Parra Sergio, Huang Xinyan, Charbeneau Raelene A, Wade Susan M, Kaur Kuljeet, Rorabaugh Boyd R, Neubig Richard R
Department of Pharmaceutical and Biomedical Sciences, Ohio Northern University College of Pharmacy, Ada, OH 45810, USA.
BMC Pharmacol Toxicol. 2014 Jun 5;15:29. doi: 10.1186/2050-6511-15-29.
Regulator of G protein signaling (RGS) proteins suppress G protein coupled receptor signaling by catalyzing the hydrolysis of Gα-bound guanine nucleotide triphosphate. Transgenic mice in which RGS-mediated regulation of Gαi2 is lost (RGS insensitive Gαi2G184S) exhibit beneficial (protection against ischemic injury) and detrimental (enhanced fibrosis) cardiac phenotypes. This mouse model has revealed the physiological significance of RGS/Gαi2 interactions. Previous studies of the Gαi2G184S mutation used mice that express this mutant protein throughout their lives. Thus, it is unclear whether these phenotypes result from chronic or acute Gαi2G184S expression. We addressed this issue by developing mice that conditionally express Gαi2G184S.
Mice that conditionally express RGS insensitive Gαi2G184S were generated using a floxed minigene strategy. Conditional expression of Gαi2G184S was characterized by reverse transcription polymerase chain reaction and by enhancement of agonist-induced inhibition of cAMP production in isolated cardiac fibroblasts. The impact of conditional RGS insensitive Gαi2G184S expression on ischemic injury was assessed by measuring contractile recovery and infarct sizes in isolated hearts subjected to 30 min ischemia and 2 hours reperfusion.
We demonstrate tamoxifen-dependent expression of Gαi2G184S, enhanced inhibition of cAMP production, and cardioprotection from ischemic injury in hearts conditionally expressing Gαi2G184S. Thus the cardioprotective phenotype previously reported in mice expressing Gαi2G184S does not require embryonic or chronic Gαi2G184S expression. Rather, cardioprotection occurs following acute (days rather than months) expression of Gαi2G184S.
These data suggest that RGS proteins might provide new therapeutic targets to protect the heart from ischemic injury. We anticipate that this model will be valuable for understanding the time course (chronic versus acute) and mechanisms of other phenotypic changes that occur following disruption of interactions between Gαi2 and RGS proteins.
G蛋白信号调节（RGS）蛋白通过催化与Gα结合的鸟嘌呤核苷酸三磷酸的水解来抑制G蛋白偶联受体信号传导。RGS介导的Gαi2调节功能丧失的转基因小鼠（RGS不敏感的Gαi2G184S）表现出有益的（对缺血性损伤的保护作用）和有害的（纤维化增强）心脏表型。该小鼠模型揭示了RGS/Gαi2相互作用的生理意义。先前对Gαi2G184S突变的研究使用的小鼠在其一生中都表达这种突变蛋白。因此，尚不清楚这些表型是由慢性还是急性Gαi2G184S表达引起的。我们通过培育条件性表达Gαi2G184S的小鼠来解决这个问题。
使用携带loxP位点的小基因策略培育条件性表达RGS不敏感的Gαi2G184S的小鼠。通过逆转录聚合酶链反应以及增强激动剂诱导的对分离的心脏成纤维细胞中环磷酸腺苷（cAMP）产生的抑制作用来表征Gαi2G184S的条件性表达。通过测量在经历30分钟缺血和2小时再灌注的离体心脏中的收缩恢复和梗死面积，评估条件性RGS不敏感的Gαi2G184S表达对缺血性损伤的影响。
我们证明了他莫昔芬依赖性的Gαi2G184S表达、对cAMP产生的抑制增强以及在条件性表达Gαi2G184S的心脏中对缺血性损伤的心脏保护作用。因此，先前在表达Gαi2G184S的小鼠中报道的心脏保护表型不需要胚胎期或慢性Gαi2G184S表达。相反，心脏保护作用在Gαi2G184S急性（数天而非数月）表达后出现。
这些数据表明RGS蛋白可能为保护心脏免受缺血性损伤提供新的治疗靶点。我们预计该模型对于理解Gαi2与RGS蛋白相互作用破坏后发生的其他表型变化的时间进程（慢性与急性）和机制将具有重要价值。
