The Function And Mechanism Of Non-coding RNA(CHKB-DT And MiR-217)in Heart Failure

Background and AimHeart Failure is the pathophysiological state which the heart is unable to pump blood at a commensurate with the requirements of the metabolizing tissues or can do so only from an elevated filling pressure.Chronic Heart failure(CHF)is the end stage of various cardiovascular diseases with high rates of hospitalization and mortality¹.Chronic heart failure can be divided into left heart failure?right heart failure and whole heart failure based on the location.The clinical manifestations are also different.The main manifestations of left heart failure are pulmonary congestion,such as cough,sputum and dyspnea,and systemic circulatory lavage deficiency,such as fatigue,dizziness and oliguria.Right heart failure is mainly manifested as systemic circulation of blood stasis.Current treatments include diuretics to improve symptoms of heart failure,inotropic drugs to improve cardiac contractility,renin-angiotensin-aldosterone inhibitors to improve cardiac remodeling,and beta-blockers to improve patient outcomes and survival².The underlying etiologies of CHF are varied and included myocardial ischemia or infarction,hypertension,inflammation and cardiomyopathy,etc.Understanding and mastering the etiology and molecular pathogenesis of heart failure systematically and comprehensively is helpful for finding better drug therapeutic targets.Cardiomyopathy is a group of myocardial diseases caused by myocardial injury,which mainly affects the systolic and diastolic functions of the heart.Cardiomyopathy is classified as:hypertrophic cardiomyopathy,dilated cardiomyopathy,restrive cardiomyopathy,right ventricular arrhythmia cardiomyopathy and unclassified cardiomyopathy²³.Dilated cardiomyopathy(DCM)is the most commen form of cardiomyopathy with morbidity up yo 1/400,which is a primary cause of CHF¹¹.DCM is characterized by left ventricular ejection fraction less than 45%or fraction shording less 25%accompared with left ventricular inter diameter more than 117%.The pathogenesis undering DCM process is variable including genetic mutations,virus's infection,and alcohol abuse,autoimmune and systemic disorders and so on²⁴.For treatment of dilated cardiomyopathy are mainly improving the hemodynamic changes in heart failure(including diuretics,renin angiotensin,beta blockers,etc.).According to new research,improving the myocardial cell energy production and the enhanced ability of myocardial cells is also one of the main measures for the treatment of dilated cardiomyopathy.Non-coding RNAs are a class of RNAs with no coding potential and are catogarized as micro RNAs(<200nt)and long non-coding RNAs(>200nt)based on the sequence length.Micro RNAs(miRNAs)are a class of short and single-stranded non-coding RNAs that act as negative regulators of target gene expression at the posttranscriptional level by interfering with m RNA translation and/or stabilization²⁵.Dysregulated miRNAs have been reported to be important in heart failure such as miR-1,miR-133,miR-208,and miR-499 and so on^15,26.Long non-coding RNAs(lnc RNA)have also been reported playing important roles in the process of heart failure by various mechanisms,such as CHAST,CHEA,etc^21,27,22.However,the function and mechanism of non-codin RNAs involved in heat failure caused by dilated cardiomyopathy is far from understanding.This study aimed to investigate the role and mechanism of miR-217 and lnc RNA CHKB-DT in dilated cardiomyopathy,which may be the novel theoretical ground for preventing and treating dilated cardiomyopathy and heart failure.Methods and Results1.CHKB-DT expression was suppressed in heart samples from dilated cardiomyopathy patients,while miR-217 expression was elevated.We found a series of differentially expressed miRNAs and lnc RNAs through microarray technology,in which CHKB-DT expression was decreased in the hearts of patients with dilated cardiomyopathy;while the expression level of miR-217 was increased,which was verified by real-time quantitative PCR in another chort population.2.CHKB-DT silencing promoted cardiac dilation and dysfunction.Under normal conditions,silencing of CHKB-DT promoted cardiac dilation but had no impact on cardiac function.Using immunohistochemical staining,echocardiography and ventricular catheter miller testing,we found that CHKB-DT silencing promoted cardiac dilation and dysfunction under pressure-overload condition.While CHKB-DT overexpression could promote myocardial hypertrophy and improve cardiac function.3.CHKB-DT silencing induced EEF1A1 protein degradation.Using RAP,RNA pull down,RNA-FISH,CO-IP and Western Blot,we found that CHKB-DT can directly bind protein EEF1A1.CHKB-DT silencing decreased the protein levels of EEF1A1 and acclebrated actinomycetin induced EEF1A1 protein degradation.And the degradation of EEF1A1 induced by low expression of CHKB-DT can be alleviated by proteasome inhibitor(MG132)rather than the lysome inhibitor chloroquine.Furthermore,silencing of CHKB-DT promoted the binding of ubiquitin with EEF1A1.4.EEF1A1 infusion alleviated ventricular dilatation and cardiac dysfunctions caused by CHKB-DT decling.By western blot,immunohistochemical staining,echocardiography,and hemodynamic examination,we found that the protein levels of EEF1A1 were decreased in heart samples from DCM patients.Lowering the expression of EEF1A1 can also cause ventricular dilatation and cardiac dysfnction,while EEF1A1infusion can alleviate ventricular dilatation and cardiac function caused by low expression of CHKB-DT.5.Knockdown of EEF1A1 constrained energy metabolism associated proteins systhesis,inhibited mitochondrial energy metabolism and impaired myocardial contraction.By western blot,mass spectrometry,etc.,lower expression CHKB-DT inhibit overall myocardial cell protein expression level.GO and KEGG analysis showed that differentially expressed proteins found in the heart tissue is mainly related to mitochondrial and energy metabolism.Using electron microscope,we found that lower expression of CHKB-DT damage the normal form of mitochondria.Besides that,silencing of CHKB-DT impaired the oxidative phosphorylation and glucolysis ultimately leading the reduction of ATP.Moreover,the uptake of glucose was reduced upon the sh-CHKB-DT intervenation in vivo by PET/CT analysis.Furthermore,the contraction of primary cardiomyocytes isolated from hearts with various treatments was also impaired.6.MiR-217 promoted cardiac hypertrophy,cardiac fibrosis and heart failure.Firstly,aorta surgery(transverse aortic contriction,TAC)induced myocardial hypertrophy and heart failure mice model was established,and recombinant adeno-associated virus technology was applied in mice to regulate the expression of miR-217 in the heart tissue.Using immunohistochemistry and immunofluorescence staining,we found that miR-217 can promote cardiac myocyte hypertrophy and myocardial fibrosis.Then using echocardiography and ventricular catheter miller testing,we found that miR-217 promote myocardial hypertrophy and impair cardiac function while miR-217 knocdown can relieve the myocardial hypertrophy and heart failure induced by TAC.7.MiR-217 targeted PTEN to activate Akt pathway.Using bioinformatics analysis,argonaute 2 immunoprecipitation,western blot and luciferase reporter gene detection,we found that PTEN is the downstream target of miR-217,and miR-217 activates the Akt pathway by down-regulating the protein level of PTEN,thereby promoting myocardial hypertrophy.Restoring PTEN expression reverses cardiac hypertrophy and cardiac function caused by miR-217.8.Cardiomyocytes derived miR-217-containing exosomes promotes cardiac fibrosis.Using exosoms isolation experiments,q PCR and Cell Counting Kit-8experiment,we found that miR-217 was exsiting in the exosomes isolated from cardiomyocytes.MiR-217-containing exosoms could be uptaking by fibroblasts and promoted cells proliferation and extracellular matrix secretion.ConclusionThe expression of CHKB-DT was remarkably decreased in heart samples from DCM patients as compared with controls;while miR-217 expression was elevated in heart samples from DCM patients.CHKB-DT silencing constrained energy metabolism related proteins synthesis by promoting EEF1A1 degradation through ubiquitin proteasome pathway thereby promoting cardiac dilation and dysfunction,while EEF1A1 reversed the cardiac phenotype induced by CHKB-DT knockdown.MiR-217 aggravated TAC-induced myocardial hypertrophy and heart failure by inhibiting the expression of PTEN to activate Akt pathway,while miR-217knockdown showed the reversed phenotype.Restoring PTEN expression alleviated miR-217 induced cardiac dysfunction.We investigated the association between non-coding RNAs and dilated cardiomyopathy caused heart failure,providing a novel theoretical ground for dilated cardiomyopathy and heart failure prevention and treatment.