Myogenin與Cyclin A2可調控心肌細胞之分化與增殖

Abstract

心肌細胞一直被認為是分化完全且不再進行分裂的細胞，但近來有研究指出：成熟心肌細胞仍然具有增殖的能力，只是由於細胞週期調控蛋白如cyclin A2表現大幅減少，導致具有增殖活性的心肌細胞比例極小。在C2C12骨骼肌細胞實驗中已被證實透過肌細胞生成素(myogenin)表達使得肌肉細胞分化時會抑制其增殖活性，但分化路徑是否同樣影響心肌細胞分裂活性則尚未明瞭，因此，本研究主要探討myogenin與cyclin A2兩者間之關聯性對於心肌細胞分化與增殖的調控機轉。 本研究之動物實驗結果顯示：在大鼠胚胎心肌細胞內大量表現的週期素cyclin A2，在出生後大幅降低；相反的，在同一時期的肌細胞生成素myogenin，表現量則是由極少量而大幅升高。再以大鼠心肌母細胞株H9c2作為研究材料，並以1 %馬血清(horse serum)培養H9c2細胞以誘導其分化，來模擬分化完全的心肌細胞，研究結果顯示：以1 %馬血清誘導分化後的H9c2細胞，其生長停止並產生肌肉組織之分化特徵”肌管(myotube)”，進一步發現，隨著細胞的分化，cyclin A2含量降低而myogenin含量上升，與動物實驗之結果相符。前人研究指出Ubiquitin-Proteasome System (UPS)可能在蛋白質的調控機轉上扮演重要角色，本論文之實驗結果也顯示：1 %馬血清誘導分化後的H9c2細胞株，經蛋白酶體抑制劑(proteasome inhibitor, MG132)作用後，可使cyclin A2上升，同時使myogenin下降。此外，以藥物MG132處理後，處於細胞週期G2-M phase中的細胞比例增加，顯示細胞分裂正在進行中；若以siRNA抑制myogenin表現後，亦可發現cyclin A2上升。綜合以上實驗結果顯示，大鼠出生後心肌細胞增殖能力大幅下降可能是由於心肌細胞走向分化路徑進而抑制細胞分裂所造成，而此現象可以被調控且逆轉。我們期待未來在臨床上能透過抑制心肌分化誘使心肌增殖能力提高， 進而應用於修復心肌受損。

Cardiomyocytes are traditionally regarded as terminal differentiated cells and being permanently withdrawn from the cell cycle. Recent studies showed that mature cardiomyocytes still have the ability to proliferate; however, the regulators of cell cycle, such as cyclin A2 expression are little to detect, so the proportion of cardiomyocytes with proliferative activity is very low. In skeletal muscle cell line, C2C12 cells, the proliferative activity was inhibited through myogenin expression during muscle differentiation; however, the effect of differentiation on cardiomyocyte proliferation remains unknown. Therefore, the purpose of this study is to explore the correlation of cyclin A2 and myogenin on the proliferation and differentiation of cardiomyocytes. In vivo studies revealed that cyclin A2 was strongly expressed in rat embryonic heart and decreased immediately after birth. In contrast, expression level of myogenin changed from trace to great amount. Moreover, the in vitro studies on rat cardiomyoblast, H9c2 cell line, demonstrated that the expression levels of cyclin A2 decreased and myogenin increased after replacing the medium with 1 % horse serum to induce the differentiation of cardiomyocytes. Previous studies indicated that ubiquitin-proteasome system (UPS) might play an important role in the regulation of expression level of proliferation and differentiation regulatory proteins, including cyclin A2 and myogenin. Our results indicated that the treatment of MG132, a reversible and cell-permeable proteasome inhibitor, resulted in an increase of cyclin A2 and a decrease of myogenin protein levels. Similar results were observed with siRNA inhibition targeting to myogenin expression. We also found that the proportion of cardiomyocytes at G2-M phase increased when treated with MG132, suggesting the cell cycle is in progression. Taking all results together, we concluded that (1) differentiation pathway dominates the growth in cardiomyocytes after birth so that cardiomyocytes have low proliferative activity, and (2) this phenomenon is reversible and can be regulated. We expect the results of present study may apply for cardiac injury repair through enhancing the cardiomyocytes proliferative activity in the future.