AP39 Pro-FUNDC1 Mediated Mitophagy Regulates Mitochondrial Dynamics To Improve HHCY-induced Myocardial Remodeling

Homocysteine（HCY）,a homologue of cysteine,is an intermediate product of methionine metabolism.Previous studies from the laboratory has found out that the high homocysteine（HHCY）induces cardiomyocyte senescence,which has composed the important factor in inducing myocardial sclerosis,vascular wall sclerosis,myocardial remodeling,and heart failure.AP39,a newly synthesized mitochondria-targeted H₂S donor,is able to inhibit cell injury and endothelial cell senescence.However,its specific regulatory mechanism remains unclear.Mitophagy is a selective autophagic process that specifically clears damaged mitochondria.It has been found that FUNDC1-mediated mitophagy may be involved in regulating mitochondrial dynamics,which is altered and closely related to cellular senescence.With the upper fundamental,we propose to apply RNA sequencing（RNA-seq）,WB,RNA interference,Immunofluorescence and other technologies to investigate whether AP39 improves HHCY-induced cardiomyocyte senescence by promoting FUNDC1-mediated mitophagy and how it regulates mitochondrial dynamics at the cellular and animal levels.The target of this study is to reveal the new mechanism of AP39 inhibition of cardiomyocyte senescence from the perspective of FUNDC1-mediated mitophagy and altered mitochondrial dynamics,and to offer a theoretical basis for whether AP39 can be a novel drug to improve HHCY-induced cardiomyocyte senescence.Methods:1.Transmission electron microscopy to observe the occurrence of mitophagy in SD rats.2.Color Doppler ultrasound diagnostic instrument for cardiac ultrasound in SD rats.3.QT-PCR assay to detect the relative expression of Col3α1,Col1α2 genes.4.Immunohistochemical observation of myocardial fiber expression in SD rats and HCY-induced expression of FUNDC1,LC3A/B,MFN1/2,DRP1,P53,P21,alpha-SMA,Collage III in myocardial tissue of SD rats.5.With the help of RNA sequencing（RNA-seq）technology,high-throughput sequencing of total RNA from H9c2 cardiomyocytes will be characterized so as to describe the transcriptional expression profile under HHCY conditions as well as identify changes in FUNDC1after HHCY treatment as well as AP39 intervention.6.Western Blot to detect expression of FUNDC1,LC3A/B,MFN1/2,DRP1,P53,P21 in rat H9c2 cardiomyocytes.7.RNA interference:designing RNA interference strands to interfere with FUNDC1 expression.8.Mitochondrial membrane potential detection kit（JC-1）to detect changes in mitochondrial membrane potential.9.β-galactosidase staining（Senescence Associated acidic-β-Gala ctosidase;SA-β-Gal）to detect cellular senescence levels.10.Immunofluorescence observation of FUNDC1 and LC3B co-localization.Results:1.Mitochondria-targeted H₂S donor AP39 improves cardiac function and cardiac index in rats with hyperhomocysteine cardiomyopathy.2.HHCY induces cardiomyocyte senescence and myocardial fibrosis in hyperhomocysteine rats,and this effect can be antagonized by AP39.3.Mitochondria-targeted H₂S donor AP39 intervention enhances mitochondrial autophagy and regulates mitochondrial division and fusion.4.Mitochondrial targeting of the H₂S donor AP39 inhibits HHCY-inducedsenescence in H9c2 cardiomyocytes.5.Altered mitochondrial dynamics are involved in HHCY-induced senescence in H9c2 cardiomyocytes,and this effect can be antagonized by mitochondria-targeted H₂S donor AP39.6.Activation of HHCY-induced FUNDC1-dependent mitochondrial autophagy in H9c2 cardiomyocytes by the mitochondria-targeted H₂S donor AP39.7.FUNDC1-siRNA significantly reverses AP39 to improve HHCY-induced mitochondrial dynamics homeostasis and cellular senescence in cardiomyocytes.CONCLUSION:Mitochondria-targeted H₂S donor AP39 promotes FUNDC1-medi ated mitophagy to improve mitochondrial dynamics of cardiomyocytes and alleviate HHCY-induced myocardial remodeling in rats.