RESUMO
Statins have beneficial pleiotropic effects beyond lipid lowering on the cardiovascular system. These cardio-protective effects are mediated through inhibition of the intracellular mevalonate pathway, by decreasing isoprenoid intermediate synthesis and the subsequent post-translational modification of small GTPases, such as Ras, Rho, and Rac. Impaired intracellular calcium handling is considered an important pathophysiologic mechanism responsible for cardiac dysfunction. Our study aimed at investigating the influence of mevalonate pathway, including its downstream small GTPases (Ras, RhoA, and Rac1) on anoxia-mediated alterations of calcium handling in H9c2 cardiomyocytes. Cultured H9c2 cardiomyocytes were exposed to acute anoxia after pretreatment with different drugs that specifically antagonize five key components in the mevalonate pathway, including 3-hydroxy-3-methylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, Rho-kinase, Rac1 and Ras farnesyltransferase. Thereafter, we evaluated the effects of the mevalonate pathway on anoxia-induced cell death, expression of the sarcoplasmic reticulum calcium release channel (ryanodine receptor 2) and its regulator FK506-binding protein 12.6, as well as functional calcium release from intracellular calcium stores. Our experiments confirmed the role of prenylated proteins in regulating cardiomyocyte dysfunction, especially via RhoA- and Ras-related signaling pathways. Furthermore, our data demonstrated that inhibition of the mevalonate pathway could ameliorate anoxia-mediated calcium handling dysfunction with the up-regulated expression of FK506-binding protein 12.6 and consequently provided evidence for FK506-binding protein 12.6 as a "stabilizer" of ryanodine receptor 2.
Assuntos
Cálcio/metabolismo , Hipóxia/metabolismo , Redes e Vias Metabólicas/efeitos dos fármacos , Ácido Mevalônico/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Animais , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Regulação da Expressão Gênica/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Hipóxia/genética , Espaço Intracelular/metabolismo , Mioblastos Cardíacos/efeitos dos fármacos , Mioblastos Cardíacos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Ratos , Proteínas de Ligação a Tacrolimo/genéticaRESUMO
Thymosinß 4 (Tß4) has been reported to exert a proangogenic effect on endothelial cells. However, little is known on the role and underlying mechanisms of Tß4 on critical limb ischemia (CLI). The present study aimed therefore to investigate the mechanisms and proangiogenic effects of Tß4 in CLI mice. Tß4 overexpression lentiviral vector was first transfected into HUVEC and CLI mice model, and inhibitors of Notch pathway (DAPT) and NFκB pathway (BMS) were also applied to HUVEC and CLI mice. Subsequently, MTT, tube formation and wound healing assays were used to determine the cell viability, angiogenesis and migratory ablity of HUVEC, respectively. Western blotting, reverse transcription, quantitative PCR, immunofluorescence and immunohistochemistry were used to detect the expression of the angiogenesisrelated factors angiopoietin2 (Ang2), TEK receptor tyrosine kinase 2 (tie2), vascular endothelial growth factor A (VEGFA), CD31 and αsmooth muscle actin (αSMA) and the Notch/NFκB pathwaysrelated factors NOTCH1 intracellular domain (N1ICD), Notch receptor 3 (Notch3), NFκB and p65 in HUVEC or CLI mice muscle tissues. The results demonstrated that Tß4 not only enhanced the cell viability, angiogenesis and migratory ability of HUVEC but also promoted the expression of Ang2, tie2, VEGFA, N1ICD, Notch3, NFκB, and phosphorylated (p)p65 in HUVEC. In addition, Tß4 promoted the expression of CD31, αSMA Ang2, tie2, VEGFA, N1ICD and pp65 in CLI mice muscle tissues. Treatment with DAPT and BMS had opposite effects of Tß4, whereas Tß4 reversed the effect of DAPT and BMS. The findings from the present study suggested that Tß4 may promote angiogenesis in CLI mice via regulation of Notch/NFκB pathways.
Assuntos
Isquemia/metabolismo , NF-kappa B/metabolismo , Neovascularização Patológica/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Timosina/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Isquemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculos/metabolismo , Neovascularização Patológica/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
AIM: We previously demonstrated that anoxia-mediated Ca2+ handling dysfunction could be ameliorated through inhibition of mevalonate pathway via RhoA- and Ras-related mechanisms in H9c2 cells. In this study, we further explored whether inhibition of mevalonate pathway is associated with cardiac remodeling and dysfunction in ischemic cardiomyopathy, and discussed the possible role of Ras, Rac and RhoA in cardiac dysfunction. METHODS: We investigated the role of mevalonate pathway in cardiac remodeling and cardiomyocyte Ca2+ handling proteins expression in a rat model of cardiac dysfunction due to myocardial infarction (MI). After MI, adult male Sprague-Dawley rats were treated with drugs that antagonize key components in mevalonate pathway, including 3-hydroxy-3-methylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, and Rho-kinase for 10 weeks. The protein expression of ryanodine receptor 2 (RyR2), sarcoplasmic reticulum Ca2+ ATPase (SERCA) 2a, phospholamban (PLB), phospho-PLB at serine-16 (PSer16-PLB), FKBP12.6, and RhoA as well as RyR2 and FKBP12.6 mRNA levels was evaluated. RESULTS: Rosuvastatin and alendronate treatment prevented myocardial remodeling, improved cardiac function and reduced infarct size. Furthermore, rosuvastatin and alendronate promoted an increase in the protein expression of SERCA2a and PSer16-PLB/PLB ratio as well as partially restored the RyR2 and FKBP12.6 gene and protein expression. Fasudil failed to exert these beneficial effects. CONCLUSIONS: These findings indicate that mevalonate pathway inhibition by rosuvastatin and alendronate prevents cardiac remodeling and dysfunction possibly through RhoA-independent mechanisms.