神經鈣粘蛋白在心肌再生的角色

Abstract

心臟疾病是造成全球死亡的最主要原因之一，依據WHO的統計，心臟疾病在全球的十大死因位居首位，在台灣則是位居第二。許多的心臟疾病都有牽涉心肌細胞的死亡，只要有些微的心肌細胞損失即可能造成心臟衰竭。一般來說，成年的哺乳類的心臟缺乏再生能力，沒有辦法利用再生的方式去修補受損的心臟。然而，之前研究顯示，出生一周內的小鼠心臟與新生兒幾乎具有完全再生的能力，可以修復受損的心臟；而在出生過後，心臟再生的能力便會逐漸下降。此外，這個心臟再生的現象也會伴隨著心肌細胞的增生，是心臟再生的機制中重要的關鍵現象。不過，目前針對心肌細胞的基因表現與心臟再生的探討並不多。因此，我們便想透過分析不同心臟再生能力的小鼠的心肌細胞，去探討其基因表現的差異，和心肌細胞增生以及心臟再生的現象進行進一步的研究。 而為了探討這種在因年齡而造成的再生能力的差異其背後的原因與機制，我們利用RNA定序 (RNAseq) 分析心臟不會再生的成鼠以及心臟會再生的新生小鼠的心肌細胞，發現除了許多和細胞週期以及細胞增生相關的基因在新生小鼠的心肌細胞表現量較高之外，亦發現Cdh2，也就是可轉譯出神經鈣粘蛋白 (N-cadherin) 的基因，在新生小鼠表現量較高，且隨著年齡上升而表現量下降，在成鼠的心肌表現量甚至只有新生小鼠的1/4，因此神經鈣粘蛋白 (N-cadherin) 很可能和新生小鼠的心臟再生功能有關。 為了理解Cdh2在心臟再生的角色。首先，在新生一天的小鼠，透過心尖切除的動物模型去模擬受損的心臟後，發現Cdh2的表現量在接近受傷區域有顯著的上升，而遠離受傷區域則是表現量下降，這和術後所造成心肌細胞的增生是吻合的，因此我們認為N-cadherin的確有可能在再生中的心臟有調控的角色。另外，N-cadherin能夠影響心肌細胞的增生。在新生第一天的小鼠所分離的心肌細胞，敲低或抑制Cdh2後，可以使心肌細胞的增生顯著的下降；然而將心肌細胞過表現Cdh2則會使心肌細胞的增生比率顯著上升。再者，一些與細胞週期相關的RNA和蛋白也在敲低Cdh2後下降、過表現Cdh2後上升。此外，在健康人類的誘導型多潛能幹細胞所誘發的心肌細胞 (hiPSC-CM) 敲低CDH2，也可以觀察到心肌細胞的增生有顯著的下降、細胞週期的基因表現上升，可以說明N-cadherin不論是在小鼠或是人類的心肌細胞都能夠去影響其增生的能力。 在機轉方面，一個與N-cadherin羧基端有結合、可促進細胞增生的轉錄因子β-鏈蛋白 (β-catenin)會隨著Cdh2的表現量而受到顯著的影響，不論是在新生小鼠的心肌細胞或是人類hiPSC-CM，β-catenin蛋白表現量會隨著N-cadherin表現上升而上升，N-cadherin表現下降而下降，然而β-catenin的信使核糖核酸 (mRNA) 的表現並沒有差異。因此，N-cadherin調控β-catenin應是透過轉錄後的機制進行調控。而在敲低 Cdh2後，β-catenin的蛋白穩定性顯著的下降，且進入核中能夠進行轉譯的β-catenin的比例下降，都驗證了此假說。此外，增加Cdh2所造成的心肌細胞的增生也可以利用β-catenin的抑制劑使其反轉，更強化了N-cadherin所調控的心肌細胞的增生，很可能就是透過β-catenin的訊息傳遞路徑所調控的假說。 在動物模型中，N-cadherin抑制劑會使新生小鼠的心臟經過受損後細胞增生的量下降；在增加Cdh2的表現後，能夠使得成年老鼠在經過心肌梗塞手術後，心臟功能惡化的速率有所減緩，這些都代表N-cadherin的確會影響在動物中的心肌細胞的增生與心臟再生。 綜合以上結果，N-cadherin可能會透過影響和它連接的β-catenin的穩定度，使得β-catenin較容易進行轉錄促進心肌細胞的增生，進一步去促進心臟的再生、心功能的修復，並能夠作為心臟疾病治療的新型策略。

Cardiovascular disease is a leading cause of death. Many cardiovascular diseases are associated with the loss of cardiomyocytes. A modest loss of cardiomyocytes could lead to heart failure and high mortality. Although the adult mammalian heart fails to regenerate after injury, it is known that newborn mice within a week have a full cardiac regenerative capacity. Besides, cardiomyocyte proliferation was accompanied by the regenerative process. The molecular determinants underlying the disparate regenerative capacity between neonatal and adult mice, however, remain incompletely understood. Although several studies showed there was some transcriptome difference during the regenerative process, the transcriptome change specifically in the cardiomyocyte was incompletely understood. Therefore, this study aimed to identify potential regulators of cardiac regeneration in the cardiomyocyte and find out their molecular mechanism. Exploiting RNA sequencing in isolated cardiomyocytes from neonatal and adult mouse heart, we identified Cdh2, which encodes the adherence junction protein N-cadherin, as a potential novel mediator of cardiac regeneration. Cdh2 expression levels were much higher in neonatal, compared with adult, cardiomyocytes and showed a strong positive correlation with that of multiple cell cycle genes. N-cadherin has been reported to be essential for embryonic cardiac development; its role in cardiac regeneration, however, remains unknown. Therefore, we attempted to determine the novel role of Cdh2 /N-cadherin in cardiac regeneration and to investigate the underlying molecular mechanisms. Comparing to sham-operated control, Cdh2 was significantly upregulated in mouse cardiac apex and border zone following apical resection on postnatal day 1 (P1) mice, which was accompanied with increased cardiomyocyte proliferation activity. In vitro, knocking down Cdh2 or inhibition of N-cadherin activity with N-cadherin inhibitor, exherin, in postnatal day 1 neonatal mouse cardiomyocytes (P1CM) significantly reduced the proliferative activity of cardiomyocytes, whereas overexpression of Cdh2 markedly increased the proliferation of P1CM. In addition, forced expression of Cdh2 resulted in significant upregulation of multiple cell cycle genes, including Ccnd1 (cyclin D1), Cdc16 (cell division cycle protein 16), and Cdk2 (cyclin-dependent kinase 2) in P1CM, while depleting expression of Cdh2 downregulated these genes. Knocking down CDH2 in human induced pluripotent stem cells-derived cardiomyocytes (iPSC-CMs) significantly reduced the proliferative activity and the expression levels of cell cycle gene CCND1 in iPSC-CMs. Mechanistically, we demonstrated that the pro-mitotic effects of N-cadherin in cardiomyocytes were mediated, at least partially, by stabilizing β-catenin, a pro-mitotic transcription factor, through direct interaction with its cytoplasmic domain and/or inactivation of GSK-β, a critical component of β-catenin destruction complex. In vivo inhibition of N-cadherin in P1 neonatal mice with exherin following apical resection led to the reduction of global cardiomyocyte proliferation, compared with PBS treated group. In addition, overexpressing Cdh2 attenuated the deterioration of cardiac function in the adult mice following myocardial infarction. Our study uncovered a previously unrecognized role of Cdh2 (N-cadherin) in cardiomyocyte proliferation and cardiac regeneration. The results presented demonstrate that increased N-Cadherin levels promotes cardiomyocyte proliferation and regeneration by post-translational stabilization and upregulation of pro-mitotic transcription factor -catenin. Enhancing cardiac expression or activity of N-cadherin, therefore, could be a potential novel therapeutic approach to promote cardiac regeneration and restore cardiac function in adult heart following injury.