| Background:Myocardial infarction(MI)is a serious hazard to human health,and its morbidity and mortality are still increasing year by year.Current reperfusion therapy significantly reduces the mortality of patients with MI,but some patients still develop heart failure(HF),which leads to increased rehospitalization rate of patients,seriously affecting the quality of life and increasing the economic burden.Mitochondrial dysfunction after myocardial ischemia and hypoxia caused by MI leads to increased apoptosis and decreased myocardial systolic function,which is an important mechanism for the occurrence and development of HF after MI.Multiple members of Kruppel-like factor(KLF)family can improve mitochondrial oxidative stress,energy metabolism,mitochondrial biosynthesis and other functions after HF.KLF family is a class of eukaryotic nuclear transcription factors,which participates in many important physiological and pathological processes through regulating the expression of related genes,such as proliferation,differentiation,apoptosis,development,tumor growth,obesity,vascular inflammation,etc.Among them,KLF2,KLF5,KLF9 and KLF15 are all involved in the occurrence and development of MI,suggesting that KLF family plays an important role in MI.KLF 14 is highly expressed in heart and can improve vascular inflammation,lipid metabolism,insulin resistance and other risk factors related to MI,suggesting that KLF14 may be associated with MI,but no study has confirmed the relationship between KLF 14 and MI.Some studies have shown that KLF 14 also regulates intracellular mitochondrial function,but the specific mechanism is unclear.Therefore,we hypothesized that KLF 14 may be involved in the occurrence and development of HF after MI by regulating mitochondrial function.Sirtuin family is a class III histone deacetylase,which can participate in the regulation of mitochondrial function by regulating the acetylation of mitochondrial protein related to mitochondrial biogenesis,mitochondrial autophagy,fatty acid oxidation,redox system.Previous studies have confirmed that some members of KLF family are involved in mitochondrial function by regulating the expression of Sirtuin family,so it is speculated that KLF 14 may regulate mitochondrial function by regulating the expression of Sirtuin family.Objective:This article is aimed to explore the changes of KLF 14 in the course of MI,the effects of KLF 14 overexpression on the cardiac function and mitochondrial function after MI and the analysis of the specific mechanism.Methods:1.To explore whether KLF14 is involved in the pathological process of MI:(1)To detect the expression of KLF 14 in ischemic tissue of mice after MI:The mRNA and protein expression of KLF 14 in normal myocardium of SHAM group and infarcted tissue of MI group were detected through real-time quantitative PCR(RT-QPCR),Western blot(WB)and immunofluorescence at different time points(3d,1w,2w,3w and 4w)after MI.(2)To detect the expression of KLF14 in H9C2 after hypoxia:After hypoxia model was established,and the expression of KLF 14 in H9C2 were compared at different time points(12h,24h,36h)by RT-QPCR and WB.2.To explore the effect of KLF 14 overexpression on cardiac function of mice after MI:(1)KLF 14 overexpression model of mice was established by injecting cardiomyo-specific KLF 14 overexpression adeno-associated virus(AAV-KLF 14)and negative virus(AAV-GFP)into tail vein for 4w respectively.(2)After the overexpression model was successfully constructed,exposing the mice to the operation of MI for 4w and detecting the cardiac function indexes including ischemia area(TTC staining),left ventricular function(echocardiography,blood BNP levels),myocardial fibrosis(HE/MASSON staining,the expression of COL1al).(3)H9C2 were transfected with Ad-KLF14 for 48h to construct KLF14 overexpression model,and transfected with negative virus(AdGFP)as control.Hypoxia models were constructed respectively,and the expression levels of apoptosis-related protein(Bax/Bcl2)were detected by WB.3.To explore the possible mechanism of overexpression of KLF14 in improving cardiac function after MI in mice:(1)Transcriptome sequence and enrichment analysis of mice myocardium;Mitochondrial function of myocardium after MI was detected:mitochondrial morphology was analyzed by transmission electron microscopy,and transcription levels of mitochondrial related genes(including mitochondrial quality control,mitochondrial biogenesis,fatty acid metabolism,electronic respiratory chain(ETC)complex)and Sirtuin family were detected by RT-QPCR.(2)Different concentrations of perhexiline(15μM,20μM,25μM),which is an activator of KLF14,were administered to H9C2 for 24h to increase the expression of KLF14.After successful intervention,SIRT7 expression level was detected by RT-QPCR.(3)H9C2 were transfected with Ad-KLF14 to construct KLF14 overexpression cell model.After 48h,trichostatin A(TSA)was then administered for 24h to inhibit SIRT7 expression and then H9C2 was exposed to hypoxia respectively.RT-QPCR was used to detect the expression of mitochondrial ETC complex after hypoxia.Results:1.To explore whether KLF14 is involved in the pathological process of MI:(1)The expression of KLF14 in myocardial tissue of mice after MI was detected:the mRNA and protein expression of KLF14 in ischemic tissue of mice after MI was significantly decreased in a time-dependent manner,and the decrease was most obvious at 4w after MI(all P<0.05).Furthermore,immunofluorescence indicated that the expression of KLF14 in ischemic tissue was significantly decreased at 4w after MI(P<0.05).(2)The expression of KLF14 in H9C2 after hypoxia was detected:the mRNA(P<0.0001)and protein(P<0.01)expression of KLF14 of H9C2 significantly decreased at 24h after hypoxia.2.To explore the effect of KLF14 overexpression on cardiac function of mice after MI:(1)RT-QPCR and WB indicated that KLF14 overexpression model of mice was successfully established(P<0.05).(2)Overexpression of KLF14 significantly improved left ventricular ejection fraction(LVEF)and shortening fraction(FS)after MI(all P<0.05),decreased blood BNP level(P<0.001),decreased myocardial ischemia area(P<0.001)and myocardial fibrosis(all P<0.05).(3)Compared with the control group,the protein expression of Bax/Bcl2 in H9C2 was significantly increased in the hypoxia group(P<0.001),while overexpression of KLF14 downregulated the protein expression of Bax/Bcl2 after hypoxia compared with the hypoxia+AdGFP group(P<0.001).3.To explore the possible mechanism of overexpression of KLF14 in improving cardiac function after MI in mice:(1)Transcriptome sequence indicated that overexpression of KLF14 significantly regulated myocardial mitochondrial function of mice after MI including fatty acid metabolism and ETC complex;Transmission electron microscopy observed abnormal mitochondrial morphology and distribution in ischemic tissue after MI,while overexpression of KLF14 could significantly improve the morphology and distribution after MI.(2)Compared with the SHAM group,only the mRNA expression of optic atrophy 1(OPA1)related to myocardial mitochondrial quality control system decreased after MI(P<0.05).while compared with the MI+AAV-GFP group,overexpression of KLF14 did not affect the mRNA expression of genes related to mitochondrial quality control system(DRP1,OPA1,MFN1,MFN2,PARKIN,FUNDC1)of mice after MI(all P>0.05).Compared with the SHAM group,only the mRNA expression of PGC-1α related to myocardial mitochondrial biogenesis decreased after MI(P<0.05).While compared with the MI+AAV-GFP group,overexpression of KLF14 did not affect the mRNA expression of genes related to mitochondria biogenesis(PGC-1α,TFAM)of mice after MI(all P>0.05).(3)Compared with the SHAM group,the mRNA expression of genes related to myocardial mitochondrial fatty acid metabolism(CPT1B,FABP4,ACADL,ACAA2,HADHB)were significantly decreased after MI(all P<0.05).However,compared with the MI+AAV-GFP group,overexpression of KLF14 only upregulated the mRNA expression of CPT1B of mice after MI(P<0.05).(4)Compared with the SHAM group,the mRNA expression levels of myocardial mitochondria ETC complex Ⅱ,Ⅲ,Ⅳ and Ⅴ were significantly decreased after MI(all P<0.05);Compared with the MI+AAV-GFP group,overexpression of KLF14 significantly upregulated the expression of myocardial mitochondria ETC complex Ⅱ～Ⅴafter MI(all P<0.05).(5)Further mechanism exploration showed that the mRNA expression of SIRT7 increased in the MI group compared with the SHAM group(P<0.05),while overexpression of KLF14 decreased SIRT7 expression compared with the MI+AAV-GFP group(P<0.05).Perhexiline interfered with H9C2 to increase the mRNA expression of KLF14,and KLF14 decreased the mRNA expression of SIRT7 in a concentrationdependent manner(all P<0.05).Trichostatin A(TSA)increased SIRT7 expression and reversed the effect of overexpression of KLF14 on mitochondrial ETC complex Ⅱ～Ⅴ expression in H9C2 after hypoxia(all P<0.05).Conclusions:1.The expression of KLF14 decreased significantly after myocardial ischemia and hypoxia in mice.2.Overexpression of KLF14 can reduce the size of myocardial infarction,improve left ventricular cardiac function,alleviate myocardial fibrosis,and reduce apoptosis after MI.The mechanism involves that improving myocardial mitochondrial function after MI through inhibiting SIRT7 expression to increase the expression of mitochondria ETC complex. |
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