利用人類胚胎幹細胞分化的心肌細胞研究由RBCK1缺陷所引發的心肌病變之分子機制

葡糖多聚醣體肌肉病變症(polyglucosan body myopathy, PGBM)是一種肝糖代謝異常所引起的罕見疾病，在骨骼肌與心肌病理切片中可發現有大量的葡萄多聚醣體(PGBs)堆積，此異常堆積體是由不正常結構的肝醣所聚合而成且其不能被α-澱粉酶(α-Amylase)酵素分解。病人會患有早發性的肌肉無力與擴張性心肌病變(dilated cardiomyopathy)。研究發現RBCK1的雙等位基因(biallelic)突變可為引發此肌肉病變症的主因之一。RBCK1的功能主要有二，第一它能進入細胞核做為轉錄因子調控下游基因，二其帶有RING-between-RING domain可被當作E3泛素連接酶 (E3 ligase)來泛素化受體使其被降解。然而這兩種截然不同的功能使得在研究RBCK1變異所引起的疾病上增加了難度，再加上是罕見疾病的原因，病人的樣本難以取得，使其致病機制之研究更加的困難。為了解決此一問題，本研究利乃用人類胚胎幹細胞作為研究模型，以CRISPR/Cas9的技術重現RBCK1變異病人的基因型。初步結果顯示，RBCK1缺陷的胚胎幹細胞能成功分化成心肌細胞且利用Periodic Acid-Schiff diastase染色(PAS-D)及穿透式電子顯微鏡來證實葡萄多聚醣堆積在RBCK1缺陷的心肌細胞。此外，心肌細胞雖然未影響肌節(sarcomere)結構，但在分化晚期的心肌細胞大小有變大趨勢，且在心肌病變指標基因NPPA、NPPB與TGFβ之表現也有明顯上升。除了在這個疾病模型上顯示出如病人心肌病的病徵外，本研究也首次發現RBCK1缺陷會明顯地影響到心肌細胞內鈣離子的調控與粒線體行有氧呼吸的能力(mitochondrial oxidative capacity)。而在機制的分析上，我發現了RBCK1缺陷並不會影響肝醣生成(glycogenesis)與肝糖降解(glycogen degradation)的相關酵素，藉此縮小葡萄多聚醣體堆積在後續研究可朝肝醣代謝的其他蛋白深入。綜合以上結果，讓我們能更全面了解RBCK1缺陷所造成的諸多心肌功能缺失，提供了治療此心肌病變的新方向，並且在未來能利用此疾病模型進一步的分析RBCK1在肝醣代謝中所扮演的角色。

關鍵字

葡糖多聚醣體肌肉病變症；擴張性心肌病； RBCK1基因；人類胚胎幹細胞；疾病模型；分化心肌細胞；鈣離子調控；肝糖代謝

並列摘要

Polyglucosan body myopathy (PGBM), a glycogen metabolism disorder, is characterized by accumulation of polyglucosan bodies in both skeletal and cardiac muscle. The polyglucosan body comprised of glycogen with abnormal configurations resists to α-amylase digestion. Patients with this disease displayed early-onset of muscle weakness and rapidly progressive dilated cardiomyopathy. Previous studies have shown that biallelic loss-of-function mutation in RBCC protein interacting with PKC1 (RBCK1) gene results in PGBM. RBCK1 has been suggested to carry out two cellular functions 1) as a transcription factor mediated by C-terminal of RBCK1, and 2) as an E3 ligase facilitated by the RING-between-RING domain. However, the molecular detail of how RBCK1 caused polyglucosan accumulation, leading to cardiomyopathy, is still unknown. PGBM is a rare disease so it is difficult to collect samples from patients for pathological mechanism study. Alternatively, I ablated RBCK1 in human embryonic stem cells (hESCs) by CRISPR/Cas9 to examine the effects of mutated RBCK1. In preliminary data, I found that RBCK1-deficient hESCs were able to differentiate into cardiomyocytes (CMs) with accumulation of polyglucosan bodies as revealed by both Periodic Acid-Schiff diastase (PAS-D) staining and transmission electron microscopy (TEM). Although the RBCKex5/ex5 KO hESC-derived CMs displayed well-organized sarcomere structure, at differentiation day 50 these RBCK1 knockout cells displayed both dilated and hypertrophic cardiomyopathy-like morphology as indicated by larger cell size, and the high expression of cardiomyopathy markers, NPPA, NPPB, and TGFβ. In addition, it was the first to discovered that RBCK1 deficiency caused severe aberrancy in the calcium handling and abnormal mitochondrial oxidative capacity. Moreover, the effects of RBCK1 deficiency were unaffected glycogenesis and glycogen degradation-related enzymes. Taken together I successfully established a cell-based disease model for pathological mechanism study that can be used for developing intervention strategies against RBCK1 deficiency-induced cardiomyopathy.

並列關鍵字

Polyglucosan Body Myopathy ； Dilated Cardiomyopathy ； RBCK1 Gene ； Human Embryonic Stem Cell ； Disease Model ； Differentiated Cardiomyocyte ； Calcium Handling ； Glycogen Metabolism

參考文獻

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利用人類胚胎幹細胞分化的心肌細胞研究由RBCK1缺陷所引發的心肌病變之分子機制

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