Role of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in Skeletal Muscle Differentiation, Function and Aging

Sarcopenia, characterized by decreased muscle mass and strength, is associated with aging. In clinics, there are few effective options for treating sarcopenia. Cystic fibrosis transmembrane conductance regulator (CFTR) is a gene encoding a chloride channel, mutations (e.g. DF508) of which result in cystic fibrosis (CF), an autosomal recessive disease with a board ranges of clinical manifestations. Although peripheral musculoskeletal dysfunction is observed in CF patients, whether and how CFTR plays a direct role in skeletal muscle function remain unknown. Recent studies suggested that CFTR in airway epithelial cells mediates senescent phenotypes including inflammation activation, increased mitochondrial stress and impaired autophagic influx, which are also known to be pivotal to the homeostasis of skeletal muscles. Therefore, we hypothesized that CFTR in skeletal muscle cells might be involved in the age-related skeletal muscle dysfunctions through regulating inflammation and/or autophagy. In this study, we first examined the skeletal muscles of different ages in a mouse model carrying CFTR mutation DF508, which causes CFTR deficiency. Using myoblast in vitro model, we tested the possible role of CFTR in myogenic differentiation and autophagy. Finally, we tested the effects of two small compounds that are known to promote CFTR expression or function, in improving skeletal function in aged wild-type mice.Our results showed that the expression of CFTR in skeletal muscles declined with age in both humans and mice. The DXA and ex vivo functional test suggested that the muscle mass and strength (twitch force) were reduced in DF508 mice compared with the paired wild-type mice, which was more obvious in the old-aged group. While the muscle typing test and mitochondrial membrane potential (MMP) assay indicated the transfer of mitochondrial activity related muscle fiber types caused by CFTR mutation. The neuromuscular junction function was not significantly altered in the tibialis anterior muscle of DF508 mice compared to the wild-type control. Besides, the expression of myogenic (reduced) and atrophy (increased) related genes were disrupted in the aging DF508 mice at transcriptional or protein level. These results suggested that CFTR deficiency was involved in the age-related skeletal muscle decline.In C2C12 myoblast cell line and primary myoblasts, CFTR expression was confirmed by Sanger sequencing. Moreover, CFTR expression was continually upregulated upon differentiation of myoblast in both mRNA and protein level. The intracellular chloride measurement confirmed the ion channel function of CFTR in the primary myoblast. Manipulation of CFTR by knockdown or over-expression changed the formation of myotube differentiated from both C2C12 and primary myoblasts. The results consistently showed that CFTR promoted the myogenic differentiation regarding to the myoblast fusion and myotube formation. In exploring the molecular mechanisms, we found that gastrocnemii from old-aged DF508 mice had lower expression of genes key to mitochondrial biogenesis (Pgc1α, Pgc1β, Tfam, Nrf1 and Nrf2) and dynamics (Mfn1, Mfn2 and Fis1), and autophagy (Atg12, Bcl-2, and Lc3β), as compared to age-matched wild-types, suggesting mitochondrial defects with CFTR deficiency. The in vitro data further confirmed that CFTR regulated the myogenic process via modulating the LC3β mediated autophagy. These results validated the role of CFTR in the skeletal muscle differentiation correlated with autophagic pathway.CFTR as a potential therapeutic target for the sarcopenia was then tested. The adenoviral over-expression of CFTR in vivo ameliorated the skeletal muscle defects (muscle mass and typing) caused by aging. We next employed the CFTR modulators (VX-809 and VX-770) which promoted in vitro differentiation of myoblasts in a dose independent manner. Besides, CFTR modulators showed anti-inflammatory activity during the polarization of macrophages and protected mitochondrial membrane potential disruption from endoplasmic reticulum stress and inflammatory cytokines. Moreover, local injection of VX-809 promoted the muscle mass and strength in wild-type mice but not in DF508, consistent with the myogenesis gene profiles including MYOG, MYOD, and MURF-1. In another batch of enormously old-aged mice (29 months) mice, local injection of VX-809 significantly promoted the skeletal muscle mass and strength. Such VX-809 treatment also reversed the abnormal expression of markers involved in the autophagy (LC3β) and inflammation (Il1-β, NFκB). However, VX770 showed no significant effect on the muscle typing and myogenic genes expression. These results suggested that CFTR is a potential therapeutic target for age-related skeletal muscle dysfunction and VX-809 may be a repurposing drug used not only for the CF patients.In summary, the present results have shown a previously undefined role of CFTR in skeletal muscle differentiation, function, and aging. Pharmaceutical drugs targeting CFTR may be a new treatment strategy for age-related skeletal muscle disorder such as sarcopenia.Key words:cystic fibrosis, skeletal muscle, myogenesis, CFTR-modulator, autophagy, anti-inflammation.肌少癥，定義為骨骼肌的重量和力量降低，和衰老密切相關並且缺乏治療手段已經在年老人群中越來越普遍。囊性纖維化跨膜傳導調節器（CFTR）突變（如DF508）會導致囊性纖維化，一種隱性常染色體遺傳病，其臨床有多種表現。盡管囊性纖維化病人有外周骨骼肌肉功能異常的表現，CFTR是否以及如何在骨骼肌肉中發揮作用仍然未知。近期研究表明CFTR能夠調節衰老表型，包括炎癥激活、線粒體應激增強、以及自噬體異常，這些生理過程都在骨骼肌穩態中發揮重要作用。因此，我們假設CFTR能夠通過調控自噬和炎癥反應參與衰老引起的骨骼肌功能缺失。本研究首先檢測了不同年齡的DF508突變鼠骨骼肌的功能，使用體外肌原細胞模型，檢測CFTR在肌原細胞分化以及自噬的作用。最後，我們檢測了兩個小分子（已知能提高CFTR表達和功能）在老齡鼠骨骼肌肉功能調節方面的效果。目前的研究表明CFTR在人和小鼠骨骼肌中的表達伴隨著年齡增長逐漸降低。並且肌肉的重量和力量（尤其是抽搐力）在DF508點突變的老鼠中下降，並且在老齡組差異更加顯著。肌肉分型檢測以及線粒體膜電位檢測顯示CFTR突變會引起線粒體相關的肌肉分型轉換。然而肌肉神經接頭的功能並沒有明顯改變。此外，DF508鼠的骨骼肌中肌生成和肌萎縮相關的靶點基因分別降低和增強。這些結果表明CFTR缺失參與在年齡引起的肌少癥中。在C2C12和原代肌原細胞中，一代測序技術明確了CFTR的mRNA水平表達。另外，CFTR的表達在蛋白和mRNA水平上隨著細胞分化持續增加。胞內的氯離子檢測證實了CFTR在原代細胞中的離子通道功能。我們在C2C12肌原細胞系和原代肌原細胞中通過突變、敲低、過表達的方式來改變CFTR的表達水平和功能，結果都影響了肌管的分化。結果一致表明，CFTR能夠通過增強肌管生產和細胞融合來調節肌原細胞分化。提示CFTR能夠調節肌管融合與形成相關的肌肉分化。進一步探索機製，我們發現多個基因在DF508鼠中表達較低，包括線粒體生物合成（Pgc1α, Pgc1β, Tfam, Nrf1 和 Nrf2）線粒體動力（Mfn1, Mfn2 和 Fis1）以及線粒體自噬（Atg12, Bcl-2, 和 Lc3β）。這些結果證實了CFTR參與在自噬相關的骨骼肌分化。隨後檢測了CFTR是否可以作為肌少癥的治療靶點。體內腺病毒過表達CFTR改善了衰老引起的骨骼肌功能缺失（重量和肌肉分型）。隨後，我們使用CFTR調節分子VX-809和VX-770，可以濃度依賴地提高肌原細胞體外分化。此外，CFTR調節分子在巨噬細胞極化過程中具備抗炎活性，並且在內質網應激和炎癥因子介導的線粒體膜電位崩壞中發揮保護作用。並此外體內局部註射VX-809促進野生鼠而不是突變鼠的肌肉質量和力量，並且相關靶點的檢測也與表型相一致，包括肌肉生成、肌萎縮、和自噬調節，如MYOG, MYOD, MURF-1和 LC3β。另外一批次更加年老的小鼠（29個月）實驗表明，局部註射VX-809顯著提高肌肉功能包括肌肉質量和力量。VX-809處理能夠減緩自噬（Lc3β）和炎癥（Il1-β, Nfκb）基因的異常表達。然而，在衰老小鼠中，VX-770沒有對肌肉分型和成肌標誌物有顯著改變。這些結果顯示CFTR是衰老引發肌少癥的潛在治療靶點，並且VX-809有不只針對囊性纖維化病人的新用途。總之，目前的數據證實了CFTR在骨骼肌分化、功能和衰老中的重要作用。靶向CFTR的藥物可能是治療衰老相關骨骼肌疾病的新策略。CHEN, Ziyi.Ph.D. Chinese University of Hong Kong 2021.Includes bibliographical references (leaves )Abstracts in English and Chinese.Title from PDF title page (viewed on ...