Extracellular Cyclophilin A Induces Cardiomyocyte Hypertrophy Via The ERK/p47phox Pathway

ObjectiveThis project will study the role of extracellular Cyclophilin A in myocardial cell hypertrophy and its potential mechanism from animal models and in vitro cell experiments.MethodsSD rats were raised and multi-point subcutaneous injected of isoproterenol to construct a pathological myocardial hypertrophy model.The myocardial hypertrophy marker genes were detected by fluorescent quantitative PCR,HE staining and Masson staining were used to assess cardiomyocyte size and fibrosis.Dihydroethidium(DHE)staining was used to observe the production of reactive oxygen species(ROS)in myocardial tissue.The expression of Cyclophilin A(CyPA)secreted into the serum was detected by ELISA.Western blot was used to detect the expression of CyPA andβ-actin in myocardial tissue.Immunohistochemistry was used to further clarify the expression and localization of CyPA in myocardial tissue.H9c2 cells were cultured in vitro and stimulated with recombinant CyPA.Myocardial hypertrophy marker genes were detected by RT-PCR,and cell surface area was measured after phalloidin staining.DHE staining and electron spin resonance spectroscopy(ESR)were used to observe ROS production in H9c2 cells.The levels of oxidative stress were observed after applying ROS inhibitors N-acetylcysteine(NAC)and nicotinamide adenine dinucleotide phosphate(NADPH)oxidase inhibitors.The protein expression levels of p-ERK1/2,p-p38,p-JNK,ERK1/2,p38,JNK,p47phox,Nox1,Nox2,Nox4,EMMPRIN,β-actin,Na~+/K~+ATPase were detected by Western blot.The confocal microscope was used to observe the location of p47phox on H9c2 cell membrane.ERK,p38,and JNK inhibitors were used to inhibit the phosphorylation of ERK,p38,and JNK,respectively,and then the indicators of cardiac hypertrophy were detected.Finally,extracellular matrix metalloproteinase inducer(EMMPRIN)small interfering RNA was used to silence EMMPRIN expression,and extracellular CyPA-mediated cardiomyocyte hypertrophy,ROS levels,and activation of MAPK/p47phox signaling pathways were detected.Results(1)Myocardial hypertrophy marker genes,cardiomyocyte size,and myocardial interstitial fibrosis increased significantly in myocardial tissue of the isoprenaline-injected group(p<0.05),and ROS levels increased significantly(p<0.05).CyPA Secretion increased,and CyPA expression increased in myocardial tissue,mainly distributed in the interstitial fibrosis around hypertrophic cardiomyocytes,rather than the cardiomyocytes themselves(p<0.05).(2)Exogenous administration of CyPA stimulated cardiomyocyte hypertrophy and oxidative stress indicators in H9c2 cells(p<0.05);both NAC(a non-specific ROS inhibitor)and VAS2870(a specific NADPH oxidase inhibitor)significantly blocked the extracellular CyPA-mediated ROS production(p<0.05);the expression of p47phox(cytoplasmic subunit of NADPH oxidase)in the total protein of H9c2 cells after CyPA stimulation did not change significantly(p>0.05),but the expression of p47phox in membrane proteins increased in a time-dependent manner under the effect of extracellular CyPA(p<0.05); VAS2870 could significantly interfere with extracellular CyPA-mediated cardiomyocyte hypertrophy(p<0.05).(3)ERK1/2,p38,and JNK were activated in the CyPA-stimulated group(p<0.05); pretreatment with PD98059(ERK1/2 inhibitor)could significantly block extracellular CyPA-mediated cardiac hypertrophy(p<0.05),while BIRB 796 (p38 inhibitor)and SP600125(JNK inhibitor)had no significant intervention(p>0.05).(4)With the intervention of PD98059,extracellular CyPA-mediated ROS production and p47phox membrane translocation were significantly inhibited(p<0.05);at the same time,the protein expression levels of Nox1,Nox2,and Nox4(the membrane-bound subunits of NADPH oxidase mainly expressed in cardiac myocytes)did not change significantly(p>0.05).(5)Exogenous administration of CyPA did not up-regulate the expression of EMMPRIN protein in H9c2 cells(p>0.05);however,after transfection of small interfering RNA to silence EMMPRIN expression,extracellular CyPA-mediated cardiomyocyte hypertrophy,ROS production,and MAPK/p47phox activation were significantly inhibited(p<0.05).Conclusions(1)Extracellular CyPA is up-regulated in isoprenaline-induced pathological myocardial hypertrophy rats.(2)Extracellular CyPA mediates H9c2 cardiomyocyte hypertrophy by activating NADPH oxidase.(3)ERK signaling pathway is involved in the regulation of extracellular CyPA-induced H9c2 cardiomyocyte hypertrophy.(4)Extracellular CyPA activates NADPH oxidase through the ERK/p47 phox signaling pathway.(5)EMMPRIN is required for extracellular CyPA-mediated cardiomyocyte hypertrophy,oxidative stress,and activation of MAPK/p47phox signaling pathways.