Ferritinophagy Promotes Sideroflexin1-dependent Mitochondria Iron Overload Mediates Apelin-13 Induced Cardiomyocytes Hypertrophy

Aim: Apelin is an endogenous ligand for APJ, a member of the G protein-coupled receptors(GPCRs) family. Apelin/APJ system plays an important role in the development of cardiovascular system formation and cardiac related diseases. In our previous studies, we have found that apelin-13 could promote cardiomyocytes hypertrophy, and autophagy participated in this process. Ferritinophagy is newly discovered selective autophagy. Overactivated ferritinophagy could induce intracellular iron overload and then might participate in cardiac hypertrophy. Thus, in our study we try to investigate the effect of apelin-13 on ferritinophagy and mitochondrial iron metabolism. In addition, we try to discuss the mechanism of ferritinophagy Sideroflexin1(SFXN1)-dependent mitochondrial iron overload mediate apelin-13 induced cardiomyocyte hypertrophy. To reveal the new mechanism of apelin-13/APJ system induced cardiac hypertrophy, and provide a new experimental basis for exploiting anti- cardiovascular drugs. Methods:1. The expression of NCOA4, FTH1, SFXN1, TRC40, Tom7, Tom70, DMT1, BNP, LC3II/I in H9c2 cells were tested by Western Blot.2. Isolated mitochondria to test mitochondrial protein expression by Western Blot.3. Design siRNA interfere chains to interfere NCOA4, SFXN1, Tom7, TRC40 expression.4. The iron deposition in H9c2 cardiac cells and heart tissue were detected by Prussian blue staining.5. The free iron content was tested by the calcein-acetoxymethyl ester(Ca-AM) prob in H9c2 cells.6. The mitochondrial chelated iron production was dectected by Flow cytometry.7. The mitochondrial chelated iron was dectected by RPA red fluorescent probe in H9c2 cells.8. The mitochondrial ROS production was dectected by Mito-SOX red fluorescent probe in H9c2 cells.9. The colocalization of NCOA4 and LC3 B was detected by immunofluorescenc in H9c2 cells e.10. The diameter and volume of H9c2 cells was tested by ScepterTM Handheld cell counter.11. The concentration of proteins in H9c2 cells was tested by BCA protein quantitation kit.12. The expression of apelin, BNP, NCOA4, SFXN1 in human autopsy cardiac hypertrophy heart tissue and two-kidney two- clip reno vascular hyp ertensive rat model heart tissue was tested by immunohistochemistry. Results:1. Apelin-13 induced free chelatable iron production in H9c2 cardiomyocytes, and further enhanced the ferric citrate(FAC) promoted free chelatable iron content. Apelin-13 induced the iron deposition, and further promoted FAC induced iron deposition in H9c2 cells.2. Apelin-13 promoted FTH1 and inhibited NCOA4 expression in a concentration(0-1μmol/L) and time(0-24h) dependent manner. APJ antagonist F13A(1 μM) blocked the effect of apelin-13 on FTH1 and NCOA4 expression in H9c2 cells. Apelin-13 promoted GFP–LC3 co-localized with NCOA4 in H9c2 cells. HCQ blocked the degradation effect of apelin-13 on FTH1, and further enhanced NCOA4 accumulation in H9c2 cells. Apelin-13 further promoted FAC induced ferritinophagy in H9c2 cells.3. NCOA4-siRNA inhibited apelin-13 induced free iron production.4. Apelin-13 stimulated mitochondrial iron(0.1μmol/L, 48h), and mitochondrial ROS production(0.1μmol/L, 6h) in H9c2 cardiomyocytes. Apelin-13(0.1μmol/L, 12h) induced H9c2 cardiomyocytes mitochondria swelling and vacuolation.5. Apelin-13 promoted the expression of SFXN1 in a concentration and time dependent manner in H9c2 cardiomyocytes. APJ antagonist F13A(1 μM) blocked the effect of apelin-13 on SFXN1 expression in H9c2 cells.6. NCOA4-si RNA blocked apelin-13 induced expression of SFXN1 in H9c2 cells.7. Apelin-13 promoted mitochondrial iron and ROS production was blocked by SFXN1-siRNA in H9c2 cardiomyocytes.8. Apelin-13 promoted the expression of Tom7 and Tom70 in a concentration and time dependent manner in H9c2 cardiomyocytes. APJ antagonist F13A(1 μM) blocked the effect of apelin-13 on Tom7 and Tom70 expression in H9c2 cells. Besides, apelin-13 promoted tail-anchor protein Tom7 anchored to mitochondria.9. Tom7-siRNA blocked apelin-13 promoted SFXN1 expression.10. Apelin-13 promoted the expression of TRC40 in a concentration(0-1μmol/L) and time(0-24h) dependent manner in H9c2 cardiomyocytes. APJ antagonist F13A(1μmol/L, 24h) blocked the effect of apelin-13 induced TRC40 expression in H9c2 cells.11. TRC40-siRNA blocked apelin-13 promoted Tom7 and SFXN1 expression in H9c2 cells.12. Apelin-13 promoted iron ion transporter divalent metal transporter 1(DMT1) expression in H9c2 cells, and this effect was blocked by APJ antagonist F13 A.13. Apelin-13 increased the diameter, volume and protein content of H9c2 cells. Apelin-13 promoted BNP expression, and this effect was inhibited by F13 A.14. NCOA4-siRNA inbibited apelin-13 promoted the volume increase and BNP expression in H9c2 cells. SFXN1-siRNA inbibited apelin-13 promoted diameter and volume of H9c2 cells and BNP expression. Tom7 si RNA,TRC40-siRNA inbibited apelin-13 promoted BNP expression in H9c2 cells.15. The apelin, BNP, SFXN1, NCOA4 proteins were strongly expressed in human autopsy hypertrophic heart tissue. And iron deposition was observed in heart of human autopsy cardiac hypertrophy specimen.16. The apelin, BNP, SFXN1, NCOA4 proteins were strongly expressed in heart tissue of two-kidney two-clip renovascular hypertensive rat model. And iron deposition was observed in heart of two-kidney two-clip renovascular hypertensive rat model. Conclusion:Ferritinophagy promotes sideroflexin1-dependent mitochondria iron overload mediates apelin-13 induced cardiomyocytes hypertrophy...