Current study demonstrated the impact of heat therapy on skeletal muscle function in a model of Duchenne muscular dystrophy (DMD). The aim of this study was to: (1) examine the impact of treatment temperature on the skeletal muscle adaptation in DBA/2J mice; and (2) determine the impact of repeated HT for 3 consecutive weeks on body composition and skeletal muscle function in D2.mdx, a model of DMD. From study 1, we revealed that HT at 39℃ for 3 weeks significantly promoted relative muscle mass of both EDL and soleus muscle in DBA/2J mice. However, from study 2, HT at 39℃ for 3 weeks does not improve muscle function or increase muscle mass in a mouse model of DMD.

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Muscular dystrophies are a heterogeneous group of genetic diseases that involve mutations in genes leading to progressive muscular weakness. Limb-Girdle Muscular Dystrophy 2B (LGMD2B) is a subset of muscular dystrophy caused by mutations in the DYSF gene, which encodes for dysferlin protein and has an incidence of 1/100,000-1/200,000 people, or 1/300 people of Libyan Jewish descent. Since there is no effective treatment that can cure or reverse effects of LGMD2B once diagnosed, our goal is to investigate and develop a protein therapy that mitigates effects of this disease in patients. Galectin-1 (Gal-1) is a small, soluble 14.5 kDa protein with a carbohydrate recognition domain capable of stabilizing the sarcolemma. The exact role that Gal-1 plays in myogenic cells is not fully understood, however, it is known that Gal-1 possesses anti-inflammatory properties and increases the terminal differentiation of committed myogenic cells. Our hypothesis is that Gal-1 treatment increases myogenic potential, improves membrane repair capability, and modulates the immune response in models of LGMD2B by stabilizing muscle integrity, leading to decreased disease manifestation. To test this hypothesis and assess the effect of Gal-1 treatment on myogenesis, anti-inflammatory modulation, and membrane repair, we designed, produced, and purified recombinant human galectin-1 (rHsGal-1) to be used in LGMD2B models. Our in vitro results indicate that after 2-3 days of treatment with 0.11μM rHsGal-1, A/J-/- myotubes enhance expression of myogenic late markers and increase in size and alignment. Additionally, after short-term treatment, rHsGal-1 improves membrane repair capability in a Ca2+ independent manner through an activated carbohydrate recognition domain (CRD) in in vitro and in vivo models of LGMD2B. We give evidence that rHsGal-1 upregulates anti-inflammatory cytokines, increases functional activity, and modulates the canonical NF-κB inflammatory pathway in dysferlin-deficient models by decreasing expression of TAK-1 and the p65 and p50 subunits in vitro and short-term in vivo treatment. Similar effects of the rHsGal-1 treatment were observed in patient-derived dysferlin-deficient human myotubes. Exploratory results show a potential decrease in muscle fat deposition in Bla/J mice. Furthermore, Gal-1 contributes to immune modulation by helping to initiate muscle regeneration by shifting M2 macrophage polarization. Together, our novel discoveries provide direct evidence that Gal-1 is a promising candidate to treat LGMD2B disease pathologies by improving expression of late-stage myogenic markers, improving membrane repair in vitro and short-term in vivo studies, promoting muscle regeneration through immune modulation, and reducing canonical NF-κB inflammation.

Previous work from our laboratory in the mdx mouse model of Duchenne muscular dystrophy (DMD) demonstrated that a fraction of muscle stem cells (i.e., satellite cells) (MuSCs) progressively lose the expression of myogenic markers during the progression of the disease. In the process of characterizing this aberrant behaviour, we serendipitously discovered that MuSCs might be separated into two distinct subpopulations based on the expression of the GPI-anchored surface protein CD90. Crucially, this separation does not correlate with a divergence from the myogenic lineage; instead, it separates the pool of MuSCs into two subpopulations, both maintaining myogenic properties in healthy muscles. These two newly identified subpopulations do not overlap with any previously reported subpopulation and may be prospectively isolated; present a different response in terms of kinetics of activation and differentiation during the regenerative process induced by acute muscle damage; show a different propensity to enter in GAlert state upon distal injury; display dissimilar pAMPK activity and contain a different amount of mitochondria; are present in different proportions in distinct muscle groups; differentially express ECM encoding genes during quiescence. Moreover, one of the two subpopulations can give rise to the other and therefore appears to be upstream in the lineage hierarchy. Notably, loss of function experiments, in which CD90 was downregulated in MuSCs, diminish the difference in activation displayed by the two subpopulations. This demonstrates that CD90 is a molecular determinant of MuSCs functional diversification. Importantly, although the two subpopulations of MuSCs are numerically similar in healthy limb muscles, one of the two subpopulations is lost with time in dystrophic mdx mice. Based on these data, we are hypothesizing that an imbalance between the two newly identified subpopulations may impair regeneration in the dystrophic muscles. These observations not only increase our knowledge of the molecular and cellular dynamics that are controlling normal and pathological muscle homeostasis but also open the possibility that restoring the proper functional equilibrium between subpopulations of MuSCs may counteract the progression of the dystrophic disease.

Glucocorticoids are the most widely prescribed medications due primarily to their anti-inflammatory and immunosuppressive actions, however, their use is not without side effects. Among these, glucocorticoids cause profound muscle atrophy, yet paradoxically are used as the first line of treatment for muscle wasting disorders such as Duchenne Muscular Dystrophy (DMD) and inflammatory myopathies. In DMD patients, glucocorticoid treatment can improve muscle strength during the first 6 months of treatment and can delay loss of muscle function by up to three years. While recent advancements have been made to understand the effect of glucocorticoids (GCs) on the myofiber, the impact of GCs on skeletal muscle stem cells (MuSCs), the adult stem cells responsible for muscle regeneration, and their role in myogenic differentiation, are relatively unknown. To study the role of glucocorticoid signalling during muscle repair, I developed a conditional null mouse (GRMuSC-/-) model in which glucocorticoid receptor (GR) expression is knocked out specifically in MuSCs (GRMuSC-/-). One-week following acute muscle injury, WT and GRMuSC-/- mice both underwent robust repair assessed by myofiber cross-sectional area (CSA) analysis. However, the GR-/- MuSCs failed to return to quiescence following repair resulting in a significant increase in average myofiber CSA at 28- and 42- days post-injury, as compared to controls. Loss of the GR led to a significant increase in the percentage of PAX7+Ki67+ cycling cells in GRMuSC-/- mice (as compared to controls) at 42 days post injury. In the uninjured contralateral limb, I observed significantly fewer MuSCs in GRMuSC-/- mice with a concomitant increase in fibers with centrally located nuclei, indicating that these PAX7+ MuSCs progressed to differentiation in the absence of direct injury. In an uninjured model, two weeks following loss of GR expression there was an increase in the percentage of BrdU+ and Ki67+ cycling cells in resting GRMuSC-/- tibialis anterior muscles as compared to WT, suggesting that the GR acts to maintain MuSC quiescence. Consistent with this, immunostaining of single EDL myofiber fibers at T2h post-dissociation revealed that loss of GR in MuSCs lead to precocious activation and subsequent proliferation of MuSCs as compared to controls. Bulk RNA-sequencing from in situ fixed MuSCs in resting muscle revealed that the gene signature of GR-/- MuSCs was consistent with cells that have exited from the quiescent state and are activated for differentiation. Despite precocious activation, GR-/- myoblasts differentiate and fuse normally, however the myotubes produced had abnormal morphology and aberrant myonuclear placement in regenerated muscle fibers in vivo.

Mutations in the gene encoding the nuclear lamina protein lamin A/C (LMNA) and the associated integral inner nuclear membrane protein emerin (EMD) give rise to similar disease phenotypes and are both classified as Emery-Dreifuss muscular dystrophy (EDMD). However, the connection between the function of these nuclear envelope proteins and disease phenotype remains elusive. Given the consistent manifestation of dilated cardiomyopathy in EDMD, my investigation focused on deciphering the roles of these nuclear envelope proteins in the heart. To better understand their functions, I generated a set of isogenic human induced pluripotent stem cell (iPSC) lines with either LMNA mutation causing lamin A/C haploinsufficiency or EMD mutation causing emerin deficiency. I differentiated these iPSCs into cardiomyocytes (iPSC-CMs) and obtained their RNA transcript and protein expression profiles. I found that both mutant lines exhibited significant overlap in transcriptome and proteome changes. Analyzing alterations at both RNA and protein levels shed light on the potential functional roles of lamin A/C and emerin in cardiomyocytes and pathogenic mechanisms. To better understand the cardiac defects caused by loss of lamin A/C. I generated mice lines with tissue-specific and temporally regulated knockout of Lmna in the heart. The mutant mice experienced lethality due to heart failure, regardless of whether Lmna was knocked out at the embryonic or mature adult heart. This demonstrates that lamin A/C has a vital role in the normal function of cardiomyocytes.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24996号 / 医博第5030号 / 新制||医||1069(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 齊藤 博英, 教授 滝田 順子, 教授 小川 誠司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

付記する学位プログラム名: 京都大学卓越大学院プログラム「メディカルイノベーション大学院プログラム」 / 京都大学 / 新制・課程博士 / 博士(医科学) / 甲第25007号 / 医科博第154号 / 新制||医科||10(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 遊佐 宏介, 教授 萩原 正敏, 教授 齋藤 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Myotonic dystrophy type 1 (DM1) is an autosomal dominant and progressive neuromuscular disorder caused by the CTG trinucleotide repeat expansion in the 3’ untranslated region of the DMPK gene. Clinical manifestations include extensive atrophy of skeletal muscle (SkM) concomitant with muscle weakness, that develops in a distal to proximal fashion. Central to muscle plasticity is the satellite cell (SC), a muscle specific stem cell that, upon activation, facilitates muscle repair and regeneration. To date, SCs have yet to be elucidated in DM1; therefore, the aim of the present study was to extensively characterize the PAX7+ SC population, along with other indices of muscle quality in SkM. DM1 patients (6 women, 5 men) performed stationary cycling 3 times per week for 12wks, with biopsies taken from the Vastus lateralis pre- (PRE) and post-endurance exercise intervention (POST). Age-matched, healthy controls (CTRL) were used for comparison of baseline measures. Type 1 and 2 myofiber-specific PAX7+ cells were significantly greater in DM1 patients (PRE), in comparison to CTRL (2.24- and 1.84-fold, respectively), with type 2 SC content further increasing following training (p<0.05). In addition, protein expression of myogenic regulatory factors PAX7 and myogenin were significantly higher in DM1 compared to CTRL, with no training effects observed. Both immunohistochemical and immunoblotting analysis showed that activated MYOD+/PAX7+ cells did not significantly differ in DM1 vs. CTRL. FISH- IF analysis of CUG repeats show that 30% of SCs in DM1 were positive for these inclusions. Muscle capillarization was significantly lower in type 2 fibers in DM1 vs CTRL, which was fully rescued with training (p<0.05). At baseline, DM1 muscle showed the presence of de novo and fat infiltrated fibres, as well as fibrosis, that were relatively non-existent in the CTRL. In vitro results show patient-derived myoblasts exhibit a proliferation defect. Furthermore, myoblasts showed impairments in both glycolysis and mitochondrial respiration, with the latter being completely normalized to CTRL in myotubes. Our novel findings display an increased, albeit non-functional, SC pool in DM1 SkM indicated by disturbances in the myogenic program and overall poor muscle quality. We show that both SCs and SkM remain responsive to exercise training, suggesting therapeutic potential. We also suggest that mitochondrial dysfunction may underpin these impairments in the myogenic program. / Thesis / Master of Science (MSc) / Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy in adults worldwide affecting 1:8000 individuals, with certain areas in northeastern Quebec having a higher prevalence of 1:600 individuals. DM1 is caused by an autosomal dominant genetic mutation that leads to muscle weakness, respiratory insufficiency, cataracts and cardiac conduction block, ultimately resulting in poor quality of life and shortened lifespan. Preliminary evidence suggests that the maintenance of muscle health can greatly improve quality of life and life-span of these individuals, making an in-depth research focus on this therapeutic intervention extremely important. Optimal muscle health is maintained by the functionality of muscle stem cells, that aid in muscle repair and facilitate adaptations in muscle following exercise interventions. These cells are shown to be dys- or non-functional in various muscular dystrophies which coincide with the observation of poor muscle health. Therefore, the aim of this study was to examine the number and functionality of muscle stem cells, and physiological factors of muscle health in DM1. In addition, we also aimed to explore whether exercise has therapeutic potential to alleviate poor muscle quality in DM1. In general, we found that DM1 patients have a higher proportion of muscle stem cells; however, they are inherently dysfunctional but did respond to exercise. Consistent with the latter observation, we found poor muscle quality metrics in DM1 patients, with aerobic training leading to improvements in muscle health. Altogether, our results provide in-depth analysis that underscores muscle dysfunction observed in DM1 and the benefits of exercise interventions.