心臟是脊椎動物中最早發育的器官之一，對基因表達的時間與空間的精確調控要求極高。在心臟肌肉細胞中，肌節（sarcomere）是其結構的基本單位，對心臟的收縮功能至關重要。每個肌節由兩條 Z 線、粗肌凝蛋白絲 (myosin thick filament) 與細肌動蛋白絲 (actin thin filament) 構成，並負責協調肌肉的收縮與放鬆。儘管肌節的長度約為1.9-2.2 微米，但其組裝涉及數百種蛋白質的協同作用。目前已知肌節基因的突變是心肌病變（cardiomyopathies）發病的主要原因之一，而心肌病變又是全球致死率較高的疾病之一。然而，肌節的組裝過程中涉及的分子機制仍然難以完全解釋，這一方面主要是由於缺乏有效的工具來深入研究人類心臟的發育過程及疾病機制，另一方面則是因為心臟疾病的多樣性及複雜性使得我們對其內在過程的理解尚不完全。

心臟發育的新模式：人類多能幹細胞與心臟疾病研究

人類多能幹細胞（Human pluripotent stem cells, hPSCs），包括胚胎幹細胞（ESCs）和誘導性多能幹細胞（iPSCs），其擁有自我更新與分化成多種細胞類型（如心肌細胞）的潛力，使其成為研究心臟發育、心臟疾病以及潛在治療方法的理想平台。在我們的實驗室，我們利用人類幹細胞衍生心肌細胞（hPSC-CMs）來研究心臟發育過程中的分子機制，以及心臟疾病的病理機制。

我們的最近研究發現，RBM24（RNA結合蛋白）在心臟發育過程中扮演重要角色。RBM24 在心臟前驅細胞階段表達量極高，並且其表達在心肌細胞分化過程中持續上升。儘管如此，RBM24如何具體調控心臟發育中的肌節組裝仍未完全理解。為了解決這一問題，我們利用CRISPR/Cas9基因編輯的技術創建了RBM24基因剔除（knockout）的人類幹細胞衍生心肌細胞模型。我們的研究結果顯示，RBM24 是心臟發育中肌節組裝的關鍵調控因子。

RBM24 在肌節組裝中的角色

我們發現RBM24 通過調控ACTN2 基因的選擇性剪接來影響肌節組裝，ACTN2 是肌節中的一種核心蛋白也是Z-line anchor protein。具體而言，在心臟發育的早期階段以及五週大的胎兒心臟中，ACTN2 的第六個外顯子（exon 6）通常不會被包含，這導致形成點狀的 α-肌動蛋白 Z 小體（Z-bodies）。這些 Z 小體是形成成熟 Z 線的前驅體。隨著心臟發育的進程，ACTN2 開始包含第六外顯子，這樣有助於形成條紋狀的 Z 線，並促進其他肌節蛋白的整合與功能性發揮。此外，RBM24 還動態調控多種核心肌原纖維形成基因的剪接，這些基因包括 ACTN2、TTN 和 MYH10，並且其剪接模式具有時間特異性。我們的研究表明，RBM24 在心臟發育過程中的作用不僅局限於單一的肌節組裝，而且涵蓋了從基因轉錄調控到蛋白質功能調節的多重層面。

基礎研究到臨床應用：非編碼 RNA 和心臟疾病的創新治療

除了肌節組裝，在我們的研究結果中，我們也發現RBM24 也對非編碼 RNA 的調控發揮著關鍵作用。非編碼 RNA（如環狀 RNA、微小 RNA 和長鏈非編碼 RNA）在基因表達後轉錄層面具有重要功能，它們可參與調控多種生物學過程，包括細胞分化、增殖以及病理反應。這些非編碼 RNA 在心臟發育過程中也扮演著重要角色，並且它們的調控對於心臟疾病的治療具有潛在應用價值。與傳統的基因治療相比，針對非編碼 RNA 的治療策略不僅可以通過小分子藥物或 RNA 相關療法來實現，這使得其更具臨床轉化的可行性。

在我們的研究中，我們發現 RBM24 不僅能夠調控肌節基因的表達，還能通過調控多種非編碼 RNA 來影響心肌細胞的發育與成熟。這一發現為治療心臟疾病提供了新的思路，即通過精確調控非編碼 RNA 的表達，來改變心肌細胞的功能性與成熟度，進而促進心臟再生與修復。

幹細胞衍生心肌細胞的臨床挑戰與發展

儘管幹細胞衍生心肌細胞（hPSC-CMs）在再生醫學中具有巨大的應用潛力，但這些細胞仍然面臨許多挑戰，尤其是它們的未成熟性。hPSC-CMs 雖然能夠分化為心肌樣細胞，但其功能與成熟度遠低於成人心肌細胞。這一未成熟的特徵限制了它們在臨床中的應用，因為這些細胞難以有效地整合進入宿主心臟組織，並發揮完全的功能。因此，促進 hPSC-CMs 成熟的研究成為提升其臨床應用的重要挑戰。

RBM24 與非編碼 RNA 在心肌細胞成熟中的角色

我們的數據顯示，RBM24 透過調控長鏈非編碼 RNA（lncRNAs）的表達，來影響心肌細胞的成熟。具體來說，我們發現某些 lncRNA 的缺失會干擾心肌細胞的正常成熟過程，這包括：1. 胎兒型肌凝蛋白（MYL2 和 MYH6）轉換為成人型肌凝蛋白（MYL7 和 MYH7）的過程受阻；2. 鈣離子處理相關基因（如 RYR2、SERCA2 和 PLN）表達異常；3. 粒線體生物合成的異常。更令人矚目的是，外源表達這些 lncRNAs 可以顯著促進心肌細胞成熟，這表明 RBM24 在調控心肌細胞成熟過程中發揮了至關重要的作用。

未來展望：加速幹細胞治療的臨床轉化

基於我們的研究，我們將繼續深入探討 RBM24 如何調控非編碼 RNA 的表達，並研究這些非編碼 RNA 是否在心肌細胞的成熟過程中發揮直接作用。這些研究成果將有助於我們更好地理解心臟發育與疾病中的內在機制，並為提升幹細胞治療提供新的策略，進一步提高 hPSC-CMs 在再生醫學中的應用潛力。

通過揭示 RBM24 和非編碼 RNA 在心臟發育與疾病中的角色，我們不僅可以推動基礎心臟生物學的發展，也為心臟病的治療提供創新的 RNA 相關療法，最終實現再生醫學領域的突破，為患者提供新的治療希望。

（113年TBF吳火獅醫學獎、台大生命科學系　蔡素宜教授）

Unraveling the Role of RBM24 in Heart Development and Regenerative Medicine

The human heart beats over 100,000 times a day, making it one of the most vital organs in the body. Yet, heart diseases remain a leading cause of death worldwide, with cardiomyopathies—often caused by genetic mutations—being particularly devastating. These disorders weaken the heart muscle, impairing its ability to pump blood effectively. Despite advances in medical science, there is no effective cure, and many patients ultimately require a heart transplant. However, donor hearts are scarce, and immune rejection poses a major challenge, leaving many patients without a viable treatment.

The heart’s engine: understanding sarcomere assembly

At the core of heart function lies the sarcomere, the fundamental contractile unit of cardiac muscle cells. Each sarcomere consists of two Z-lines, thick myosin filaments, and thin actin filaments, forming a highly organized structure essential for muscle contraction. Although a single sarcomere is only about 1.9-2.2 µm long, its assembly requires the precise coordination of hundreds of proteins. Mutations in sarcomeric genes are a leading cause of cardiomyopathies, yet the molecular mechanisms governing sarcomere assembly remain poorly understood. This gap in knowledge has been largely due to the lack of robust tools for studying human cardiac development and disease.

A new model for studying heart development

Human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, provide an invaluable platform for modeling heart development, studying sarcomere formation, and identifying new therapeutic strategies. In our laboratory, we use human stem cell-derived cardiomyocytes to investigate the molecular mechanisms underlying cardiac diseases.

Through this approach, we identified RBM24, an RNA-binding protein, as a key regulator of heart development. RBM24 is highly expressed during the cardiac progenitor stage, with its levels continuously increasing as cardiomyocytes mature. However, its precise function remained unclear. To address this, we used CRISPR/Cas9 gene editing to generate RBM24 knockout cardiomyocytes. Our findings revealed that RBM24 plays a critical role in sarcomere assembly during cardiac differentiation.

RBM24’s role in sarcomere formation

RBM24 regulates the alternative splicing of ACTN2, a key structural protein in the sarcomere. In early heart development, ACTN2 lacks exon 6, resulting in the formation of punctate α-actinin Z-bodies, which serve as precursors to mature Z-lines. As development progresses, exon 6 is incorporated, allowing the proper alignment of sarcomeric proteins and the transition to fully striated Z-lines. Additionally, RBM24 modulates the splicing of other essential myofibrillogenesis genes, including TTN and MYH10, in a stage-specific manner.

This study is the first to elucidate the stepwise process of sarcomere assembly using the premyofibril model. By establishing RBM24 as a master regulator of sarcomere formation and an upstream modulator of multiple cardiomyopathy-related genes, our work provides new insights into heart development and potential therapeutic strategies for treating cardiomyopathies.

Bridging basic research with clinical applications

To extend these findings toward clinical applications, our unpublished data suggest that RBM24 also regulates the alternative splicing of sarcomeric genes through non-coding RNAs, including circular RNAs, microRNAs, and long non-coding RNAs. Non-coding RNAs are crucial regulators of gene expression and offer a promising avenue for therapeutic intervention. Unlike traditional gene therapies, which often involve complex and permanent DNA modifications, non-coding RNA-based therapies can be developed into small-molecule drugs or RNA-based treatments, making them more feasible for clinical translation. By targeting these regulatory pathways, we may develop innovative treatments for cardiomyopathies and other heart diseases.

The challenge of immature stem cell-derived cardiomyocytes

Since no effective treatment currently exists for cardiomyopathy, many patients progress to end-stage heart failure and require a heart transplant. However, donor shortages and immune rejection remain major obstacles, often leading to fatal delays. To address this crisis, scientists are exploring human pluripotent stem cell-derived cardiomyocytes as a potential resource for cell therapy. These lab-grown heart cells could, in theory, be transplanted to repair damaged heart tissue.

However, a major barrier to their clinical application is their immaturity—stem cell-derived cardiomyocytes resemble fetal heart cells rather than adult cardiomyocytes, limiting their ability to integrate into the heart and restore function. Finding ways to enhance cardiomyocyte maturation is therefore a top priority in regenerative medicine.

RBM24 and non-coding RNAs: a path to maturation

Beyond its role in sarcomere assembly, our unpublished findings indicate that RBM24 also plays a critical role in cardiomyocyte maturation by regulating long non-coding RNAs. We identified a specific long non-coding RNA whose deletion disrupts key aspects of maturation, including the transition from fetal to adult myosin isoforms, the expression of calcium-handling genes, and mitochondrial biogenesis.

Remarkably, ectopic expression of this long non-coding RNA significantly enhanced cardiomyocyte maturation, suggesting that RBM24-mediated non-coding RNA regulation is a key driver of this process.

Future directions and impact on regenerative medicine

Building on these findings, we will systematically investigate how RBM24 regulates non-coding RNA expression and how these non-coding RNAs contribute to cardiomyocyte maturation.

The insights gained from this research will deepen our understanding of the molecular mechanisms underlying sarcomere assembly and cardiomyocyte maturation. More importantly, they will lay the foundation for improving stem cell-based therapies, offering new strategies to enhance cardiomyocyte function and increase the therapeutic potential of stem cell-derived heart cells.

By uncovering the role of RBM24 and non-coding RNAs, our work not only advances fundamental cardiac biology but also opens new possibilities for RNA-based therapies. These findings could lead to innovative treatments for cardiomyopathies and heart failure, bringing us closer to realizing the full potential of regenerative medicine.

(2024 Wu Ho-Su TBF Medical Award, Professor Su-Yi Tsai)