每條骨骼肌都是由不同收縮速度及代謝功能的肌纖維細胞(或肌管)依不同比率平行組合而成，骨骼肌佔約40%的身體質量所以是最大的代謝器官。粒線體和過氧化氫體是參與肌管氧化代謝的主要胞器。在粒線體基質發生的三羧酸循環(TCA循環)與嵌在粒線體內膜上的電子傳遞鏈(ETC)合作以產生ATP，但從ETC滲漏的電子卻是造成活性氧物質(ROS)在細胞內產生的主要原因。過氧化氫體參與非常長碳鏈、不飽和及支鏈脂肪酸的β-氧化，並透過α-氧化代謝含有3-甲基或2-羥基的羧酸。過氧化氫體另一個重要的功能，是移除在粒線體和過氧化氫體中氧化物質所伴隨產生的ROS。雖然這兩個胞器有各自獨立的功能，但它們在許多代謝途徑，特別是脂肪酸β-氧化及ROS代謝，確實是有緊密的交互作用。大量的研究均顯示，它們的生合成及功能，深受轉錄共同活化因子PGC-1α所調控。而我們的研究亦發現轉錄因子Bhlhe40能負向調控PGC-1α的啟動子活性，並抑制在目標基因啟動子上的PGC-1α活性，使得粒線體和過氧化氫體的數量和功能皆受到抑制。在本次研究中，我們將聚焦在在肌肉生成時粒線體和過氧化氫體兩者之間的相互作用如何受到Bhlhe40的調控。首先，兩個胞器在肌肉生成過程中之動態平衡(生合成和自噬作用)及代謝功能，將在野生型以及基因改造型(Bhlhe40過量表現(C2C12-Bhlhe40)，基因敲落(C2C12-shBhlhe40)或拮抗(C2C12-VBH135))的肌原母細胞中作詳實觀察。之後，帶有粒線體或過氧化氫體相關基因突變的肌原母細胞(C2C12-MITOm and C2C12-PEXm)也會被建立，並找出個別胞器突變對於另一方的影響。兩種胞器相關的重要基因之表現和功能將會在前述的野生型和基因改造型細胞株進行研究，以了解Bhlhe40和胞器缺陷對這些基因的影響。Bhlhe40和PGC-1α被召集到這些基因的調控區域的情況也將會被詳細研究，以便更進一步驗證它們的參與。因此，本研究的具體目標可簡述如下：(一)研究粒線體和過氧化氫體在肌肉生成時的動態平衡及功能(二)研究粒線體或過氧化氫體缺陷時對另一胞器的影響(三)分析粒線體或過氧化氫體缺陷對胞器特定基因(organelle-specific)的表現及功能之影響(四)研究在野生型及胞器缺陷細胞株中，Bhlhe40如何調控organelle-specific 基因 ;Skeletal muscle (SKM) is the largest metabolic organ and constitutes about 40% of the adult body mass in mammals. Each SKM is composed of parallel mature myocyte fibers (or called myotubes) that are generally classified as mitochondria-rich slow-twitch (oxidative/type I) or mitochondria-poor fast-twitch (glycolytic/type IIa, IIx, and IIb) fibers. Mitochondria (MITO) and peroxisomes (PEX) are the major organelles involved in the oxidative metabolism of myotubes. The tricarboxylic acid (TCA) cycle happens in the matrix while the reducing equivalents (FADH2 and NAD(P)H) generated from TCA cycle are used to generate ATP via electron transport chain (ETC) harbored in the inner membrane. Electron leakage from the ETC is the major source of reactive oxygen species (ROS) in the cell. Peroxisomes participate in the β-oxidation of very long chain, unsaturated, and branched fatty acids. Additionally, they can also metabolize carboxylates containing a 3-methyl or 2-hydroxyl group via α-oxidation. Another important function of PEX is removing the harmful reactive oxygen species (ROS) concomitantly generates from the oxidation of substrates in MITO and PEX. Although MITO and PEX perform independent roles in metabolism, they do collaborate in many metabolic processes, especially fatty acid β-oxidation (FABO) and ROS metabolism. For instance, acetyl-CoA generated from FABO in peroxisomes is transported into MITO for further metabolism in TCA cycle, and ROS leaked from MITO can be handled by the scavenging systems in peroxisomes. Abundant studies have shown that the biogenesis and function of both organelles are critically regulated by the transcriptional coactivator PGC-1α to support oxidative metabolism. Furthermore, our recent studies found the transcription factor Bhlhe40 negatively regulates PGC-1α promoter activity and directly interacts with PGC-1α on target gene promoters to repress PGC-1α coactivational activity. Consequently, both the number and activity of MITO and PEX are repressed by Bhlhe40. In this study, we will focus on delineating the mechanisms by which Bhlhe40 regulates the functional crosstalk between PEX and MITO during myogenesis. Firstly, the homeostasis (biogenesis and autophagy) and metabolic functions of both organelles during myogenesis will be documented in wildtype and genetically modified myoblasts where Bhlhe40 is either over-expressed (C2C12-Bhlhe40), knockdowned (C2C12-shBhlhe40) or antagonized (C2C12–VBH135). Then, myoblasts carrying targeted mutation of either organelle (C2C12-MITOm or -PEXm) will be created to reveal its effects (homeostasis and functions) on the other. The expression and functions of genes important to either organelle will be examined in wildtype and genetically modified myoblasts to reveal the impacts of Bhlhe40 and/or organelle defects on them. The recruitment of Bhlhe40/PGC-1α to their regulatory elements will also be examined to provide further evidence for their involvement. Therefore, the specific aims of this study can be summarized as follow:1. Examining the homeostasis and functions of MITO and PEX during myogenesis.2. Examining the effects of defective PEX or MITO on the other.3. Analyzing the effects of defective PEX or MITO on organelle-specific genes’ expression and functions.4. Examining the regulation of organelle-specific genes by Bhlhe40 in wildtype and organelle-defective myoblasts.
