Effects And Mechanism Of RGS5 On Cardiomyocyte Apoptosis During Cardiac Ischemia-Reperfusion In Mice

Backgeound Ischemia-reperfusion (IR) injury plays a significant part in coronary artery disease and cardiac transplantation, and has been shown to lead to cardiomyocyte apoptosis. Apoptotic cell death plays a pivotal part in myocardial IR injury. It is generally known that signaling pathways such as c-Jun NH^-terminal kinase 1/2 (JNK1/2) and p38 promote cardiac IR-induced apoptosis, and these signaling pathways are activated by stimulating G protein-coupled receptors (GPCRs) which are regulated by both G(q) and G(i/o) proteins under control of the regulator of G-protein signaling (RGS) proteins. Regulator of G-protein signaling 5 (RGS5), a negative regulator of G protein-mediated signaling, plays a role in embryonic development, wound healing and reproductive cycle maintenance. RGS5 is highly expressed in different cells of murine and human adult heart, and actively involves in angiogenesis, vascular pericyte maturation and myocardium repolarization, through which it involves in the pathogenesis of hypertension, atherosclerosis, arrhythmia and heart failure. However its function of RGS5 in cardiac IR-induced apoptosis remains unclear. This study aims to clarify the effect and mechanism of RGS 5 on cardiomyocyte apoptosis during cardiac IR in mice.Part one Effect of RGS5 on cardiomyocyte apoptosis during in vivo cardiac IR in miceObjective This study was undertaken to discover the function of RGS5 in cardiomyocyte apoptosis during in vivo cardiac IR in mice.Methods An in vivo cardiac I/R (30min/24h) model was examined in cardiac-specific human RGS 5 expressing transgenic mice (TG), RGS 5-knockout mice (KO), and wild type mice (WT). After experiments, the left ventricles of all hearts were examined by hemodynamic measurements, pathological and molecular assays.Results Our results showed that during in vivo cardiac IR the TG mice exhibited a remarkable inhibition of cardiomyocyte apoptosis compared with WT or KO mice, as evidenced by lower TUNEL staining levels, less expression levels of Bax and higher expression levels of Bcl-2 in RGS5 TG mice. Furthermore, in comparison with WT mice after IR, the TG mice displayed a significant alleviation of abnormal cardiac function, as determined by greater reduction of the left ventricular end-diastolic pressure (LVEDP), but a more significant increase of hemodynamic parameters including left ventricular end-systolic pressure (LVESP), the difference of LVESP and LVEDP (?LVP), and the maximal rate of increase and decrease of left ventricular pressure (±LVdp/dtmax).Conclusion This study confirms that RGS5 inhibits cardiomyocyte apoptosis during in vivo cardiac IR in mice.Part two Effect of RGS5 on cardiomyocyte apoptosis during in vitro cardiac IR in miceObjective This study was undertaken to investigate the effect of RGS5 on cardiomyocyte apoptosis during in vitro cardiac IR in mice.Methods A Langendorff perfusion I/R (15/30 min) model was applied to the isolated hearts of cardiac-specific human RGS5 expressing transgenic mice (TG), RGS5-knockout mice (KO), and wild type mice (WT). After experiments, the left ventricles of all hearts were evaluated by pathological and molecular analysis, and hemodynamic detection.Results Our results revealed that in comparison with WT or KO mice after IR, the TG mice displayed a significant alleviation of abnormal cardiac functions, as determined by a more significant increase of hemodynamic parameters including +LVdp/dtmax, - LVdp/dtmax, LVESP, and ALVP, but greater reduction of the LVEDP. We also discovered that in contrast to WT or KO mice, the TG mice inhibited IR-induced cardiomyocyte apoptosis, as indicated by a more obvious reduction in both TUNEL and Bax-positive cardiomyocytes with an increase in Bcl-2 positive cells.Conclusion The study shows that RGS5 protects cardiomyocytes against apoptosis during in vitro cardiac IR in mice.Part three Effect of inhibition of both p38 and JNK1/2 signaling pathways on cardiomyocyte apoptosis during in vitro cardiac ischemia-reperfusion in RGS5-knockout miceObjective This study aimed to discover the effect of inhibition of both p38 and JNK1/2 signaling pathways on cardiomyocyte apoptosis during in vitro cardiac ischemia-reperfusion in RGS5-knockout mice.Methods A Langendorff perfusion I/R (15/30 min) model was applied to the isolated hearts of wild type mice (WT), cardiac-specific human RGS5 expressing transgenic mice (TG), and RGS5-knockout mice (KO). And inhibitors of both p38 and JNK1/2 signaling pathways were used to RGS5-KO mice. After experiments, the left ventricles of all hearts were detected by pathological and molecular analysis, and hemodynamic detection. Western blotting analysis was used to detect the extent of protein expression of p38 MAP kinase and JNK1/2. Cardiomyocyte apoptosis was evaluated by pathological and molecular analyses, and hemodynamic analysis.Results We found that the extent of phosphorylation of both JNK1/2 and p38 was reduced in IR-induced TG hearts compared with IR-induced WT or KO hearts. We also found that in line with the observation of nuclear atrophy or chromatin condensation, TUNEL staining and pro-apoptotic markers such as Bax were more distinctly blocked, and yet the extent of expression of anti-apoptotic markers such as Bcl-2 was enhanced with SP600125 (a JNK inhibitor) and SB203580 (a p38 MAPK inhibitor) treatment than with sodium chloride solution (0.9% NaCl, NS) treatment in RGS5-KO mice in response to IR. Furthermore, in comparison with the NS-perfusion group in RGS5-KO mice, hemodynamic markers after SP600125 and SB203580-perfusion in the IR group such as+LVdp/dtmax,-LVdp/dtmax, LVESP, and ALVP, were significantly improved, but LVEDP was reduced.Conclusion These studies show that RGS5 protects cardiomyocytes against apoptosis during cardiac IR in mice through inhibition of both JNK1/2 and p38 signaling pathways. These findings may lead to improved approaches for the treatment of cardiac IR-induced apoptosis by targeting signal transduction pathways related to RGS5.