摘要:肌節是收縮肌肉的基石,它由數百種蛋白質精確組裝而成。當編碼肌節蛋白的基因有突變時就可能導致心肌病變,其是全世界發病率和死亡率的主要原因。儘管肌節的功能和結構已得到很好的研究,但肌節形成的精確過程仍有待闡明。目前顯示分子伴侶在調節蛋白質折疊,維持其穩定和防止損傷中扮演重要作用。最近,我們實驗室成功地製造人類幹細胞之心肌報導細胞株MYH6:mCherry,我們發現UNC45B在mCherry陽性群體中高度表達。UNC45B可調節肌球蛋白裝配和肌球蛋白頭部區域折疊。將Unc45b的量敲低在斑馬魚模型,會導致斑馬魚缺乏粗肌絲且心室無法跳動。此外,Unc45b在小鼠中剔除(KO)會導致心臟纏繞缺陷(heart looping)。這些發現證明Unc45b不僅在肌球蛋白裝配中具有功能,而且在早期心臟發育也可能扮演重要的功能。然而,UNC45B在人類心臟發育中的作用仍不清楚,仍需要進一步調查UNC45B突變是否會導致心肌病變。
動物模型已被廣泛用於研究心臟發育。然而,模式生物與人類之間的差異通常限制了從動物模型獲得的結果應用到人類系統。人多能幹細胞(hPSCs)衍生的心肌細胞可做為研究心臟發育和作為病理學機制研究的疾病模型。因此,在這個計劃中,我們將使用CRISPR / Cas9技術在hPSCs中剔除UNC45B,以闡明UNC45B在肌節形成和早期心臟發育中的作用。
在我們的初步數據中,我們成功獲得UNC45B剔除細胞株。有趣的是,UNC45B KO hESC衍生的心肌細胞是不會跳動。 透射電子顯微鏡分析進一步顯示在剔除之心肌細胞中沒有Z-線只有混亂無組織的肌絲。結果是出乎意料的,因為在斑馬魚中降低Unc45b時仍然可以觀察到Z線。因此,我們將進一步深入探討UNC45B的敲除表型,並檢查UNC45B是否也參與早期心臟分化階段(Aim1)。接下來,我們想確定UNC45B在心臟發育中的分子機制(Aim2)。這些結果將有助於我們了解UNC45B在肌節形成中的功能,並可能闡明肌節形成的過程。此外,我們希望進行化學篩選以鑑定可以拯救KO表型的小分子(Aim3)。這一發現可能為心肌病治療提供了一種潛在的策略。令人興奮的是,從我們的小型初篩,我們發現用藥物伴侶組合治療,可以使約5-10%的肌節結構得到改善。雖然第一次篩選中只能獲得5-10%的KO-CMs肌節結構改善,但這結果確實令我們確立我們的篩選策略是朝著正確的方向發展。接下來,我們將確定更多化學化合物是否有更好的救援條件。此計畫的主要目標是(1)確定分子伴侶UNC45B在心肌細胞發育中的分子機制,(2)了解人類心臟細胞的發育和(3)鑑定小分子用於未來心肌病變的治療。
Abstract: The sarcomere is a building block of contraction muscle, and it is precisely assembled by hundreds of proteins. It has been shown that mutations in the genes coding sarcomere proteins result in cardiomyopathy, leading cause of morbidity and mortality worldwide. Although the function and structure of sarcomere have been well studied, the precise process of sarcomere formation remains to elucidate. Molecular chaperones have been shown to play an important role in modulating protein folding, maintaining protein stability and preventing protein damage. Recently, we have successfully generated a human cardiac MYH6:mCherry reporter line and identified a molecular chaperone, UNC45B, that is highly expressed in MYH6:mCherry positive population. Unc45b is known to modulate myosin assembly and myosin head domain folding, and knockdown of Unc45b in Zebrafish results in lack of thick filaments and no beating activity in ventricular. Furthermore, knockout (KO) of Unc45b in mice causes early cardiac defects in heart looping. These findings suggest that Unc45b not only has a function in myosin assembly, but also may have an additional function in early cardiac development. However, the role of UNC45B in human cardiac development remains unclear and whether mutations in UNC45B are causing cardiomyopathy still needs to be investigated.
Animal models have been used to study cardiac development. However, differences between model organisms and humans often limit the translation of results obtained from animal models to the human system. Human pluripotent stem cells (hPSCs)-derived cardiomyocytes (CMs) provide great potential for studying cardiac development and serve as a disease model for pathological mechanism studying. Hence, in this proposal, we will ablate UNC45B in hPSCs using the CRISPR/Cas9 technique to elucidate the role of UNC45B in sarcomere formation and cardiac cell development.
In our preliminary data, we have successfully obtained several UNC45B knockout lines. Interestingly, no beating was observed in UNC45B KO hESC-derived CM. TEM analysis further showed disorganized filaments without Z-lines in KO cell lines. The results were unexpected as Z-line still can be observed when Unc45b was knocked down in zebrafish. Therefore, we will further characterize the UNC45B knockout phenotypes and examine whether UNC45B involves in early cardiac differentiation stage as well (Aim1). Next, we want to determine the molecular mechanism of UNC45B in cardiac cell development (Aim2). These results will help us understand UNC45B function in sarcomere formation and may elucidate the process of sarcomere formation. Besides, we want to perform a chemical screening to identify small molecules which can rescue KO phenotypes (Aim3). This finding may provide a potential strategy for cardiomyopathy treatment. Excitingly, from our small primary screening, we found that treatment with combinations of chemical chaperones can rescue around 5-10% sarcomere structure. Although only 5-10% KO-CMs’s sarcomere structure can be rescued in the first screening, this result is truly encouraging that our screening strategy is in the right direction. Next, we will identify better rescuing conditions with more chemical compounds. The goals of this proposal are to (1) to determine the molecular mechanisms of molecular chaperon UNC45B in cardiac cell development, (2) to understand human cardiac cell development and (3) to identify small molecules for potential cardiomyopathy treatment.