인슐린 저항성 특이 골격근육 유래의 분비 단백질체 발굴 및 체계적인 연구 : Systematic Study on Skeletal Muscle Secretome under Insulin Resistance

윤종혁 포항공과대학교 일반대학원 2012 국내박사

Skeletal muscle plays major function in the regulation of energy metabolism in the whole body. Secretomic studies to identify secretory proteins from metabolic cells/tissues have detected several candidate cytokines but skeletal muscle-derived cytokines have not been studied seriously yet. This study sought to provide lists of skeletal muscle-derived secretory proteins that have the potential to play a role in insulin resistance and to characterize insulin resistance through systematic analysis of the secretomes from different causes of insulin resistance. By novel label-free methods for secretomics using G-statistics, this study found 33 insulin-modulated skeletal muscle secretory proteins. After establishment of cell-based insulin resistant condition by TNF-alpha and palmitate, subsequent studies provided the lists of insulin resistant-modulated skeletal muscle secretory proteins using reliable quantitative methods: 28 TNF-alpha-modulated secretory proteins and 42 palmitate-modulated secretory proteins. This study found candidate myokines by clear verification. In insulin-treated secretome study, MMP-2 and PAI-1 were found that are highly related with metabolic disease especially with atherosclerotic complications. In TNF-alpha-treated secretome study, clusterin, nucleobindin-2, IGFBP-4 and DJ-1 were found. Clusterin and nucleobindin-2 are novel factors to explain roles skeletal muscle in anti-atherogenic responses and food intake, relatively. Annexin A1 was screened from palmitate-treated secretome study, which able to explain insulin resistance of skeletal muscle by autocrine pathway. From individual secretome analysis, several of novel skeletal muscle-derived insulin resistant-modulated secretory proteins that have not yet been reported as the proteins related with metabolic disease were discovered: 26 of insulin-modulated, 23 of TNF-alpha modulated and 23 of palmitate-modulated secretory proteins. In systematic analysis, TNF-alpha secretome study combined analysis with transcriptome of skeletal muscle of diabetic fatty rat suggested TNF-alpha-mediated inflammation is an important cause of diabetic skeletal muscle phenotypes. In systematic study of the secretomes from three different insulin resistant conditions, 7 proteins were common and 3 proteins of these were commonly modulated by the given insulin resistant conditions (Alpha-enolase, Nucleobindin-1, Metalloproteinase inhibitor 2). Those are identified as the common insulin resistance-dependent secretory proteins. Systematic bioinformatics analysis found common terms in biological process and molecular function of functional annotation that become foothold to further characterization of insulin resistance of skeletal muscle. This study revealed that insulin, TNF-alpha and palmitate-treated skeletal muscle secrete a variety of cytokines disparately and the extracellular environment affects cytokine secretion by skeletal muscle tissue. Therefore, the lists of identified proteins provides useful information for designing further metabolic disease studies, such as studies to identify potential communication mediators involving endo-/para-/auto-crines, as well as biomarkers for irregular states like obesity induced insulin resistance.

Implications of Chronic Infection on Skeletal Muscle Immunity, Regeneration, and Function

Jin, Richard Mark State University of New York at Buffalo ProQuest D 2020 해외박사(DDOD)

Skeletal muscle function is indispensable for activities critical to an organism’s long-term survival such as voluntary movement, posture, and respiration. Given its importance, several mechanisms exist to ensure the maintenance of proper skeletal muscle function despite exposure to a variety of insults from pathogens to mechanical injury. Among these mechanisms include the robust regenerative potential of the tissue and skeletal muscle immunity. Notably, tissue regeneration and immunity are intricately intertwined. The cellular events that coordinate skeletal muscle repair to acute sterile injuries have been well-studied. However, how chronic inflammatory settings–a hallmark of persistent infection and progressive myopathies–interfere with tissue immunity and the regenerative process remains an outstanding question in the field. To address this gap in knowledge, I use the obligate intracellular protozoan parasite, Toxoplasma gondii, to establish a chronic infection in the skeletal muscle. In these studies, I (1) investigate how chronic T. gondii infection remodels the immunologic landscape of skeletal muscle, (2) determine whether changes to skeletal muscle immunity and function due to chronic infection could be therapeutically alleviated and (3) test how chronic infection-induced inflammation affects the regenerative capacity of skeletal muscle.In the first part of these studies, I show that T. gondii infection leads to loss of muscle function accompanied by ongoing muscle damage and persistent Th1 inflammation. Surprisingly, we demonstrate that Tregs–in contrast to their well-established tissue protective role in other inflammatory settings–become pathogenic during chronic infection and facilitate the injurious accumulation of inflammatory macrophages. In the second part of this study, I show that therapeutic administration of IL-10 and amphiregulin during chronic infection can augment the proportion restorative macrophages in the skeletal muscle and improve overall skeletal muscle fitness. Finally, in the third part of these studies, I show the regenerative capacity of skeletal muscle is disrupted during chronic T. gondii infection. Compared to the current paradigm of skeletal muscle repair, my results reveal macrophages responding to injury exhibit vast heterogeneity defined by unique transcriptional states, not confined to traditional “inflammatory” and “restorative” nomenclature. Furthermore, dysfunctions in skeletal muscle repair during chronic infection are associated with the inability of macrophages to progress toward reparative transcriptional states. Collectively, I demonstrate long-term remodeling of skeletal muscle immunity during chronic T. gondii infection leads to reductions in the function and reparative capacity of skeletal muscle. Tissue-immunity must balance the ability to fight infections, heal injuries, and ultimately maintain tissue homeostasis. My findings highlight how chronic inflammation due to infection can continually tip this balance and destabilize the preservation of tissue homeostasis. A greater understanding of the mechanisms underlying how chronic inflammatory conditions, such as persistent infection or progressive myopathies, alter tissue immunity will embolden the development of targeted therapeutics for these diseases.

Maintenance of Type 2 Glycolytic Myofiber with age via Mib1-Actn3 axis

서지윤 서울대학교 대학원 2021 국내박사

Skeletal muscle accounts for 40 % of body weight and plays fundamental roles in locomotion, energy reservoir, and maintenance of whole-body homeostasis and health. However, with age, the skeletal muscle undergoes progressive changes involving alteration of skeletal muscle homeostasis and plasticity. This debilitating feature of aging is age-associated muscle atrophy, also known as sarcopenia. Sarcopenia is a multifactorial disease and a debilitating condition associated with loss of muscle mass and function with age that contributes to limitation of mobility and locomotion. However, the underlying mechanisms of how intrinsic muscle changesith age are largely unknown. The myofibers are classified into slow-twitch (type 1) and fast-twitch (type 2) myofibers. The components and characteristics of myofibers mediate their susceptibility or resistance to physiological and pathological changes in skeletal muscle. In sarcopenia, there are selective alteration of numbers and size of type 2 glycolytic myofibers. Although recent studies have reported some mechanisms associated with preferential atrophy of type 2 glycolytic myofiber with age, it remains largely unclear why type 2 glycolytic myofibers are affected with age. Mind Bomb-1 (Mib1) is a well-known E3 ubiquitin ligase implicated in Notch signaling. Mib1 is essential in the activation of Notch signaling. In skeletal muscle, Mib1 plays an essential role in the establishment of a reserve pool of adult muscle stem cells via Notch signaling. Albeit Mib1 is highly expressed in skeletal muscle and has putative binding partners besides Notch signaling, the Notch-independent role of Mib1 in skeletal muscle is largely unknown. Herein I report that, with age, Mib1 plays important role in skeletal muscle maintenance. The disruption of Mib1 in myofibers (Mib1ΔMF) results in the alteration of type 2 glycolytic myofibers, muscle atrophy, impaired muscle function, and abnormal characteristics in skeletal muscle accompanying aging. Mechanistic analyses revealed that Mib1 binds to and regulates -actinin 3 (Actn3) via proteasomal degradation-dependent degradation pathway. After chronic exercise, Mib1ΔMF mice showed muscle atrophy even at a young age. In Mib1-deficient skeletal muscles, Actn3 was accumulated with age or after chronic moderate exercise. However, when Actn3 level was downregulated, chronic exercise-induced muscle atrophy was ameliorated. Importantly, the Mib1 and Actn3 levels showed clinical relevance in human skeletal muscles accompanied by a decrease in skeletal muscle function with age. Together, these findings revealed the significance of the Mib1-Actn3 axis in skeletal muscle maintenance with age and suggest the therapeutic potential for the treatment or amelioration of age-related muscle atrophy. 골격근은 체중의 40% 를 차지하며 운동, 에너지 저장소, 전신 항상성 및 건강 유지에 중요한 역할을 한다. 하지만 나이가 들면서 골격근은 항상성과 가소성의 변화를 동반한 점진적인 변화를 겪는다. 특히 노화의 가장 큰 특징으로는 노인성 근감소증을 들 수 있다. 노인성 근감소증은 다인성 질환으로, 노화에 따른 근육량과 근기능이 감소가 되면서 운동성 및 이동성 그리고 일상생활 기능의 저하가 발생하게 된다. 하지만, 노인성 근감소증을 일어나면서 어떠한 메커니즘으로 인해 근육의 내인성 변화가 일어나는지에 대해서는 아직까지 잘 밝혀져 있지 않다. 근섬유는 느린 연축 섬유 (제 1형 근육 섬유)와 빠른 연축 섬유 (제 2형 근육 섬유)로 이루어져 있다. 근섬유의 구성과 특성은 골격근의 생리적 및 병리학적 변화에 대한 근섬유의 민감성 또는 저항성을 조절한다. 노인성 근감소증은 제 2형 해당성 근육섬유 특이적으로 개수와 근단면적 크기를 감소시킨다. 최근 연구들에 의해 선택적으로 일어나는 제 2형 해당성 근육섬유의 위축증에 대한 연구와 그 기전들이 보고된바가 있지만, 아직까지도 노화에 따른 제 2형 해당성 근육섬유의 선택적 위축증에 대해서는 잘 밝혀져 있지 않다. Mind Bomb-1 (Mib1)은 E3 유비퀴틴 라이게이즈로 Notch 신호 기작의 활성화에 중요한 역할을 하는 것으로 잘 알려져 있다. 골격근에서는 Mib1이 Notch 신호 기작을 통해 성체 근육 줄기세포 군집을 형성하고 일생동안 유지하는데 중요한 역할을 한다고 알려져있다. Mib1은 골격근에서 발현이 높으며 Notch 신호 기작 외의 다른 결합할 수 있는 단백질들이 있다고 추정되지만, 골격근에서 Notch 신호 기작을 제외한 Mib1의 독립적인 기능은 아직까지 알려져있지 않다. 본 연구에서는 노화 과정에서 Mib1이 골격근에 중요한 역할을 하는 것을 밝혔다. 근섬유 특이적으로 Mib1을 적중시킨 마우스 모델을 분석한 결과 노화에 따른 제 2형 해당성 근육섬유의 변화, 근위축증, 근기능 저하, 그리고 병리학적인 변화가 보이는 것을 확인했다. 메커니즘 분석을 통해 Mib1이 -Actinin (Actn3)와 결합하고, 프로테아좀 단백질 분해 경로를 통해 Actn3를 조절한다는 것을 밝혔다. 또한 지속적인 운동을 통해 근섬유 적중 Mib1 결손 마우스에서 노화가 발생되지 않는 성체시기에서도 근위축증이 발생된다는 것을 발견했다. Mib1이 적중된 골격근에서는 나이가 들어가거나 지속적인 운동에 의해 Actn3가 축적되는 것을 발견했다. Actn3가 축적되는 것을 막기 위해 Actn3 발현 정도를 하향 조절시키는 경우, 지속적인 운동에 의해 유도된 근위축증이 개선되는 것을 확인하였다. 무엇보다도 Mib1과 Actn3 발현 레벨 패턴이 임상적 관련성을 보여주는 연구를 통해, 골격근에서 Mib1-Actn3 축의 중요성을 밝혔다. 결론적으로, 본 연구는 Mib1-Actn3 축이 노화에 따른 골격근을 유지시키는데 중요한 역할을 한다는 것을 밝혔으며, 더 나아가 노인성 근감소증을 완화 또는 치료하기 위한 치료제로서 큰 가능성을 보인다는 것을 보여준다.

프로테오믹스 기술을 이용한 골격근에서 비만 관련 바이오 마커 발굴 연구 : Proteomics analysis of skeletal muscle in rats to discover novel obesity biomarkers

김동현 Graduate School Daegu University 2011 국내석사

1. 캡사이신을 이용한 골격근에서 항 비만 활성 연구 본 연구에서는 쥐에게 고지방 식이를 통하여 비만을 유도한 뒤 골격근에서 캡사이신의 항 비만 활성을 확인 하였다. 약 8주간 고지방식이와 캡사이신을 경구 투여한 결과, 캡사이신을 처리하지 않은 군에 비하여 처리한 군의 체중이 약 8% 가량 감소 하였다. 비만관련 바이오마커 발굴을 위한 쥐 비 장근의 단백질을 2차원 전기영동으로 분석한 결과, 총 36개의 발현 량 차이를 보이는 단백질을 발견 하였으며, 이 중 27개의 단백질을 동정 하였다. 동정된 27개의 단백질은 캡사이신의 항 비만 활성과 관련하여 기존에 보고되지 않았던 것으로, 고지방식이에 의한 비만을 억제하는 것에 일부 기여한 것으로 판단 되었다. 골격근에서 에너지 소비와 관련된 단백질들 (AMPK, ACC, CPT1, UCP3)의 발현 량을 비교 분석한 결과, 캡사이신의 처리에 의해서 골격근에서 지방의 분해가 대조 군에 비하여 활발하게 진행되고 있는 것을 확인하였다. 또한 골격근 세포 주 (L6)를 이용하여 동물실험에서 얻은 결과를 확인한 결과, 캡사이신이 골격근에서 에너지 소비와 관련된 단백질들을 직접 활성화 시키는 효과를 나타낸 것으로 확인 되었다. 2. 비만이 잘 유도 되는 쥐와 비만이 잘 유도 되지 않는 쥐의 골격근 단백 체 분석연구 지금까지의 연구에 의하면, 동일한 고지방 식이에 의해서 사람과 실험동물이 비만이 잘 유도 되는 경우 (obesity-prone, OP)와 잘 유도 되지 않는 경우 (obesity-resistant, OR)가 나타나지만 아직 명확하게 그 원인이 밝혀진 바가 없지만, 에너지 대사 효율의 변화가 비만 및 항 비만에 영향을 주는 것으로 추정되고 있다. 따라서 본 연구에서는, 프로테오믹스 기술을 이용하여 OP와 OR 쥐의 골격근에서 단백질 발현 량을 비교 분석하여 비만 및 항 비만에 관련된 바이오마커를 발굴 하고자 하였다. 약 8 주간 쥐에게 동일한 고지방 식이를 한 결과, OP 군이 OR 군에 비하여 체중이 약 25% 가량 증가하였다. 쥐의 비 장근을 이용하여 2 차원 전기영동을 한 결과, 26 개의 단백질을 발견 하였으며, 이 중 23 개의 단백질을 동정하였다. 23 개의 동정된 단백질들은 근육의 수축과 이완 그리고 지방 산화와 관련된 단백질로, OR 군에서 근육의 활동성과 지방의 산화의 효율의 증가한 것으로 나타났다. 또한 근육의 활동성 증가와 더불어 근육의 타입의 변화가 (백 근육 → 적 근육) 항 비만 활성에 중요한 역할을 한다는 결론을 도출하였다. Chapter 1. Differential expression of skeletal muscle proteins in high fat diet fed rats in response to capsaicin feeding In the present study, the effects of capsaicin on expression of skeletal muscle proteins in Sprague-Dawley rats fed with a high-fat diet (HFD) were investigated. Rats were fed a HFD with or without capsaicin treatment for 8 weeks. After HFD feeding, capsaicin-treated rats weighed an average of 8% less than those of the HFD control group. Gastrocnemius muscle tissue from lean and obese rats with or without capsaicin treatment was arrayed using 2-DE for detection of HFD associated markers. Proteomic analysis using 2-DE demonstrated that 36 spots from a total of approximately 600 matched spots showed significantly different expression; 27 spots were identified as gastrocnemius muscle proteins that had been altered in response to capsaicin feeding, and 6 spots could not be identified by mass fingerprinting. Expression of various muscle proteins was determined by immunoblot analysis for determination of molecular mechanisms, whereby capsaicin caused inhibition of adipogenesis. Immunoblot analysis revealed increased UCP3 protein expression in HFD-fed rats, whereas UCP3 contents were reduced with capsaicin treatment. Compared with the HFD control group, capsaicin treatment increased phosphorylation of AMPK and ACC. To support this result, we also analyzed in vitro differential protein expression in L6 skeletal muscle cells. These data suggest that the AMPK-ACC-malonyl-CoA metabolic signaling pathway is one of the targets of capsaicin action. To the best of our knowledge, this is the first proteomic study to report on analysis of diet-induced alterations of protein expression that are essential for energy expenditure in rat muscle. Chapter 2. Changes in expression of skeletal muscle proteins between obesity-prone and obesity-resistant rats induced by a high-fat diet A primary goal in obesity research is to determine why some people become obese (obesity-prone, OP) and others do not (obesity-resistant, OR) when exposed to high-calorie diets. The metabolic changes that cause reduced adiposity and resistance to obesity development have yet to be determined. We thus performed proteomic analysis on muscular proteins from OP and OR rats in order to determine whether other novel molecules are involved in this response. To this end, rats were fed a low or high-fat diet for 8 weeks, and were then classified into OP and OR rats by body weight gain. OP rats gained about 25% more body weight than OR rats, even though food intake did not differ significantly between the two groups. Proteomic analysis using 2-DE demonstrated differential expression of 26 spots from a total of 658 matched spots, of which 23 spots were identified as skeletal muscle proteins altered between OP and OR rats by peptide mass fingerprinting. Muscle proteome data enabled us to draw the conclusion that enhanced regulation of proteins involved in lipid metabolism and muscle contraction, as well as increased expression of marker proteins for oxidative muscle type (type I), contributed to obesity-resistance; however, anti-oxidative proteins did not.

The Effects of Exercise on Skeletal Muscle Metabolism in Cisplatin-Administered Rats

배준현 서울대학교 대학원 2021 국내박사

The survival rate of cancer patients in Korea has been observed to be increasing recently due to the development of cancer-related medical technology. The usage rate of anti-cancer drugs is on an upward trend with these medical technologies. In particular, an anti-cancer drug called Cisplatin is widely used by many cancer patients. This anti-cancer agent is part of a family of alkalizing agents, which are mainly used to treat testicular cancer, bladder cancer, prostate cancer, ovarian cancer, head and neck cancer, lung cancer, and cervical cancer. The mechanism for treatment using Cisplatin has been shown to be through attacking a specific area within the DNA in the cell and then inhibiting the resulting DNA, RNA, and protein synthesis. However, it has been recently reported that there are various side effects in cancer patients who are being treated with cisplatin-related drugs. To date, the most severe observed side effects reported of Cisplatin are: renal toxicity, ototoxicity, neurotoxicity, and muscle loss. In particular, it has been reported that 80% of cancer patients to whom Cisplatin has been administered have experienced muscle wastage and decreased muscle function, and the mortality rate has increased. The current dosage of Cisplatin being used in clinical practice is 1 mg per body weight, and despite its many side effects, it is the first line of treatment for these patients. Many previous studies have reported that exercise is necessary to prevent muscle loss and functional deterioration in cancer patients who underwent chemotherapy. It has also been reported that resistance exercise positively affected muscle metabolism in cancer patients who underwent chemotherapy. Recent studies showed that anticancer drugs have been reported to decrease the generation of mitochondria, decrease energy metabolism, and decrease the expression of autophagy. Moreover, exercise is an effective means of prevention to counteract these issues, resulting in a higher metabolism to address these factors in cancer patients. However, there is insufficient evidence to compare changes in mitochondrial biogenesis, muscle wasting metabolism, and autophagy in relation to different types of exercise after undergoing chemotherapy. Therefore, in this dissertation, over a period of eight weeks, the resistance and aerobic exercise in the cisplatin-administered rat model was used to investigate the expression level of proteins in the proteasome, morphological change in muscles, and altered energy metabolism, muscle atrophy, and autophagy expression. The study was divided into two chapters. These chapters covered protein expression levels using proteomics, morphologically altered muscle, protein expression analysis of autophagy, energy metabolism, and muscle atrophy. In the first study, there were 1018 proteins observed through proteomic analysis. This increased ubiquitin ligase-related expression, repairing the improvement of damaged proteins, and improved mitochondrial ATP synthesis-related proteins significantly increased cisplatin-administered skeletal muscle during the eight weeks of aerobic exercise. In addition, it was confirmed that the decrease in ADP-ribose increased in the group without exercise. In the second study, it was noted that in the morphological changes observed in the muscle treated with Cisplatin, the muscle's cross-sectional area and the number of muscle cells significantly increased in the SOL muscle which had undergone exercise. Furthermore, the expression of proteins related to autophagy, energy metabolism, and muscle loss in muscle tissue showed a tendency to increase through exercise. In particular, AMPK and PGC-1α related to energy metabolism were associated with the increased expression of FOXO3a autophagy-related proteins. Thus, combining the results of this study and reports referenced in previous papers, exercise post-administration of Cisplatin will be effective in preventing muscle loss by increasing mitochondrial generation and improving autophagy-related factors. 최근 의료기술 발달로 인해 국내 암환자들의 생존율이 높아지고 있다. 이러한 의료기술 중 항암제의 사용률이 지속적으로 증가 되는 추세를 보이고 있다. 특히 Cisplatin이라는 항암제가 많은 암환자들에서 사용 되고 있다. 본 항암제는 알칼리화제에 속하는 항암제로서 주로 고환암, 방광암, 전립선암, 난소암, 두경부암, 폐암, 자궁경부암 치료에 많이 사용되고 있다. 치료의 기전은 세포 내 DNA 내 특정 그룹을 공격하고, 이로 인한 DNA, RNA, 단백질 합성을 저해하여 항암 효과를 보여준다. 하지만, 최근 들어 Cisplatin을 투여 한 암환자들 중에서 여러가지 부작용이 나타남을 보고 하였다. 특히 콩팥독성, 내이독성, 신경독성, 근육감소들이 나타났다. 특히 Cisplatin을 투여한 암환자들 중 80%가 근육 감소를 경험하고 근기능의 저하와 사망률을 높이는 것으로 보고하였다. 현재 임상에서 사용되는 Cisplatin의 용량은 몸무게당 1mg의 용량 이며, 많은 부작용이 있음에도 불구하고 환자들의 치료를 위해 가장 많이 사용되고 있는 실정이다. 많은 선행연구에서는 항암제를 투여한 암환자들에서 근육 감소와 기능 저하를 예방하기 위해 운동이 필요 하다고 보고하였다. 특히 저항성 운동이 항암제를 투여한 암환자들에서 근육대사 증가에 긍정적인 영향을 준다는 것으로 보고 되어졌다. 하지만 최근 연구들에서 항암제가 미토콘드리아의 발생 감소, 에너지 대사 감소, 그리고 자가포식 발현의 감소로 보고 되어졌다. 이러한 감소를 증가 시킬 수 있는 방법은 운동이며, 특히 에너지 대사를 높인 운동 방법이 암예방에 효과가 있는 것으로 보고 하였다. 하지만 항암제를 투요 후 운동의 종류에 따른 미토콘드리아 발생, 근육 감소 대사, 자기포식 변화 비교에 관한 근거들이 부족하다. 따라서 본 논문에서는 Cisplatin을 투여한 Rat모델에서 8주간 운동 종류에 따른 근육 내 발현되는 단백질 종류, 근육 내 조직학적 변화, 단백질 수준에서 에너지 대사, 근육 감소, 자가포식 발현의 변화를 확인하고자 한다. 이를 위해 총 2가지의 연구로 나누어 Cisplatin을 투여한 근육에서 단백질 발현체 분석 및 근육 내 조직학적 변화, 조직내 자가 포식, 에너지 대사, 근육 감소의 단백질 수준 발현 량 조사를 하였다. 첫번째 연구에서는 단백칠체학의 분석을 통해 8주간의 유산소성 운동을 적용한 근육에서 1018개의 단백질 중 손상된 단백질 개선, ubiquitin ligase 관련 발현 증가, 미토콘드리아 ATP합성 관련 단백질 개선 유의미하게 증가가 나타났다. 특히 운동을 하지 않은 그룹에서 ADP-ribose 감소가 증가됨을 확인 하였다. 두번째 연구에서는 cisplatin을 투여한 근육 내 조직학적 변화에서는 운동을 적용한 SOL 근육에서 근육의 단면적 크기, 근육세포의 숫자가 크게 유의미하게 증가 하였다. 근육 조직내 자가포식, 에너지 대사, 근육 감소 관련 단백질의 발현이 운동을 통해 증가 되는 경향을 보였으며, 특히 에너지 대사와 관련 AMPK 및 PGC-1α가 FOXO3a 자가포식 관련 단백질 발현 증가와 관련 있는 것으로 나타났다. 본 연구 결과와 기존의 선행논문들의 보고를 종합해 보면, Cisplatin을 투여 후 운동이 미토콘드리아 발생 증가와 자가포식 관련 인자들의 개선을 통해 근육 감소 예방 효과가 있을 것이다.

단백질 및 유전자 분석을 통한 골격근의 비만 관련 바이오마커 발굴에 관한 연구 : Proteomic and genomic analysis of skeletal muscle for mining biomarkers for gender difference in obesity

오태석 대구대학교 대학원 2012 국내석사

Chapter 1. Gender dimorphism in skeletal muscle proteome between lean and diet-induced obese rats The purpose of this study was to develop a global view of gender-dependent protein abundance changes in skeletal muscle (soleus and gastrocnemius) of lean and HFD-induced obese rats. For examination of differential expression of proteins between gender and diet, 2-DE-based proteomic analysis of skeletal muscle were conducted, and approximately 390 (soleus) and 400 (gastrocnemius) individual protein spots were detected, ranging from 15 to 240 kDa mass between pH 3 and 10. A total of 48 proteins among 790 spots were identified with high confidence by MALDI-TOF/MS and database searches. Our gender-specific proteome comparison showed that male and female rats present different patterns of proteome regulation, for instance for the proteins involved in muscle contraction, carbohydrate and lipid metabolism, oxidative phosphorylation, as well as detoxification and antioxidant defenses. In particular, abundance of many myofibrillar filamentous proteins that are responsible for generating the physical movement of muscles was significantly higher in males in both normal and HFD diet. Some of them indicated reduced abundance upon HFD feeding in both genders, suggesting that high fat loading is associated with perturbations of myofibrillar network. Most of the glycolytic proteins identified here was less abundant in females, which could be consistent with either a greater reliance on lipid oxidation in females or a fiber type gender dimorphism. Apart from proteomic study, our Western blot analysis revealed that higher abundance of FAS and lower protein levels of UCPs, pAMPK, GLUT4, SOD2, catalase in HFD male rats might play a pivotal role in increased body weight gain together with lower metabolic, thermogenic, and antioxidative capacities. In conclusion, most of the candidate proteins identified herein by differential proteomics were previously unrecognized in gender dimorphism of skeletal muscle. The gender dimorphism found in this proteomic study could point towards a higher tendency of male rats to undergo metabolic syndrome manifestation associated with higher reliance on lipid as an energy fuel, lower antioxidative capacity, decreased energy expenditure despite of higher contractile protein expression. Our data can serve as basis for specific evidence-based interventions allowing prevention, treatment of obesity by matching the different needs of women and men such as development of gender medicine. Chapter 2. DNA microarray analysis reveals differential gene expression in the soleus muscle between male and female rats exposed to a high fat diet It is well recognized that diet-induced dysfunctions in skeletal muscle are closely related with many metabolic diseases, such as obesity and diabetes. In the present study, we identified global changes in gender-dependent gene expressions in the soleus muscle of lean and obese rats fed a high fat diet (HFD), using DNA microarray analysis. Prior to microarray analysis, the body weight gains were found to be higher in male HFD rats than the female HFD rats. To better understand the detailed phenotypic differences in response to HFD feeding, we identified differential gene expression in soleus muscle between the genders. To this end, we extracted and summarized the genes that were up- or down-regulated more than 1.5-fold between the genders in the microarray data. As expected, a greater number of genes encoding myofibrillar proteins and glycolytic proteins were expressed higher in males than females when exposed to HFD, reflecting greater muscular activity and higher capacity for utilizing glucose as an energy fuel. However, a series of genes involved in oxidative metabolism and cellular defenses were more up-regulated in females than males. These results allowed us to conclude that compared to males, females have greater fat clearing capacity in skeletal muscle through the activation of genes encoding enzymes for fat oxidation. In conclusion, our microarray data provide a better understanding of the molecular events underlying gender dimorphism in soleus muscle, and will provide valuable information in improving gender awareness in the health care system. 1. 비만이 될 때 성별에 따른 근육 단백질 발현의 차이 본 연구에서는 쥐가 고지방 식이에 의해 비만이 유도될 때 골격근에서 남녀 간의 근육 단백질의 변화 양상을 조사하였다. 골격근에서 2차원 전기영동 방법을 이용하여 분석한 결과 790개의 단백질 중 48개의 단백질이 큰 차이를 보였다. 특히 근육활동에 관련된 단백질들은 일반 식이와 고지방 식이를 처리한 수컷 쥐 모두에서 발현 양이 많았다. 대부분의 동정된 당분해 관련 단백질은 암컷 쥐에서 발현 양이 적었고, 이것은 암컷이 지방을 우선적으로 에너지원으로 사용했다는 것을 암시한다. 뿐만 아니라 면역분석법을 통해 고지방 식이를 처리한 수컷 쥐의 골격근의 특정 단백질 (UCPs, pAMPK, GLUT4, SOD2, catalase)의 발현 양을 비교하였고, 해당 단백질의 변화가 체중 증가뿐만 아니라 대사율, 열생성, 항산화 능력이 낮아진 것에 영향을 준 것으로 추측된다. 요약하면, 수컷 쥐는 고지방 식이에 노출되었을 때 근육 수축 단백질이 많이 발현되었음에도 불구하고 낮은 항산화 능력과 에너지 소비율을 보였고 따라서 암컷 쥐에 비해 비만이 더 잘 유도되었다. 이러한 결과는 비만의 예방과 치료에 있어서 남녀 간의 차이에 따라 차별된 접근법이 필요하다는 증거를 제시할 것으로 사료된다. 2. 고지방 식이에 노출 된 쥐의 골격근에서 성별에 따라 차이를 보이는 유전자의 분석 식이에 기인한 근육의 기능장애는 비만과 당뇨와 같은 대사질환과 밀접한 연관이 있다. 본 연구에서는 고지방 식이를 섭취한 쥐의 골격근에서 성별에 따라 차이를 보이는 유전자를 DNA microarray 기술을 사용하여 분석하였다. 고지방 식이를 취한 수컷 쥐가 암컷 쥐에 비해 체중증가율이 높았다. 고지방 식이에 따른 이러한 차이점을 보다 잘 이해하기 위해서 골격근에서 성별에 따라 발현 차이를 보이는 유전자를 발굴하였다. 그 중 1.5 배 이상의 차이를 보이는 유전자를 따로 선별하였다. 예상했던 것과 같이 근섬유 단백질과 당분해 관련 단백질을 지정하는 유전자가 고지방 식이 후 암컷 쥐보다 수컷 쥐에서 발현 양이 높았다. 그리고 이것은 수컷 쥐의 근육활동과 당분해 능력이 높다는 것을 반영한다. 하지만 산화적 대사와 세포 방어에 관련된 유전자들은 암컷 쥐에서 발현 양이 높았다. 이러한 결과를 토대로 수컷 쥐에 비해 암컷의 근육에서 지방 산화에 관련된 효소를 지정하는 유전자가 활성화 되었다는 사실을 유추할 수 있다. 본 연구 결과는 골격근에서 성별에 따라 차이를 보이는 유전자의 변화를 이해하는데 도움을 줄 것으로 기대된다.

단백체 및 유전체 발현분석법을 이용한 돼지 품종의 근육 내 대사 능력 및 스트레스 반응성 차이에 관한 연구

김남국 건국대학교 2006 국내박사

Human Skeletal Muscle Units for the Repair of Volumetric Muscle Loss

Wroblewski, Olga Maria ProQuest Dissertations & Theses University of Mich 2022 해외박사(DDOD)

Volumetric muscle loss (VML) is a common pathological condition caused by traumatic loss of skeletal muscle that exceeds the muscle’s regenerative capabilities and results in functional impairment. Current standards-of-care fail to fully recover contractile function. To address these limitations, our laboratory has developed scaffold-free tissue engineered skeletal muscle units (SMUs) for the treatment of VML. Isolated skeletal muscle stem cells (satellite cells) and fibroblasts are cultured into a confluent cell monolayer before being rolled into a cylindrical 3D construct. SMUs are biocompatible, incorporate into surrounding muscle tissue upon implantation, and have shown efficacy to partially repair a 30% VML in rat and sheep models. Ideally, SMUs could be engineered from small autogenic muscle biopsies, alleviating the limitations of donor site morbidity and immune rejection seen in current VML treatments. There are two key challenges that must be resolved to successfully translate our technology to a human cell-sourced model. To date, it has been difficult to grow human cell-sourced SMUs with contractile function. Secondly, many satellite cells are required for SMU fabrication. Any methodology that can optimize the number of cells obtained in a human skeletal muscle biopsy and enhance the functional properties of the resultant muscle tissue will advance SMUs towards clinical use. The work described in this thesis addresses these challenges.Human epidermal growth factor (hEGF) has shown promise enhancing myobundle formation and contractile function in vitro, but the impact of hEGF treatment on SMU fabrication had yet to be evaluated. We investigated the effects of hEGF on SMU fabrication, structure, and biomechanical function. Our results indicated that hEGF treatment was an effective means to enhance contractile function in human cell-sourced SMUs as evidenced by the 30 times higher force generated by SMUs treated with 7.5nM hEGF. The higher force was primarily due to increases in SMU myosin content.Due to the small numbers of satellite cells present in skeletal muscle, we also sought to optimize our methodologies so that fewer satellite cells are required for effective SMU fabrication. By altering the timing of our fabrication protocol and allowing cell cultures to reach >90% confluency in media that promotes proliferation, we found that we could lower starting cell-seeding density by 90% compared to ovine models to 1,000 cells/cm2 with no detrimental impact to monolayer development or SMU function.To further expand the capabilities of satellite cells from a single autogenic skeletal muscle biopsy, we evaluated the impact of in vitro cell proliferation (increasing cell number by cell passaging) on human primary skeletal muscle cells within an engineered skeletal muscle tissue environment. While cell passaging decreased the percentage of Pax7-positivecells in the total cell population from 17% to >10%, the size and contractile function of skeletal muscle constructs formed were not different from those created with unpassaged cells. With a cell-seeding density of 1,000 cells/cm2, a single passage can increase the total cell yield from a human skeletal muscle biopsy fiftyfold compared to cells harvested without a passage.Overall, this work significantly contributed to the field of skeletal muscle tissue engineering by advancing fabrication methodologies to develop SMUs of appropriate structure and function for human application. We addressed key limitations in human cell-sourced skeletal muscle tissue engineering by optimizing cell culture conditions to increase the cell yield from a single skeletal muscle biopsy and also promoting SMU biomechanical function.

The interaction of diet and exercise on skeletal muscle adaptations in rats

이종삼 School of Medical Sciences RMIT University 2002 해외박사

Physical exercise and dietary manipulations are major factors that induce significant metabolic, biochemical and cellular changes in skeletal muscle. In this thesis the interaction of exercise and diet on a variety of metabolic, enzymatic, mitogenic and genetic adaptations in rat skeletal muscle and liver was investigated. In order to determine the interaction of diet and training on skeletal muscle and liver adaptations and their metabolic consequences for endurance (Chapter three), eighty rats performed a baseline treadmill run to exhaustion at 16 m·min^(-1) (RUN1). Animals were then divided into one of two dietary conditions: high-carbohydrate (CHO) or high-fat (FAT). Each dietary group was then divided into one of four subgroups: sedentary control that performed no training (NT); low-intensity running (8 m·min^(-1); LOW) and two groups who trained at their maximal voluntary running speed (28 m·min^(-1); VMAX). Training volume was identical for LOW and VMAX (1,000 m·session^(-1)) and animals ran 4 times·wk^(-1) for 8 wk. To assess the interaction of the higher intensity exercise with diet, a second endurance test (RUN2) was undertaken after 6 wk at both 16 m·min^(-1) and 28 m·min^(-1). It was hypothesised that a high-fat diet in combination with low-intensity training would evoke the greatest metabolic adaptations for fat metabolism in skeletal muscle and improve endurance running capacity to a greater extent than when either low or more intense training was undertaken on a high carbohydrate diet. Compared to CHO, FAT increased the activities of citrate synthase, β-hydroxyacyl-CoA dehydrogenase and carnitine palmitoyl-transferase (P<0.01). NT animals ran 77% longer at 16 m·min^(-1) after FAT than CHO (239±28 vs. 135±30 min, P<0.05). There was no effect of diet on run time for LOW when rats were tested at 16 m·min^(-1) (454±86 vs. 427±75 min for CHO and FAT). However, VMAX rats fed FAT ran longer than CHO at 28 m·min^(-1) (100±28 vs. 58±11 min, P<0.05). In contrast to the original hypothesis, there was no additive effect of a high-fat diet on endurance performance when rats performed low-intensity training. Indeed, running performance was only enhanced by a high-fat diet after the more intense training programme. Although exercise capacity can be greatly influenced by dietary manipulation (Chapter three) a chronic high-fat diet is associated with poor health prognosis. Thus, whereas exercise improves carbohydrate metabolism by increasing insulin sensitivity and glucose transporter (GLUT)-4 protein in skeletal muscle, a high-fat diet reduces glucose tolerance and insulin sensitivity. Of interest is how glucose metabolism might be altered when undertaking regular exercise (which promotes glucose transport) in the face of a high-fat diet (which reduces glucose transport). It was hypothesised that exercise would partially (but not completely) preserve the increases in glucose metabolism (i.e., GLUT-4 content) despite a diet-induced impairment as a result of fat feeding. Therefore in the second experiment (Chapter four), the interaction of exercise and diet on GLUT-4 protein and messenger ribonucleic acid (mRNA) expression in both type I (soleus) and type II (extensor digitorum longus, [EDL]) skeletal muscle was determined. Forty eight rats were randomly assigned into one of two dietary conditions: a high-fat or a high-carbohydrate diet. Animals in each dietary condition were then randomly allocated to one of two subgroups: a sedentary control group that performed NT, and a group that undertook 8 wk of treadmill running training (28 m·min^(-1) for 1 km·session^(-1), 4 times·wk^(-1)). GLUT-4 protein expression in NT rats was similar in both muscles after 8 wk of either CHO or FAT. However, there was a training-induced increase in GLUT-4 protein in the soleus in rats fed either CHO or FAT (P<0.05) and in the EDL in animals fed CHO (P<0.05). Exercise training only increased GLUT-4 gene expression in animals fed CHO (soleus: 100% ↑ P=0.009, EDL: 142% ↑ P=0.002). FAT trained rats had a significant decrease in mRNA in the EDL (↓ 45%, P<0.05) but not the soleus (↓ 14%, NS), but the exercise-induced increases in GLUT-4 protein were largely preserved. Exercise is a complex physiological stimulus that activates multiple biochemical and biophysical aspects of cellular function. The effects of exercise on metabolic/morphological responses depend on the intensity, duration and frequency of this stimulus. Exercise/contraction activates specific molecular signalling pathways that transduce extracellular impulses into intracellular responses. One specific pathway is the mitogen-activated protein (MAP) kinase pathway. Activation of the MAP kinase signalling cascade has been proposed as a possible pathway whereby extracellular signals are transmitted to their intracellular targets. In the final experiment (Chapter five), the effect of a chronic programme of either low- or moderate- to high-intensity running (as described in the first experiment) on the activation of the p38 MAP kinase and the extracellular-signal regulated protein kinase (ERK) 1 / 2 pathways was investigated. It was hypothesised that a chronic programme of either low- or moderate- to high-intensity treadmill running would result in differential activation of the ERK 1 / 2 and p38 MAP kinase pathways in rat skeletal muscle. A novel feature of this study was that the intensity of training sessions was calculated so that animals covered the same distance and had similar glycogen utilisation, despite the fact that rats undertaking the less intense protocol spent almost four times as long running. A single bout of either low- or moderate- to high-intensity exercise following 8 wk of training led to a 2-fold increase in the phosphorylation of ERK 1 / 2 (P<0.05) and a 2-3 fold increase in p38 MAP kinase (P<0.01) compared to sedentary values. The ERK 1 / 2 phosphorylation in muscle sampled 48 h after the last exercise bout was similar to sedentary values, while p38 MAP kinase phosphorylation was 70-80% lower than sedentary. One bout of exercise increased total ERK 1 / 2 and p38 MAP kinase expression with the magnitude of this increase being independent of prior exercise intensity or duration. The ERK 1 / 2 expression was increased 3-4 fold in muscle sampled 48 h after the last exercise bout irrespective of the prior training programme (P<0.05), but p38 MAP kinase expression was significantly lower than sedentary values (P<0.05). It was concluded that either the activation threshold for exercise-stimulation of the MAP kinase pathway in rat soleus muscle is independent of the intensity and duration of either the last exercise bout or the prior training regimen, or that the total energy turnover (i.e., muscle glycogen utilisation during the exercise bout) determines the phosphorylation of ERK 1 / 2 and p38 MAP kinases. Taken collectively, these studies show that exercise and diet elicit a multitude of metabolic effects in skeletal muscle including changes in substrate metabolism, gene expression and protein synthesis. These adaptations are the result of both the cummulative (but independent) effects of chronic diet and/or exercise exposure (i.e., increases in enzyme activity), and the acute response to a single bout of exercise (i.e., activation of intracellular signalling cascades). The results of the studies in this thesis have provided new insight into the complex interaction of diet and exercise on a variety of physiological and cellular functions in rat skeletal muscle.

Optimization of Mouse Embryo- and Skeletal Muscle-derived Fibroblasts Isolation and Comparison of Their Functional Genes Expression for Tissue Engineering

최주광 충북대학교 2021 국내석사

섬유아세포는 생체조직에서 발견되는 대표적인 결합조직 세포이며, 특히 골격근 섬유아세포는 전반적인 신체 곳곳에 분포해있다. 조직공학기법에서 세포를 생체 내로 주입하는 방법 중 하나인 근육내 주사법의 경우, 주입한 세포와 처음으로 상호작용하게 될 세포는 골격근 섬유아세포가 된다. 그러나, 각기 다른 다양한 조직으로부터 유래한 섬유아세포에 비해 골격근 유래 섬유아세포의 분리방법은 명확히 알려진 것이 없다. 따라서, 본 연구에서는 마우스 골격근 섬유아세포의 분리방법을 최적화하고, 일반적으로 다양하게 활용되는 마우스 배아 섬유아세포와의 형태학적 모양과 배가시간, 골격근에 생물학적 영향을 미칠 수 있는 기능성 유전자의 발현수준 차이를 조사하였다. 조직공학적으로 의료용 세포를 사용할 때 세포의 저장이 필수적이므로, 본 연구는 세포의 근원과 저장기간에 따라 기능성 유전자의 발현수준 변화여부를 조사함으로써, 조직공학에서의 골격근 섬유아세포 효용성을 높이고자 하였다. 배아 섬유아세포의 분리는 마우스 배아의 머리와 내부장기를 제거한 후, 조직을 절단하고 트립신을 사용한 효소분해법으로 진행되었다. 또한, 골격근 섬유아세포의 분리는 마우스의 골격근 조직을 절단한 후, 콜라겐 분해효소 type Ⅱ를 사용한 효소분해법으로 진행되었다. 조직에서 분리한 일차 섬유아세포의 양성대조군으로써 섬유아세포 세포주 (McCoy)를 사용하였다. 분리된 두 섬유아세포는 형태학적으로 유사했지만, McCoy를 기준으로 배아 섬유아세포와 골격근 섬유아세포의 배가시간은 각각 7.5 ± 4.4 시간, 16.3 ± 3.3 시간이 느린 것으로 조사되었다 (p < 0.0001). 분리된 배아 섬유아세포 및 골격근 섬유아세포의 기능성 유전자 발현정도 (vimentin, fibroblast specific protein-1; FSP-1, fibronectin, α-smooth muscle actin; α-SMA)를 조사한 결과는 다음과 같았다. Vimentin, FSP-1의 유전자 발현수준은 분리된 섬유아세포간에 유의적인 발현차이는 나타나지 않는 것으로 조사되었다. Fibronectin의 유전자 발현수준은 McCoy를 기준으로 골격근 섬유아세포에서 유의적인 발현차이가 나타나지 않았지만, 배아 섬유아세포에 비해 골격근 섬유아세포에서 0.6 ± 0.1 배로 유의하게 낮은 것으로 조사되었다 (p < 0.01). α-SMA의 유전자 발현에 대한 밀도차이는 McCoy를 기준으로 배아 섬유아세포 및 골격근 섬유아세포에서 각각 13.9 ± 0.9 배, 11.1 ± 0.8 배로 유의하게 높은 것으로 조사되었다 (p < 0.0001). 이는, 섬유아세포의 근원에 따른 세포의 기본적 형태에는 큰 차이가 없으나, 섬유아세포의 근원에 따른 유전적 변화 및 골격근에 영향을 미칠 수 있는 생물학적 기능에는 차이가 있다는 것을 의미한다. 또한, 장기간 저장기간에 따른 (12 주) 배아 섬유아세포에서 fibronectin의 유전자 발현수준은 저장기간이 경과함에 따라 (8 – 12 주), 저장하지 않은 세포와 비교시 1.5 ± 0.2 배로 유의하게 증가하는 것으로 조사되었다 (p < 0.05). 이는, 배아 섬유아세포의 경우, 장기간 저장과정이 fibronectin 관련 연구 결과에 영향을 줄 수 있음을 의미한다. 본 연구에서는 광범위하게 활용될 수 있는 골격근 섬유아세포의 효율적인 분리방법을 확립하였으며, 마우스 배아 섬유아세포와 골격근 섬유아세포와의 생물학적으로 기능적인 차이를 제시하였다. 본 연구는 연구목적에 따른 세포선정을 위한 기초자료로 제공될 수 있다. 또한, 확립된 골격근 섬유아세포 분리법은 향후 연구에서 골격근 섬유아세포의 응용성 및 효용성 극대화에 활용될 수 있을 것으로 기대된다. Fibroblasts are representative connective tissue cells found in biological tissues, and in particular, skeletal muscle-derived fibroblasts are distributed throughout the body. In the case of intramuscular injection, which is one of the methods of injecting cells into vivo in tissue engineering techniques, the cells that will first interact with the injected cells are skeletal muscle-derived fibroblasts. However, compared to fibroblasts derived from various different tissues, there is no clearly known method of isolating fibroblasts derived from skeletal muscle. Therefore, in this study, we optimized the method of isolating mouse skeletal muscle-derived fibroblasts, investigated the morphological shape, doubling time and the difference in expression levels of functional genes that may have biological effects on skeletal muscle, compared to commonly used mouse embryonic fibroblasts (MEFs). Since storage of cells is essential for the medical use of the cells for tissue engineering, this study aimed to improve the utility of skeletal muscle-derived fibroblasts in tissue engineering by investigating whether the expression level of functional genes changes depending on the source and storage period of the cells. Isolation of MEFs was performed by removing the head and internal organs of mouse embryos, chopping the tissues, and enzymatic digestion using trypsin-EDTA. Then, isolation of skeletal muscle-derived fibroblasts was performed by enzymatic digestion using collagenase type II after chopping the skeletal muscle tissue of the adult mouse. Fibroblast cell line (McCoy) was used as a positive control for the characterization of primary fibroblasts. Though two isolated fibroblasts were morphologically similar, the doubling times of MEFs and skeletal muscle-derived fibroblasts were 7.5 ± 4.4 hours and 16.3 ± 3.3 hours slower, respectively compared to McCoy (p < 0.0001). The results of functional gene expression levels (vimentin, fibroblast specific protein-1; FSP-1, fibronectin, α-smooth muscle actin; α-SMA) in MEFs and skeletal muscle-derived fibroblasts were as follows. The gene expression levels of vimentin and FSP-1 were found to have no significant difference between MEFs and skeletal muscle-derived fibroblasts. The gene expression level of fibronectin was found to be significantly lower in skeletal muscle-derived fibroblasts than MEFs, 0.6 ± 0.1 times (p < 0.01), although there was no significant difference in skeletal muscle-derived fibroblasts compared to McCoy. The difference in density for gene expression of α-SMA was significantly higher in MEFs and skeletal muscle-derived fibroblasts, 13.9 ± 0.9 times and 11.1 ± 0.8 times, respectively, compared to McCoy (p < 0.0001). This means that there is no significant difference in the basic morphology of cells according to the source of fibroblasts, but there are differences in genetic changes and biological functions that may affect skeletal muscle according to the source of fibroblasts. In addition, the gene expression level of fibronectin in MEFs according to the long-term storage period (12 weeks) was significantly increased by 1.5 ± 0.2 times as compared to the non-stored cells as the storage period proceed (8 – 12 weeks) (p < 0.05). This means that a long-term storage can affect the results of fibronectin-related studies in the case of MEFs. In this study, an efficient isolation method for skeletal muscle-derived fibroblasts that can be widely used has been established, and the biologically functional differences between MEFs and skeletal muscle-derived fibroblasts were observed. This study can provide as basic data for the selection of optimal fibroblasts for tissue engineering according to the each research purpose. Furthermore, the established skeletal muscle-derived fibroblasts isolation method is expected to be utilized to maximize the applicability and utility of skeletal muscle-derived fibroblasts in the future studies.