探討rrbp1基因缺失對於胞內鈣離子恆定及心臟功能之機制

Abstract

簡介：從先前的SAPPHIRe研究計畫中發現，位於人類染色體20上的基因和胰島素阻抗及高血壓代謝症候群有高度相關，經莊醫師團隊進一步研究發現其與染色體20上的核餹體結合蛋白1(RRBP1)基因的單核甘酸多態性有關。目的：本篇論文旨在找出RRBP1基因對於心血管功能的影響。 材料與方法： 為了研究RRBP1對於心血管功能的影響，我們建立了一個幹細胞模型，使用了rrbp1-/-及wild type (WT)的老鼠胚胎幹細胞並分化成心肌細胞來做後續研究。Fura2AM為胞內鈣染劑，DAF為胞內一氧化氮指示劑。慢病毒載體用來使rrbp1-/-的心肌細胞表現過量的人類RRBP1。Real-time qPCR用來測量NOS的轉錄差異。 實驗結果： 我們觀察到在rrbp1-/-的心肌中其胞內鈣離子的處理和WT組有差異，包括較高的胞內鈣離子含量、較慢的肌漿網釋放和回收鈣離子能力以及肌漿網的鈣離子洩漏。而rrbp1-/-的細胞中也含有較高的一氧化氮含量，在以NOS抑制劑(L-NAME和1400W)治療後，其鈣離子處理能力都有恢復和WT一致。由Real-time qPCR 結果顯示rrbp1-/-老鼠心臟細胞中表現較多的eNOS及iNOS。KN93，一種Ca/calmodulin依賴性蛋白激酶抑制劑，僅能回復肌漿網釋放鈣離子的能力，而無法回復回收鈣離子的能力及降低胞內的一氧化氮。過量表現hRRBP1於rrbp1-/-的心肌細胞中也可以回復胞內鈣離子的處理能力。 總結： RRBP1缺失可能導致eNOS、iNOS及nNOS的表現量提高，進而製造出較多的一氧化氮，導致肌漿網上RyR及SERCR等鈣離子通道後轉譯修飾改變，造成其控制鈣離子進出之功能不正常，導致靜止期胞內鈣離子濃度增加、肌漿網釋放和回收鈣離子速度減慢、以及鈣離子由肌漿網上RyR2洩漏增加。而靜止期胞內鈣離子濃度提昇，會經由活化CAMKII，進一步導致肌漿網上RyR2釋鈣能力異常。

Introduction: From fine mapping the gene locus on chromosome 20 found in the SAPPHIRe study, multiple SNP of RRBP1 was identified to be highly associated with hypertension and insulin resistant in human. The present study aims to investigate the roles of RRBP1 in regulating cardiovascular function. Material and Method: To clarify the role of RRBP1 in cardiovascular diseases, rrbp1-/- mouse embryonic stem cell (ESC) was generated and the cardiovascular cells derived from ESC were used to identify the mechanism contributed to the different functional properties between wild type (wt) and rrbp1-/- cells. Fura2AM was used as intracellular calcium indicator, and DAF as nitric oxide indicator. Lentivirus vectors were used to deliver human RRBP1 gene (hRRBP1) into rrbp1-/- myocytes. Real-time qPCR was performed to determine transcriptional alterations of NOSs. Results: We demonstrated different calcium handling properties in rrbp1-/- myocytes, including the elevated basal calcium levels, the slowed rates of both SR calcium release and cytosolic calcium return to baseline in calcium transients of cardiac cells, and diastolic SR Ca2+ leak. We further observed that the NO level was high in rrbp1-/- cells and calcium handling properties were restored in the presence of NOSs inhibitors (L-NAME and 1400W). Real-time RT-PCR demonstrated that the transcriptional levels of eNOS, iNOS and nNOS were both higher in rrbp1-/- myocytes. KN93, a Ca/calmodulin dependent kinase (CAMKII) inhibitor, could only restore SR calcium releasing velocity, but not in calcium decay rate and intracellular NO level. Overexpression of hRRBP1 in rrbp1-/- myocytes could also restore all intracellular calcium handling properties. Conclusion: Taken together, rrbp1 deficiency could increase the expression of iNOS, eNOS and nNOS to produce more intracellular NO, which may result in the post-translational modification on cardiac RyR2 and SERCA2 to lead to the slowed rates of SR calcium release and cytosolic calcium return to basal level, the diastolic SR calcium leak as well as the increase of diastolic calcium levels. The elevated diastolic calcium level could further activate CAMKII to cause the abnormality of cardiac RyR2 in regulating calcium release.