrophy in animal models.Secondly,RNA sequencing was used to analyze the transcriptional data of the heart tissue,therefore TNF pathway was found to be down-regulated by dantrolene.Thirdly,western blot was used to detect the relative expression and activity of key proteins associated with Ca²⁺-dependent pathway,TNF pathway,in order for the target mechanism of drug intervention.Gene interference technology and pathway inhibitor were used to explore and verify the mechanism of dantrolene.Part Ⅰ:animal experimentPurpose:To determine the effect of dantrolene on stress overload-induced cardiac hypertrophy in vivo.Methods:Male C57BL/6 mice（8 weeks old）were randomly underwent TAC to establish cardiac hypertrophy model or sham surgery,and dantrolene（30 mg/kg）or vehicle were administered on the second day after surgery.4 weeks after surgery,we performed transthoracic echocardiography to assess the LV function and structure.Later,invasive hemodynamic examination was administered.After the heart was removed,heart weight/body weight and heart weight/tibia length were calculated.Left ventricular tissues were used for histological study,the left ventricular cross-sections were stained with Hematoxylin-Eosin staining,Masson staining and Wheat Germ Agglutinin staining.The expression levels of the markers for hypertrophy were determined by real-time qPCR and western blot.Results:Hypertrophy model was successfully established by TAC.Dantrolene significantly attenuated cardiac hypertrophy,mainly shown by decreased HW/BW,decreased HW/TL,reduced rate of fibrosis area of Masson staining and cardiomyocyte size,thinner left ventricular post wall,lower relative expression levels of the genes hypertrophy markers.However,no significant changes were observed in hemodynamic index.Contemporarily,EF and FS showed no significant changes between four groups.Part Ⅱ:RNA sequencingPurpose:To seek for more evidence for the targets and mechanisms of dantrolene,through the transcriptional analysis via RNA sequencing.Methods:RNA sequencing was performed,A false discovery rate<0.05 and fold change>2 or<0.5 were set as the cut-off criteria for identifying DEGs.GO analysis together with the enrichment KEGG pathway analysis were employed for DEGs to predict gene functions and calculate the frequency distribution of functional categories.Key signaling pathways involved in the regulation of dantrolene were identified.According to the result of RNA-seq,we performed real-time qPCR of certain DEGs for validation.Results:184 down-regulated genes were found when the TAC+dantrolene group was compared with the TAC+control according to RNA sequencing,including Creb3l3,IL18R1 and Ccl5,which were related to TNF-αpathway.The relative expression of DEGs in qPCR is consistent with the result in RNA-seq.Part Ⅲ:Mechanism explorationPurpose:To explore the effects of dantrolene on cardiac hypertrophy and inflammation-related signaling pathway proteins in vivo and in vitro,and to screen out and verify possible targets of dantrolene.Methods:In vivo section,western blot was used to detect Calcineurin pathway-,CaMKII pathway-,MAPKs pathway-,AKT pathway-and TNF-αpathway-related proteins in heart tissue.In vitro section,PE was used to create a pathological humoral environment for neonatal rat cardiomyocytes.Ppp3ca（calcineurin）overexpression plasmid or FK506（a Calcineurin/NFAT inhibitor）or were administered to up-regulate or down-regulate calcineurin.Subsequently we used western blot for the detection of Calcineurin pathway-,AKT pathway-,MAPKs pathway-and TNF-αpathway-related proteins in cardiomyocytes.Results:The protein expression levels of Calcineurin pathway were shown by western blot analysis to be lower in treated group than in the untreated group,both in vivo and vitro examination.Dantrolene can also reduce the phosphorylation of the key proteins in AKT signaling pathway,MAPKs signaling pathway and TNF-αsignaling pathway.The above changes were similar to the effect of FK506 and attenuated by Ppp3ca overexpression.Conclusion:Dantrolene,the RyR2 inhibitor,may mainly inhibit Calcineurin/NFAT3,thereby down-regulating gene transcription of hypertrophy,reducing inflammation and immune response,inhibiting downstream TNF-α/NF-κB/NLRP3 pathways,alleviating cardiomyocyte apoptosis,and attenuating cardiac hypertrophy.