Mitochondrial Serine/One-carbon Metabolism Pathway Mediates Apelin-13 Induced Cardiomyocytes Hypertrophy

Aim:Apelin,an endogenous ligand of G protein-coupled receptor-APJ,plays an important role in the formation of the cardiovascular system and the development of related diseases.According to our previous research,we have found that Apelin-13 can induce cardiomyocyte hypertrophy,and the ferritinophagy-mitochondrial iron ion transporter Sideroflexin1(SFXN1)participates in the process of cardiomyocyte hypertrophy induced by Apelin-13.Recent studies report that SFXN1 can transfer serine into the mitochondria and participate in the mitochondrial one-carbon metabolism.It is reported in the literature that SFXN1 located in mitochondria can transport both iron ions and serine from cytoplasm into mitochondria.When iron ions are transported,the iron autophagy-SFXN1-mitochondrial iron overload pathway is involved in the occurrence and development of Apelin-13-induced cardiomyocyte hypertrophy.Apelin/APJ promotes SFXN1 mitochondrial iron transport and at the same time it is likely to lead to the reduction of SFXN1mitochondrial serine transport,resulting in the disorder of one-carbon unit metabolism in the cell and finally causing the development of cardiac hypertrophy.Hence,in this study we try to investigate the effect of Apelin-13on serine metabolism and one-carbon metabolism.In addition,we try to discuss the mechanism of ferritinophagy Sideroflexin1(SFXN1)-dependent mitochondrial iron overload mediate Apelin-13 induced cardiomyocyte hypertrophy.Revealing a new mechanism of myocardial hypertrophy induced by Apelin-13/APJ system,and provides a new experimental basis for developing anti-cardiovascular drugs.Methods:1.ELISA Kit:Detecting the concentration of serine and tetrahydrofolate(THF)in H9c2 cardiomyocytes’mitochondria.2.Western Blot:detecting the effect of Apelin-13 on the expression of serine de novo synthesis pathway key enzyme PHGDH(Phosphoglycerate dehydrogenase),PSAT1(Phosphoserine aminotransferase 1),and PSPH(Phosphoserine phosphatase),mitochondrial one-carbon metabolism key enzyme SHMT2(Serine hydroxymethyl transferase 2),cytoplasmic one-carbon metabolism key enzyme SHMT1(Serine hydroxymethyl transferase 1),methionine cycle key enzyme MAT2A(Methionine adenosyltransferase),cytoplasmic tetrahydrofolate metabolism key enzyme MTHFD1(methylenetetrahydrofolate dehydrogenase 1),mitochondrial tetrahydrofolatemetabolismkeyenzymeMTHFD1L(methylenetetrahydrofolate dehydrogenase 1L)and MTHFD2(methylenetetrahydrofolate dehydrogenase 2)in H9c2 cardiomyocytes,and detecting the effect of the high concentration serine medium(10x)and PHGDH overexpression on the expression of BNP and ANP treated with Apelin-13.3.DNA overexpression:designing PHGDH overexpression plasmid to overexpress the PHGDH in H9c2 cardiomyocytes’mitochondria.4.High concentration serine medium(10x):use high concentration serine medium(10x)to culture the H9c2 cardiomyocytes.5.The diameter and volume of H9c2 cells was tested by Scepter~TMM Handheld cell counter.6.The concentration of proteins in H9c2 cells was tested by BCA protein quantitation kit.Results:1.Apelin-13 decreased the concentration of serine and THF in mitochondria of H9c2 cardiomyocytes at a dose-dependent(0-1μmol/L)manner.2.Apelin-13 decreased the expression of SHMT2,MTHFD1L,and MTHFD2 in mitochondria of H9c2 cardiomyocytes at a dose and time dependent manner,and the APJ antagonist F13A antagonized its effect on SHMT2,MTHFD1L,and MTHFD2.3.Apelin-13 decreased the expression of SHMT1 and MTHFD1 in cytoplasm of H9c2 cardiomyocytes at a dose and time dependent manner,and the APJ antagonist F13A antagonized its effect on SHMT1 and MTHFD1.4.Apelin-13 decreased the expression of MAT2A in H9c2cardiomyocytes at a dose and time dependent manner,and the APJ antagonist F13A antagonized its effect on MAT2A.5.Apelin-13 decreased the expression of PHGDH,PSAT1,and PSPH in H9c2 cardiomyocytes at a dose and time dependent manner,and the APJ antagonist F13A antagonized its effect on PHGDH,PSAT1,and PSPH.6.HS(high concentration serine medium 10x)down-regulated the expression of myocardial hypertrophy marker protein BNP and ANP in H9c2cardiomyocytes induced by Apelin-13.7.HS(high concentration serine medium 10x)significantly inhibited the increase in diameter,volume and protein content of H9c2 cardiomyocytes induced by Apelin-13.8.PHGDH overexpression down-regulated the expression of myocardial hypertrophy marker protein BNP and ANP in H9c2 cardiomyocytes induced by Apelin-13.9.PHGDH overexpression significantly inhibited the increase in diameter,volume and protein content of H9c2 cardiomyocytes induced by Apelin-13Conclusion:Mitochondrial serine/one-carbon metabolism pathway mediates Apelin-13 induced cardiomyocytes hypertrophy...