Valsartan Regulates TGF-?/Smads And TGF-?/p38 Pathways Through LncRNA CHRF To Improve Doxorubicin-induced Heart Failure

Background and Objective:Cardiovascular disease is one of the most common chronic diseases in clinic;it causes high morbidity and mortality worldwide.Heart failure is the final stage of all kinds of heart disease,and its myocardial pathological changes are often the coexis-tence of myocardial hypertrophy,apoptosis and fibrosis.When hearts suffer stress,the "cardiac remodeling" characterized by typical pathological cardiac hypertrophy oc-curs,resulting in heart failure(HF).Patients with HF experience the progressive deterioration of cardiac function.Doxorubicin(DOX),a chemotherapy medication used to treat cancer,is commonly used to establish cardiomyopathy in animal models and acts as an important factor in dilated cardiomyopathy-like congestive HF in clinic.However,this widely applicable and effective anti-cancer drug's utility is limited partly due to its cardiotoxicity and ability to induce severe HF.At present,common treatments mainly slow down the functional decline but cannot prevent or reverse the HF reduced by DOX.In addition,although the pathogenic factor of HF has been determined,the complex biology and underlying molecular mechanisms have not yet been fully elucidated.Long non-coding RNAs(lncRNAs)are RNA transcripts with more than 200 nucleotides and without protein-coding potential Numerous studies have confirmed that lncRNAs play a critical role in cell proliferation and differentiation,chromatin modification,and also serve as "miRNA sponges".Currently,compared with lnc-RNAs well-reported dysregulation in various cancers,their function in heart disease has seldom been investigated.lncRNA myosin heavy chain-associated RNA trans-cript(MHRT)prevented DOX-induced cardiac apoptosis by positively regulating nuclear factor erythroid 2-related factor(Nrf2)expression.The mitochondrial Inc-RNA uc022bqs.1(LIPCAR)was quickly down-regulated after a myocardial in-farction but up-regulated during later stages of myocardial infarction;it acts as a novel biomarker in HF patients.A recent report also indicated that a lncRNA cardiac hypertrophy-related factor(CHRF)is directly bound to miRNA-489 and regulated MyD88 expression in cardiac hypertrophy,impling that CHRF play a role in heart disease.A growing body of evidences link elevated levels of TGF-? super-family ligands to the progression of HF.TGF-?1 is a locally generated cytokine that has been im-plicated as a major contributor to myofibroblast proliferation and tissue fibrosis in diverse organ systems.Numerous reports have demonstrated the functional out-comes of TGF-?1 in HF,and these results have shown that TGF-? antagonism could inhibit fibrotic processes and provide salutary cardiac effects.Recently,CHRF has been shown to regulate MyD88 and SMAD3 by targeting miR-489,and overexpression of CHRF led to increased expression of TGF-?1 protein levels in silica-induced pulmonary fibrosis(Wu et al.2016).Valsartan(VAL)is a kind of anti-hypertensive drug and licensed for the treatment of patients with symptomatic HF(Chaplin 2016),and it plays an important role in the process of HF injury.For example,compared to a placebo added to a prescribed therapy,VAL slowed progressively deteriorating quality of life in HF patients.In the PARADIGM-HF trial(Prospective Comparison of angiotensin receptor neprilysin inhibitor(ARNI)with angiotensin converting enzyme inhibitor(ACEI)to determine impact on global mortality and morbidity in heart failure),the risk of death from cardiovascular causes from worsening HF was significantly reduced with VAL compared to the ACE inhibitor enalapril in patients with chronic HF.Therefore,VAL represents effect approach in the treatment of HF;it is commonly used in clinical practice and is known to have heart-lung protective effects.The current study sought to determine the expression and function of CHRF and TGF-?1 pathways in the tissues and cells of patients with HF.It also sought to detecte the protective effect of VAL in regulating HF malignant processes and the underlying mechanisms involved.Methods:1.To construct the mouse model of HF,eighteen C57BL/6 male mice,aged 8 weeks,were purchased from Zhengzhou Univercity Research Center of Laboratory Animals.These mice were randomly divided into three groups(n = 6 in each group):Group 1 was used as a control and mice were treated with an equivalent volume of sterile saline solution;mice in Group 2 were intraperitoneally injected with 2.5 mg/kg of DOX six times in 2 weeks;animals in Group 3 were also intraperitoneally injected with 2.5 mg/kg of DOX six times in 2 weeks,but then were treated with 30 mg/kg/days of VAL for 4 weeks by oral gavage.Cardiac function was evaluated via left ventricular systolic pressure(LVSP),left ventricular end-diastolic pressure(LVEDP),and the maximal rate of the increase of left ventricular pressure(+dp/dt),the maximal rate of the decrease of left ventricular pressure(-dp/dt)max parameters detection.Quantitative real-time PCR(qRT-PCR)analysis was used to detect CHRF expression.The proteins in the heart tissues were determined by western blot an-alysis.West blot analysis was used to detect expression of TGF-?1 and caspase-3.Colorimetric assay was employed to analyze caspase-3 activity.To evaluate the caspase-3 activity,primary cardiomyocyte cell lysates were prepared subsequent to various designated treat-ments.Samples were measured with Western blot analysis.All data were expressed as the mean ± SEM of three independent experiments.2.Primary cardiomyocyte cells were isolated,were randomly divided into four groups:control,DOX,VAL,DOX+VAL.After transfect,the cells in Group DOX were treated with 2 ?M DOX,the cells in group VAL were treated with 1 ?M VAL,and the cells in group DOX+VAL were treated with 2?M DOX after pretreatment of 1?M VAL for 12 hours,and then cultured in 37 C incubator for 16 hours to establish a HF model in vitro.The same method was used to detect expression of CHRF and TGF-?1.To evaluate the caspase-3 activity as metioned above.Cell apoptosis of caspase-3 was determined via a(TUNEL)staining assay.3.Primary cardiomyocyte cells were isolated,the cells in different groups were trans-fected or co-transfected with pc-DNA-CHRF,si-CHRF,or the corresponding controls using Lipofectamine 2000(Invitrogen,USA)in the light of the manufacturer's instruct-tions.Then detect expression of CHRF?TGF-?1 and the caspase-3 activity in the same way as motioned above.4.The mouse myocardial cell lines HL-1 used in this study were obtained from Ameri-can Type Culture Collection(USA).The cells in different groups were transfected or cotransfected with si-CHRF,pc-DNA TGF?1,or the corresponding controls using Lipofectamine 2000(Invitrogen,USA)in the light of the manufacturer's instructions.The proteins such as TGF-?1?SMAD2?SMAD3 and p38 in cultured cells were deter-mined by western blot analysis.The mouse myocardial cell lines HL-1 used in this study were obtained from American Type Culture Collection(USA).The cells in different groups were transfected or cotransfected with pcDNA,pcDNA-CHRF,pcDNA-CHRF+DMSO,pcDNA-CHRF + SB-202190,si-NC,si-SMAD2,or the corre-sponding controls using Lipofectamine 2000(Invitrogen,USA)in the light of the manufacturer's instructions.After transfected for 48 hours,the cells in all groups were treated with 2?M DOX after pretreatment of 1 ?M VAL for 12 hours Cell apoptosis was determined via a(TUNEL)staining assay.All data were expressed as the mean ± SEM of three independent experiments.?-actin was used as the control in the western blot and GAPDH acted as the control in the qRT-PCR.5.Twenty-four C57BL/6 Mice were randomly divided into four groups:control,VAL,VAL + Ad-control,and VAL + Ad-CHRF.After injection of adenovirus vectors,mice were treated with VAL by oral gavage.Four weeks later,LVSP,LVEDP,and ± dp/dt max parameters were detected to assess the mice's cardiac function.All data were expressed as the mean ± SEM of three independent experiments.Results:1.Compared with the control mice,the levels of left ventricular systolic pressure(LVSP),left ventricular end-diastolic pressure(LVEDP),and ± dp/dt max parameters were aberrant in the HF mice,implying seriously dysregulated cardiac function(P<0.05).In addition,the activity of caspase-3 and cleaved caspase-3 were significantly increased in the HF mice compared to the control group(Group 1,n = 6)(P<0.05).Compared with the control group,CHRF expression was greatly boosted in the hearts of the HF mice(P<0.05).Western blotting results showed that TGF-?1 expression was also markedly increased in the heart tissues of the HF mice compared to the control mice(P<0.05).The activity of caspase-3 and cleaved caspase-3 were significantly increased in the HF mice compared to the control group(P<0.05).However,VAL at a dosage of 30 mg/kg/d significantly mitigated the adverse DOX-induced effects noted above(P<0.05).All data were expressed as the mean ±SEM of three independent experiments.2.Compared with the control,CHRF was significantly up-regulated in the DOX group.In addition,the protein level of TGF-?1 was higher in the DOX treatment group than in the control group,and the activity of caspase-3 and cleaved caspase-3 were markedly increased in the DOX group compared to the control group TUNEL assay results also indicated that DOX treatment significantly promoted the apoptosis of primary myocardial cells Importantly,all these DOX-induced changes were effectively reversed by VAL.All data were expressed as the mean ± SEM of three independent experiments.3.Si-CHRF transfection partly reversed the DOX-induced up-regulation of CHRF.The TGF-?1 protein level and activity of caspase-3 and cleaved caspase-3 clearly decreased in the cells transfected with si-CHRF compared with the si-control group(P<0.05).In addition,the number of apoptosis cells was also synchro-nously reduced in the CHRF interference group compared to the cells transfected with the si-control(P<0.05).Overexpression of CHRF(with pcDNA-CHRF transfected)reversed the VAL's inhibitory effect on CHRF.4,In HL-1 cells transfected with si-CHRF,the levels of TGF-?1,p-SMAD2,p-SMAD3,and p-p38 were significantly decreased compared with the si-control(P<0.05);however,expression of the above-mentioned proteins was clearly reversed by pcDNA-TGF-?1,while the increased TGF-?1,p-SMAD2,p-SMAD3,and p-p38 protein expressions in the pcDNA-TGF-?1 group were further reversed by si-CHRF(P<0.05).HL-1 cells were transfected with pcDNA,pcDNA-CHRF,pcDNA-CHRF + si-NC(si-negative control)or DMSO,pcDNA-CHRF + si-SMAD2/3,or SB202190(p38 inhibitor,5 ?M,24 h),then the cells were treated with 1?M of VAL for 12 h,followed by 2 ?M of DOX.The results showed that inhibiting of SMAD2/3 or p38 reversed the up-regulation of cell apoptosis induced by pcDNA-CHRF.These results demonstrate that CHRF regulated the expression of TGF-?1 and its target genes through TGF-?1/Smads and TGF-?1/p38 pathways.5.Compared with the control,the levels of LVSP,+ dp/dt max,and-dp/dt max were elevated by VAL,but reversed by Ad-CHRF injection(P<0.05),while the LVEDP level was sharply decreased by VAL but reversed by Ad-CHRF injection(P<0.05).Conclusion:1.The expression of CHRF and TGF-?1 in the hearts of DOX-induced HF models was abnormally expressed and cardiac function also seriously dysregulated,VAL relieved those effects.It is confirmed that VAL is effective in the treatment of DOX-induced heart failure and is related to CHRF and TGF-beta 12.The expression of CHRF and TGF-?1 in mice primary myocardial cells was imbalance,DOX treatment significantly promoted the apoptosis of primary myocardial cells,but all these things can be improved by VAL.These data indicate that VAL,CHRF,TGF-?1,and cell apoptosis might be related to the development of HF.3.Primary myocardial cells were transfected with si-CHRF and pcDNA-CHRF,and the levels of TGF-?1 and caspase-3 activity were determined.All these results show that CHRF influenced the expression of TGF-?1 and cell apoptosis.4.HL-1 cells were transfected or co-transfected with si-CHRF and pcDNA-TGF-?1,and the protein expressions of TGF-?1,p-SMAD2,p-SMAD3,and p-p38 were detected by western blot.HL-1 cells were transfected with pcDNA,pcDNA-CHRF,pcDNA-CHRF + si-NC(si-negative control)or DMSO,pcDNA-CHRF +si-SMAD2/3,or SB202190(p38 inhibitor,5 ?M,24 h),then the cells were treated with DOX and VAL.The results demonstrate that CHRF regulated the expression of TGF-?1 and its target genes through TGF-?1/Smads and TGF-?1/p38 pathways.5.while cardiac function clearly improved after treatment with VAL,those effects were reversed by Ad-CHRF.All the data suggest that overexpression of CHRF reversed VAL's cardiac protective effects.6.This study has confirmed for the first time that VAL functions as a protective agent to regulate TGF-?/Smads and TGF-?/p38 pathways through CHRF to improve DOX-induced HF.The significant correlation among VAL,CHRF,and TGF-?1 pathways in HF was highlighted for the first time.