The Effect Of Carvacrol,an Antagonist Of TRPM7,on Cardiac Hypertrophy And Its Mechanism

Objective:Hypertrophic cardiomyopathy is a congenital or acquired disease characterized by marked ventricular hypertrophy and diastolic dysfunction,not accompanied by an increase in afterload(eg,aortic valve stenosis,aortic stenosis,systemic hypertension).Symptoms include chest pain,difficulty breathing,syncope,and sudden death.The systolic murmur that can be enhanced during Valsalva maneuvers is a typical manifestation of hypertrophic obstructive cardiomyopathy and is the late stage of many pathological changes in cardiovascular disease.Chronic hypertrophic remodeling involves cardiac dysfunction,which will lead to heart failure and eventually death.Transient receptor voltage(Transient Receptor Potential,TRP)channels are located on the cell membrane of various types of cells and are a group of non-selective cation channels.The TRP channel was originally discovered in Drosophila photoreceptors and was named because it carried a mutant gene of Drosophila photoreceptor cells and triggered a transient response to light.Based on its homologous sequence classification,TRP channels can be divided into six subfamilies,namely TRPC,TRPV,TRPM,TRPA,TRPL and TRPP5.Studies have shown that the TRPM subfamily,especially TRPM7,is involved in important physiological processes in heart disease,such as mediating calcium ion signaling,mediating magnesium ion transport,negatively regulating Ang II,and differentially regulating vascular function.Studies have shown that TRPM7 plays an important role in heart diseases such as hypertension,ischemic cardiomyopathy,and arrhythmia.Carvacrol is an essential oil component that can be used as a non-specific inhibitor of transient receptor potential Melastatin subfamily 7(TRPM7).It is the main product of many aromatic plants,including oregano,thyme,etc.Blood /reperfusion(I / R)injury has a protective effect.The role of carvacrol in cardiomyocyte apoptosis and hypertrophy has not been fully demonstrated.The research on the role of TRPM7 in myocardial hypertrophy and heart failure is scarce,and the conclusions on the effect of fibrosis are conflicting.In this study,we will investigate the role and mechanism of the TRPM7 inhibitor carvacrol in cardiac hypertrophy.Methods:1.Identification of differentially expressed genes(DEGs)We obtained the expression profiles of HCM from the GEO database(Gene Expression Omnibus,http://www.ncbi.nlm.nih.gov/geo/).All data were analyzed on the GPL1261.GEO2 R is a GEO online analysis tool.Based on this tool,GEO2 R is a GEO online analysis tool.Based on this tool,gene differential expression analysis can be performed on some GEO sample data.We applied GEO2 R to compare normal healthy group and HCM group.Common up-or downregulated DEGs in the selected datasets were extracted and p < 0.05 and |log FC| > 1 were set as the cut-off criteria.The original probe-level data in Series Matrix Files were converted into gene symbol based on the downloaded platform annotation files.2.GO and pathway analysisSignificantly DEGs with as merging into a gene list were then submitted to the Database for Annotation,Visualization,and Integrated Discovery(DAVID)for functional annotations.We also conducted The Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis in our study.We set p < 0.05 as the cut-off criteria.3.Protein-Protein Interaction(PPI)NetworkThe STRING database(https://string-db.org/)is a database that stores information about direct and indirect interactions between known and predicted proteins.We upload apoptosis-related genes to the online STRING database to obtain protein interaction information.4.AnimalsThe 5 groups rat(n = 8,half male and half female)(8–10 weeks)weighting 150 ±5 g in this study were individually housed under a standard environment.All rats were housed under controlled temperature in a 12-h light/dark cycle with enough food and water.We have established a model of HCM using methods that are completely consistent with the previous study 19.The control group was intraperitoneally injected with normal saline for seven days;the vehicle HCM group,the low dose carvacrol group,the high dose of carvacrol group and the propranolol group were intraperitoneally injected with Isoprenaline(ISO)(10 mg / kg / day)for 7 days to induce cardiac hypertrophy.After the 7-day period,we dissolved carvacrol in propylene glycol for intraperitoneal injection.Both the control group and the vehicle HCM group were administrated with normal saline and propylene glycol for 14 days,respectively.The low dose carvacrol group(25mg / kg / day)and that of high dose(100mg / kg / day)was administrated for 14 days,respectively 20.Additionally,the propranolol group was administrated with propranolol(12.5mg / kg)as a positive drug control group.5.Western BlotProtein samples were collected using RIPA buffer(Beyotime,Shanghai,China)and were sonicated at 4°C for 30 s after it was incubated on ice for 30 min.After centrifugation at 12000 g for 10 minutes at 4°C,the supernatant was collected for BCA protein quantification(Beyotime,Shanghai,China).Western Blot analysis was performed as described previously.Protein samples were run on 15% SDS-PAGE and transferred to PVDF membranes.Membranes were then incubated overnight at 4℃ in TTBS containing the primary antibodies: rabbit anti-Bax(1:800;Abcam,USA),rabbit anti-GAPDH(1:800;Abcam,USA),rabbit anti-Cleaved-Caspase 3(1:200;Abcam,USA),rabbit anti-Bcl 2(1:250;Abcam,USA).After several washes in TTBS,membranes were incubated in horseradish peroxidase conjugated goat anti-rabbit Ig G(1:10000;Santa Cruz,USA)for 2 h at RT.Immunoreactive bands were visualized using an enhanced chemiluminescence(ECL)kit.The levels of protein were evaluated by measuring optical densities of the protein bands using Scion Image for Windows image-analysis software.For target proteins,bands in each lane were normalized to GAPDH to control for any variation in protein loading.6.Real-time RT-PCRThe rats were sacrificed by injecting excessive chloral hydrate.Then,the left ventricles of rats were collected for further RNA detection.Total RNA was isolated using the TRIzol(Invitrogen,Carlsbad,CA,USA)assay,the concentrations and purities of which were quantified using an ultraviolet spectrophotometer.Follow this procedure to reverse transcribe RNA and keep at-80℃ until using 21.Specific primer was designed for SYBR Green I real-time RT-PCR(Table 1).The real-time RT-PCR assay was carried out using different RT-PCR reagents and thermocycling conditions.The real-time RT-PCR was carried out on an ABI Prism(?) 7500(Applied Biosystems,CA).All assays applied two reactions mixtures placed in the wells of96-well plates(Applied Biosystems).A 10-μL volume of the reaction mixture contained 5 μl of Taq enzyme,1 μl of c DNA,0.2 μl of Dye l enzyme,0.4 μl of forward and reverse primers,and 3 μl of RNA free water.The thermal cycling profile for the uniplex RT-PCR was 95 °C for 30 s(1 cycle),95 °C for 5 s followed by 60 °C for 34 s(40 cycles)and 95 °C for 15 s,60 °C for 1 min,95°C for 15 s(1 cycle).Specimens were considered positive when the Ct value was < 3522.The expression levels of TRPM7 gene was normalized to GAPDH.Relative m RNA expression level was analyzed by the 2-ΔΔ cycle threshold(CT)method.7.HE StainingHE staining was conducted according to routine protocols 23.Briefly,after deparaffinization and rehydration,5?μm longitudinal sections were stained with hematoxylin solution for 5?min followed by 5?dips in 1% acid ethanol(1% HCl in70% ethanol)and then rinsed in distilled water.Then the sections were stained with eosin solution for 3?min and followed by dehydration with graded alcohol and clearing in xylene.The mounted slides were then examined and photographed using an NIKON Eclipse ci and NIKON digital sight DS-FI2(Tokyo,Japan).8.Statistical AnalysisStatistical data were expressed as mean ± SEM(standard error of mean).Statistical significance of differences was evaluated using assessed by Student’s t-test and one-way ANOVA followed by Tukey test(Graph Pad Software,San Diego,CA),and p < 0.05 was considered statistically significant.Result:1.The analyses of identification of differentially expressed genesWe identified the differentially expressed genes between the HCM group and the normal group based on the dataset GSE18801 in the GEO database.According to the criteria of | log FC |> 1 and p ≤ 0.05,we identified 761 DEGs,including 507up-regulated genes and 254 down-regulated genes.2.GO and KEGG pathway analysis for DEGs in HCMWe applied the STRING tool to construct gene-gene interactions,which also hides the disconnected nodes in the network.The results showed the analyzed number of nodes(29),expected number of edges(12),the number of edges(25),average node degree(1.72),average local clustering coefficient(0.463),and PPI enrichment0.000659 in negative regulation of apoptotic process;number of nodes(27),expected number of edges(7),the number of edges(17),average node degree(1.26),average local clustering coefficient(0.433),and PPI enrichment 0.000933 in apoptotic process;number of nodes(16),expected number of edges(1),the number of edges(1),average node degree(0.125),average local clustering coefficient(0.125),and PPI enrichment 0.722 in negative regulation of neuron apoptotic process;number of nodes(16),expected number of edges(2),the number of edges(6),average node degree(0.75),average local clustering coefficient(0.312),and PPI enrichment 0.0117 in positive regulation of apoptotic process.3.Gene network analysis for DEGs in HCMWe submitted the apoptosis related genes in the differentially expressed genes to the STRING database to obtain a protein-protein interaction network.4.The effect of carvacrol on apoptosis in a rat model of HCMThe protein expression levels of Cleaved-Caspase 3,Bax,and Bcl-2 related to apoptosis were checked in a rat model of HCM by Western Blot.The results showed that the protein of Cleaved-Caspase 3 was significantly higher than that in HCM group compared to control(n=4,p <0.05).Additionally,high dose of carvacrol significantly reduced the protein expression of Cleaved-Caspase 3 in comparison to control group(n=4,p <0.05).5.The effect of carvacrol on a TRPM7 expression in a rat model of HCMIn our study,we further examined the expression of the TRPM7 at the m RNA level in a rat cardiac hypertrophy model.The results from real-time RT-PCR demonstrated that the m RNA expression level of TRPM7 was significantly higher in HCM group compared to control(n=4,p <0.05).It is noting that both low dose of carvacrol and high dose of carvacrol decreased the m RNA expression level of TRPM7 compared to HCM group(n=4,p <0.08).6.Effect of carvacrol on morphology of cardiomyocytes in HCM rats.Obvious fibrotic changes were observed by HE staining and Masson staining in a rat model of HCM(Fig.6 and Fig.7).However,high dose of carvacrol ameliorated the infiltration of inflammatory cells and the scope of myocardial fibrosis.Thus,carvacrol may ameliorate apoptosis morphological changes in HCM.Conclusion:1.We analyzed the gene expression profile of HCM from Gene Expression Omnibus,and identified differentially expressed genes through GO,KEGG and PPI,indicating that apoptosis is very important in HCM.2.Carvacrol reduces the expression of Cleaved-Caspase 3 in HCM rat model.3.Carvacrol reduces the m RNA expression of TRPM7 in HCM rat model.4.Carvacrol delays myocardial fibrosis in HCM rats.In summary,our research shows that carvacrol can improve HCM cardiomyocyte apoptosis and fibrosis,and provide effective therapeutic candidates and new therapeutic targets for HCM.