Study On The Mechanism Of LBQ657 Delaying Cardiomyocyte Senescence Based On LncRNA And MRNA Expression Profiles

Background And ObjectiveAging can lead to the decline of the tissues and organs of the body.One of the important manifestations of aging in the heart is heart failure,and the aging of cardiomyocytes is closely related to the development of heart failure.Our previous study also showed that exogenous testosterone treatment delayed cardiomyocyte aging and ameliorated heart failure-related gene expression abnormalities in aged male mice.Sacubitril/Valsartan(LCZ696)is composed of angiotensin receptor blocker valsartan and enkephalidin inhibitor sacubitril,by antagonizing angiotensin receptor and enhance natriuretic peptide function,natriuresis,enlargement,organ protection,has been sufficient clinical research confirmed its curative effect in the treatment of heart failure,has been recommended by many guidelines for the first-line treatment of heart failure.The main metabolic active product of sacubitril in vivo is LBQ657.Our previous study found that LBQ657 can improve the senescence state of cardiomyocytes to some extent,and the presence of changes of SIRT1,p53,and other factors affecting cell metabolism and apoptosis were observed,but the study of the mechanism is still limited.lnc RNA can further regulate the translation process of its downstream proteins by regulating m RNA.The determination of lnc RNA and m RNA expression profiles facilitates further investigation of the relevant mechanisms.In this study,D-galactose-induced cardiomyocytes were used to establish a senescent cell model to explore the effects of LBQ657 on senescent cardiomyocytes and analyze the possible mechanisms of action by high-throughput sequencing and lnc RNA-m RNA co-expression network.Methods(1)Mouse HL-1 cardiomyocytes: cultured in complete medium.(2)Experimental group:(1)control group(group N): HL-1 cardiomyocytes,normal culture;(2)senescence model group(group A): D-galactose treatment induced senescence;(3)LBQ657 treatment group(group L): D-galactose induced senescence and LBQ657 treatment.(3)The rate of aging in all three cardiomyocytes was assessed by usingβ-galactosidase staining.(4)By measuring the levels of SOD(superoxide dismutase)and MDA(malondialdehyde)to understand the changes in cardiomyocyte ability to resist oxidative stress.(5)The levels of MYH-7(myosin heavy chain 7),ACTA-1(α-actin-1),MYH-6(myosin heavy chain 6)and SERCA-2(myoplasmic reticulum / ER Ca2 +-ATP enzyme-2)were measured by q PCR to understand the effects of enzymes and protein expression involved in cardiomyocyte aging and dysfunction.(6)Differentially expressed lnc RNAs and m RNAs were detected by high-throughput sequencing technology.(7)Differential m RNA function(GO,KEGG)enrichment analysis.(8)Construction of the lnc RNA-m RNA co-expression network.Results(1)The rate of cardiomyocyte senescence was increased in the aging model group compared with the control group,The difference was statistically significant(P <0.01);The rate of cardiomyocyte senescence was decreased in the LBQ657 treated group compared with the aging model group,The difference was statistically significant(P <0.01).(2)Compared with the control group,The SOD levels of cardiomyocytes were significantly decreased in the aging model group,The MDA levels of cardiomyocytes were significantly increased in the aging model group,The difference was statistically significant(P <0.01);Compared with the aging model group,The SOD levels of cardiomyocytes were increased in the LBQ657-treated group,The MDA levels of cardiomyocytes were decreased in the LBQ657-treated group,The difference was statistically significant(P <0.01,P <0.05).(3)Compared with the control group,The expression levels of MYH-6and SERCA-2 were significantly decreased in the aging model group,The expression levels of MYH-7 and ACTA-1 were significantly increased in the aging model group(P <0.05).Compared with the aging model group,The expression levels of MYH-6 and SERCA-2 were increased in the LBQ657-treated group,The expression levels of MYH-7 and ACTA-1 were significantly decreased in the LBQ657-treated group(P <0.05).(4)Compared with the control group,The aging model group had 1294 differentially expressed lnc RNAs,716 upregulated,578 downregulated and7491 differentially expressed m RNAs,4885 and 2606 downregulated.Compared to the aging model group,The LBQ657-treated group had 811 differentially expressed lnc RNAs,with 393 upregulated and418 downregulated and 2950 differentially expressed m RNAs,including 1556 and 1394 downregulated.(5)Compared with aging model group and control group,significantly enriched GO function is mainly related to metabolic process,apoptosis and cell proliferation regulation;significantly enriched KEGG pathway is mainly related to cell cycle,p53 signaling pathway and apoptosis.Compared with LBQ657 intervention group and aging model group,the significantly enriched GO function is mainly related to the regulation of macromolecular metabolic process,endogenous apoptosis signaling pathway and cell proliferation regulation;the significantly enriched KEGG pathway is mainly related to cell apoptosis,PPAR signaling pathway and p53 signaling pathway.(6)From the findings of the cluster heat map and lnc RNA-m RNA co-expression network,In comparison with the control group,Gene expression of Cdkn1 b and Bbc3 was significantly up-regulated in aging model cardiomyocytes(P <0.05);In comparison with the aging model group,Cdkn1 b and Bbc3 gene expression were significantly downregulated in cardiomyocytes in the LBQ657-treated group(P <0.05).There were five lnc RNA with co-expression relationship with Cdkn1 b and five with co-expression relationship with Bbc3.Conclusion(1)LBQ657 can effectively delay D-galactose-induced senescence of HL-1 cardiomyocytes in mice.(2)The mechanism of LBQ657 in delaying cardiomyocyte senescence may have the involvement of m RNA including Cdkn1 b,Bbc3 and lncRNA.