| Objective:Cardiac hypertrophy is a complex pathophysiological process, which interferes with a large number of factors and signaling pathways and results in high incidence of adverse cardiovascular events including heart failure and sudden death. However, the molecular mechanism underlying the development of cardiac hypertrophy is largely unknown. The involvement of several RGS families in the pathophysiological process of cardiac hypertrophy is well documented. RGS 12 is the largest, typical multi-domain member of the RGS family. This multi-domain architecture contributes to the regulatory role of RGS 12 in diverse signaling pathways. The present study aims to elucidate the effect of RGS 12 on cardiac hypertrophy and its associated mechanism.Methods:Section one: The WT mice and neonatal rat cardiomyocytes (NRCMs) from Sprague-Dawley rat were used in this section. The aortic binding surgery or Ang II administration was performed to established in vivo or in vitro hypertrophied model,respectively. Subsequently, the western blots assays were performed to determine the protein levels of RGS 12 in myocardium, cardiomyocytes and fibroblast at different time points.Section two: The RGS12-KO and WT mice were used in this section. The aortic binding surgery was performed to established in vivo hypertrophied model. After 4 weeks of aortic binding surgery, histological analyses were performed to determine the HW/BW, HW/TL and LW/BW; the H&E, WGA and PSR stainings were performed to calculate the cardiomyocyte cross area and the extent of collagen deposition; the echocardiography measurements were performed to evaluate cardiac function in mice; the real-time PCR were performed to assess the mRNA levels of hypertrophic and fibrotic markers.Section three: The RGS12-CTG and CRMC mice were used in this section. The aortic binding surgery was performed to established in vivo hypertrophied model.After 4 weeks of aortic binding surgery, histological analyses were performed to determine the HW/BW, HW/TL and LW/BW; the H&E, WGA and PSR stainings were performed to calculate the cardiomyocyte cross area and the extent of collagen deposition; the echocardiography measurements were performed to evaluate cardiac function in mice; the real-time PCR were performed to assess the mRNA levels of hypertrophic and fibrotic markers.Section four: The AdshRGS12 or AdRGS12 adenoviruses were used to knockdown or up-regulate RGS12 expression in NRCMs, respectively. NCRMs infected with AdshRNA and AdGFP were used as controls. The in vitro hypertrophied model was established through Ang Ⅱ administration. After challenge with Ang II or PBS for 48 hours, NRCMs were immunostained with a-actinin to measure the cell surface area. Moreover, the real-time PCR were performed to assess the mRNA levels of hypertrophic markers.Section five: In both in vivo and in vitro experiments, western blots assays were performed to measure the protein levels of phosphorylated and total MAPK signaling molecules in each group. To identify whether RGS12 directly regulate MEKI/2,isolated NRCMs treated with PBS or Ang Ⅱ, are used to perform co-immunoprecipitation experiments. In the rescue experiments, RGS12-CTG and CRMC mice were injected with the MEK1/2-specifc inhibitor U0126 for 4 weeks after aortic binding surgery. After 4 weeks of aortic binding surgery, histological analyses were performed to determine the HW/BW, HW/TL and LW/BW; the H&E,WGA and PSR stainings were performed to calculate the cardiomyocyte cross area and the extent of collagen deposition; the echocardiography measurements were performed to evaluate cardiac function in mice.Results:Section one: The elevated protein levels of cardiac hypertrophic markers were observed in both in vivo cardiac hypertrophy and in vitro cardiomyocyte enlargement models. Compared with the sham operation,RGS12 expression was up-regulated in pressure overload-induced hypertrophied mouse hearts. Moreover, the increase in RGS12 expression was more significant in mouse hearts subjected to 8 weeks of aortic binding compared with that in mouse hearts after 4 weeks of aortic binding.Consistent with in vivo data, RGS12 expression increased progressively in cultured NRCMs treated with Ang Ⅱ for 24 and 48 hours, compared with the PBS-treated controls. Nevertheless, Ang Ⅱ treatment have no impact on RGS12 expression in fibroblast.Section two: In response to sham operation, there is no difference in extent of cardiac hypertrophy, fibrosis dysfunction and associated gene expression between WT and RGS12-KO mice. After 4 weeks of aortic binding, the HW/BW, HW/TL and LW/BW in RGS12-KO mice were less than those in WT mice. In addition, the heart size, cardiomyocyte cross area and cardiac fibrosis in RGS12-KO mice were lower than those in WT mice. Moreover, left ventricular expansion and cardiac dysfunction were alleviated by RGS12-KO compared with the control group. Furthermore,compared with WT group, PCR assays revealed that RGS12 deficiency preserved the pressure overload-induced elevation in mRNA levels of cardiac hypertrophic markers.Section three: Upon sham operation, the extent of cardiac hypertrophy, fibrosis dysfunction and associated gene expression was comparable between CRMC and RGS12-CTG mice. 4 weeks after aortic binding, the HW/BW, HW/TL and LW/BW in RGS12-CTG mice were higher than those in CRMC mice. In addition, the heart size,cardiomyocyte cross area and cardiac fibrosis in RGS12-CTG mice were more pronounced than those in WT mice. Moreover, left ventricular expansion and cardiac dysfunction were exaggerated by RGS12 overexpression compared with the CRMC controls. Furthermore, compared with CRMC group, PCR assays revealed that RGS12 overexpression accelerated the pressure overload-induced elevation in mRNA levels of cardiac hypertrophic markers.Section four: In response to PBS administration, the cardiomyocyte surface area and associated gene expression was comparable between AdshRGS12, AdshRNA,AdRGS12 and AdGFP group. 48 hour after Ang II treatment, the cardiomyocyte surface area, determined by immunofluorescence staining, was decreased in AdshRGS12 group but increased in AdRGS12 group compared with their control groups, respectively. Additionally, compared with their respective controls, the Ang II-induced up-regulation of cardiac hypertrophic markers were alleviated by AdshRGS 12 but exaggerated by AdRGS 12.Section five: The total expression of MAPK signaling molecules was not altered by pro-hypertrophic stresses, whereas the phosphorylated levels of MAPK signaling molecules was elevated in hypertrophied models. Among them, neither the absence nor the overexpression of RGS12 altered the stresses-induced phosphorylation of JNKI/2 and P38. In contrast, the phosphorylated levels of MEK1/2 and ERK1/2 was further elevated by RGS12 overexpression but alleviated by RGS12 deficiency.Meanwhile, our experiments demonstrated that RGS12 could interact with MEK1/2 weakly at baseline, but this interaction was significantly enhanced by Ang II administration. U0126 offset the hypertrophic growth and subsequent adverse remodeling in both CRMC and RGS12-CTG hearts, as evidenced by: (1) decrease in the HW/BW, HW/TL and LW/BW; (2) alleviated hypertrophic growth, cardiomyocyte enlargement and collagen deposition; (3) improved left ventricular expansion and cardiac dysfunction. More importantly, these indices of pathological hypertrophic response were comparable between U0126-treated CRMC and U0126-treated RGS12-CTG hearts.Conclusion:RGS12 expression is increased in the development of pathological cardiac hypertrophy. The up-regulated RGS12 contributes to pathological cardiac hypertrophy by binding to and accelerating MEK1/2-ERK1/2 signaling. Therefore, RGS12 may represent a potential therapeutic target for pathological cardiac hypertrophy and heart failure. |
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