Prepubertal skeletal muscle growth requires Pax7-expressing satellite cell-derived myonuclear contribution.

The functional role of Pax7-expressing satellite cells (SCs) in postnatal skeletal muscle development beyond weaning remains obscure. Therefore, the relevance of SCs during prepubertal growth, a period after weaning but prior to the onset of puberty, has not been examined. Here, we have characterized mouse skeletal muscle growth during prepuberty and found significant increases in myofiber cross-sectional area that correlated with SC-derived myonuclear number. Remarkably, genome-wide RNA-sequencing analysis established that post-weaning juvenile and early adolescent skeletal muscle have markedly different gene expression signatures. These distinctions are consistent with extensive skeletal muscle maturation during this essential, albeit brief, developmental phase. Indelible labeling of SCs with Pax7CreERT2/+ ; Rosa26nTnG/+ mice demonstrated SC-derived myonuclear contribution during prepuberty, with a substantial reduction at puberty onset. Prepubertal depletion of SCs in Pax7CreERT2/+ ; Rosa26DTA/+ mice reduced myofiber size and myonuclear number, and caused force generation deficits to a similar extent in both fast and slow-contracting muscles. Collectively, these data demonstrate SC-derived myonuclear accretion as a cellular mechanism that contributes to prepubertal hypertrophic skeletal muscle growth.

Wt1 and ß-catenin cooperatively regulate diaphragm development in the mouse.

The developing diaphragm consists of various differentiating cell types, many of which are not well characterized during organogenesis. One important but incompletely understood tissue, the diaphragmatic mesothelium, is distinctively present from early stages of development. Congenital Diaphragmatic Hernia (CDH) occurs in humans when diaphragm tissue is lost during development, resulting in high morbidity and mortality postnatally. We utilized a Wilms Tumor 1 (Wt1) mutant mouse model to investigate the involvement of the mesothelium in normal diaphragm signaling and development. Additionally, we developed and characterized a Wt1(CreERT2)-driven ß-catenin loss-of-function model of CDH after finding that canonical Wnt signaling and ß-catenin are reduced in Wt1 mutant mesothelium. Mice with ß-catenin loss or constitutive activation induced in the Wt1 lineage are only affected when tamoxifen injection occurs between E10.5 and E11.5, revealing a critical time-frame for Wt1/ ß-catenin activity. Conditional ß-catenin loss phenocopies the Wt1 mutant diaphragm defect, while constitutive activation of ß-catenin on the Wt1 mutant background is sufficient to close the diaphragm defect. Proliferation and apoptosis are affected, but primarily these genetic manipulations appear to lead to a change in normal diaphragm differentiation. Our data suggest a fundamental role for mesothelial signaling in proper formation of the diaphragm.

Muscle-specific functional deficits and lifelong fibrosis in response to paediatric radiotherapy and tumour elimination.

BACKGROUND: As paediatric cancer survivors are living into adulthood, they suffer from the age-related, accelerated decline of functional skeletal muscle tissue, termed sarcopenia. With ionizing radiation (radiotherapy) at the core of paediatric cancer therapies, its direct and indirect effects can have lifelong negative impacts on paediatric growth and maintenance of skeletal muscle. Utilizing our recently developed preclinical rhabdomyosarcoma mouse model, we investigated the late effects of paediatric radiation treatment on skeletal muscles from late adolescent (8 weeks old) and middle-aged (16 months old) mice. METHODS: Paediatric C57BL/6J male mice (3 weeks old) were injected with rhabdomyosarcoma cells into their right hindlimbs, and then fractionated irradiation (3 × 8.2 Gy) was administered to those limbs at 4 weeks old to eliminate the tumours. Radiation-alone and tumour-irradiated mice were assessed at either 8 weeks (3 weeks post-irradiation) or 16 months (14 months post-irradiation) of age for muscle physiology, myofibre characteristics, cell loss, histopathology, fibrosis, inflammatory gene expression, and fibrotic gene expression. RESULTS: Mice that received only paediatric radiation demonstrated reduced muscle mass (-17%, P < 0.001), muscle physiological function (-25%, P < 0.01), muscle contractile kinetics (-25%, P < 0.05), satellite cell number (-45%, P < 0.05), myofibre cross-sectional area (-30%, P < 0.0001), and myonuclear number (-17%, P < 0.001). Paediatric radiation increased inflammatory gene expression, increased fibrotic gene expression, and induced extracellular matrix protein deposition (fibrosis) with tumour elimination exacerbating some phenotypes. Paediatric tumour-eliminated mice demonstrated exacerbated deficits to function (-20%, P < 0.05) and myofibre size (-17%, P < 0.001) in some muscles as well as further increases to inflammatory and fibrotic gene expression. Examining the age-related effects of paediatric radiotherapy in middle-aged mice, we found persistent myofibre atrophy (-20%, P < 0.01), myonuclear loss (-18%, P < 0.001), up-regulated inflammatory and fibrotic signalling, and lifelong fibrosis. CONCLUSIONS: The results from this paediatric radiotherapy model are consistent and recapitulate the clinical and molecular features of accelerated sarcopenia, musculoskeletal frailty, and radiation-induced fibrosis experienced by paediatric cancer survivors. We believe that this preclinical mouse model is well poised for future mechanistic insights and therapeutic interventions that improve the quality of life for paediatric cancer survivors.

Endurance exercise attenuates juvenile irradiation-induced skeletal muscle functional decline and mitochondrial stress.

BACKGROUND: Radiotherapy is commonly used to treat childhood cancers and can have adverse effects on muscle function, but the underlying mechanisms have yet to be fully elucidated. We hypothesized that endurance exercise following radiation treatment would improve skeletal muscle function. METHODS: We utilized the Small Animal Radiation Research Platform (SARRP) to irradiate juvenile male mice with a clinically relevant fractionated dose of 3× (every other day over 5 days) 8.2 Gy X-ray irradiation locally from the knee to footpad region of the right hindlimb. Mice were then singly housed for 1 month in cages equipped with either locked or free-spinning voluntary running wheels. Ex vivo muscle contractile function, RT-qPCR analyses, resting cytosolic and sarcoplasmic reticulum (SR) store Ca2+ levels, mitochondrial reactive oxygen species levels (MitoSOX), and immunohistochemical and biochemical analyses of muscle samples were conducted to assess the muscle pathology and the relative therapeutic impact of voluntary wheel running (VWR). RESULTS: Irradiation reduced fast-twitch extensor digitorum longus (EDL) muscle-specific force by 27% compared to that of non-irradiated mice, while VWR post-irradiation improved muscle-specific force by 37%. Radiation treatment similarly reduced slow-twitch soleus muscle-specific force by 14% compared to that of non-irradiated mice, while VWR post-irradiation improved specific force by 18%. We assessed intracellular Ca2+ regulation, oxidative stress, and mitochondrial homeostasis as potential mechanisms of radiation-induced pathology and exercise-mediated rescue. We found a significant reduction in resting cytosolic Ca2+ concentration following irradiation in sedentary mice. Intriguingly, however, SR Ca2+ store content was increased in myofibers from irradiated mice post-VWR compared to mice that remained sedentary. We observed a 73% elevation in the overall protein oxidization in muscle post-irradiation, while VWR reduced protein nitrosylation by 35% and mitochondrial reactive oxygen species (ROS) production by 50%. Finally, we found that VWR significantly increased the expression of PGC1α at both the transcript and protein levels, consistent with an exercise-dependent increase in mitochondrial biogenesis. CONCLUSIONS: Juvenile irradiation stunted muscle development, disrupted proper Ca2+ handling, damaged mitochondria, and increased oxidative and nitrosative stress, paralleling significant deficits in muscle force production. Exercise mitigated aberrant Ca2+ handling, mitochondrial homeostasis, and increased oxidative and nitrosative stress in a manner that correlated with improved skeletal muscle function after radiation.

Inhibition of inflammatory CCR2 signaling promotes aged muscle regeneration and strength recovery after injury.

Muscle regeneration depends on a robust albeit transient inflammatory response. Persistent inflammation is a feature of age-related regenerative deficits, yet the underlying mechanisms are poorly understood. Here, we find inflammatory-related CC-chemokine-receptor 2 (Ccr2) expression in non-hematopoietic myogenic progenitors (MPs) during regeneration. After injury, the expression of Ccr2 in MPs corresponds to the levels of its ligands, the chemokines Ccl2, 7, and 8. We find stimulation of Ccr2-activity inhibits MP fusion and contribution to myofibers. This occurs in association with increases in MAPKp38δ/γ signaling, MyoD phosphorylation, and repression of the terminal myogenic commitment factor Myogenin. High levels of Ccr2-chemokines are a feature of regenerating aged muscle. Correspondingly, deletion of Ccr2 in MPs is necessary for proper fusion into regenerating aged muscle. Finally, opportune Ccr2 inhibition after injury enhances aged regeneration and functional recovery. These results demonstrate that inflammatory-induced activation of Ccr2 signaling in myogenic cells contributes to aged muscle regenerative decline.

Radiation-Induced Damage to Prepubertal Pax7+ Skeletal Muscle Stem Cells Drives Lifelong Deficits in Myofiber Size and Nuclear Number.

During prepubertal development, muscle stem cells (satellite cells, SCs) actively contribute to myofiber growth. Because some SCs are active during this time, they may be particularly susceptible to damage. Using a Small Animal Radiation Research Platform (SARRP), we investigated the effects of local fractionated radiation treatment on prepubertal SCs. Immediately after this regimen, there was a reduction in SC number. Although surviving SCs had deficiencies in function, some myogenic potential remained. Indeed, some muscle regenerative capacity persisted immediately after irradiation. Lastly, we assessed the long-term consequences of radiation-induced SC loss during prepuberty. We observed a reduction of myofiber size and corresponding loss of nuclei in both fast- and slow-contracting muscles 14 months post-irradiation. Notably, prepubertal SC depletion mimicked these lifelong deficits. This work highlights the susceptibility of prepubertal SCs to radiation exposure. We also reveal the importance of prepubertal SC contribution to the lifelong maintenance of skeletal muscle.

Chemoradiation impairs myofiber hypertrophic growth in a pediatric tumor model.

Pediatric cancer treatment often involves chemotherapy and radiation, where off-target effects can include skeletal muscle decline. The effect of such treatments on juvenile skeletal muscle growth has yet to be investigated. We employed a small animal irradiator to administer fractionated hindlimb irradiation to juvenile mice bearing implanted rhabdomyosarcoma (RMS) tumors. Hindlimb-targeted irradiation (3 × 8.2 Gy) of 4-week-old mice successfully eliminated RMS tumors implanted one week prior. After establishment of this preclinical model, a cohort of tumor-bearing mice were injected with the chemotherapeutic drug, vincristine, alone or in combination with fractionated irradiation (5 × 4.8 Gy). Single myofiber analysis of fast-contracting extensor digitorum longus (EDL) and slow-contracting soleus (SOL) muscles was conducted 3 weeks post-treatment. Although a reduction in myofiber size was apparent, EDL and SOL myonuclear number were differentially affected by juvenile irradiation and/or vincristine treatment. In contrast, a decrease in myonuclear domain (myofiber volume/myonucleus) was observed regardless of muscle or treatment. Thus, inhibition of myofiber hypertrophic growth is a consistent feature of pediatric cancer treatment.

Selective Sexual Dimorphisms in Musculoskeletal and Cardiopulmonary Pathologic Manifestations and Mortality Incidence in the Tumor Necrosis Factor-Transgenic Mouse Model of Rheumatoid Arthritis.

OBJECTIVE: To examine and quantify the sexual dimorphism in pathologic features manifested in the musculoskeletal and cardiopulmonary systems and incidence of mortality in the tumor necrosis factor-transgenic (TNF-Tg; Tg3647 strain) mouse model of inflammatory erosive arthritis. METHODS: Kaplan-Meier survival estimates were determined in male and female Tg3647 mice and sex-matched wild-type (WT) littermate mice. Longitudinal and cross-sectional pathologic outcomes in the musculoskeletal and cardiopulmonary systems were assessed via ultrasound, micro-computed tomography, grip strength measurements, histologic and serologic analyses, flow cytometry, and skeletal muscle physiologic measures. RESULTS: Compared to male Tg3647 mice (n = 30), female Tg3647 mice (n = 34) had significantly shorter lifespans (P < 0.001) and exhibited the following pathologic features (n = 4-6 per group; P < 0.05 versus male Tg3647 littermates): gross deficits in body mass and muscle weight, early-onset inflammatory arthritis with severity of end-stage arthritis that was as severe as that seen in male transgenic mice, and early onset and increased severity of inflammatory interstitial lung disease (ILD). Histologically, the ILD observed in Tg3647 mice was characterized by inflammatory cell accumulation and pulmonary arteriole thickening, which was concomitant with the presence of right ventricular hypertrophy, a feature that was also more severe in the female compared to male Tg3647 mice (P < 0.05). No sexual dimorphisms in TNF-induced deficient grip strength, axial skeletal growth, or bone loss were found. Globally, the extent of the pathologic changes observed in female Tg3647 mice was greater than that observed in male Tg3647 mice when each group was compared to their sex-matched WT littermates. CONCLUSION: These findings indicate that TNF selectively drives the early onset of arthritis and progression of pathologic changes in the cardiopulmonary system in female Tg3647 mice. These results in the Tg3647 mouse identify it as a suitable model to better understand the mechanisms underlying sexual dimorphism and cardiopulmonary disease in the setting of inflammatory arthritis and other connective tissue diseases.

Castration induces satellite cell activation that contributes to skeletal muscle maintenance.

BACKGROUND: Sarcopenia, the age-related loss of skeletal muscle, is a side effect of androgen deprivation therapy (ADT) for prostate cancer patients. Resident stem cells of skeletal muscle, satellite cells (SCs), are an essential source of progenitors for the growth and regeneration of skeletal muscle. Decreased androgen signaling and deficits in the number and function of SCs are features of aging. Although androgen signaling is known to regulate skeletal muscle, the cellular basis for ADT-induced exacerbation of sarcopenia is unknown. Furthermore, the consequences of androgen deprivation on SC fate in adult skeletal muscle remain largely unexplored. METHODS: We examined SC fate in an androgen-deprived environment using immunofluorescence and fluorescence-activated cell sorting (FACS) with SC-specific markers in young castrated mice. To study the effects of androgen deprivation on SC function and skeletal muscle regenerative capacity, young castrated mice were subjected to experimental regenerative paradigms. SC-derived-cell contributions to skeletal muscle maintenance were examined in castrated Pax7CreER/+; ROSA26mTmG/+ mice. SCs were depleted in Pax7CreER/+; ROSA26DTA/+ mice to ascertain the consequences of SC ablation in sham and castrated skeletal muscles. Confocal immunofluorescence analysis of neuromuscular junctions (NMJs), and assessment of skeletal muscle physiology, contractile properties, and integrity were conducted. RESULTS: Castration led to SC activation, however this did not result in a decline in SC function or skeletal muscle regenerative capacity. Surprisingly, castration induced SC-dependent maintenance of young skeletal muscle. The functional dependence of skeletal muscles on SCs in young castrated mice was demonstrated by an increase in SC-derived-cell fusion within skeletal muscle fibers. SC depletion was associated with further atrophy and functional decline, as well as the induction of partial innervation and the loss of NMJ-associated myonuclei in skeletal muscles from castrated mice. CONCLUSION: The maintenance of skeletal muscles in young castrated mice relies on the cellular contributions of SCs. Considering the well-described age-related decline in SCs, the results in this study highlight the need to devise strategies that promote SC maintenance and activity to attenuate or reverse the progression of sarcopenia in elderly androgen-deprived individuals.

Loss of adult skeletal muscle stem cells drives age-related neuromuscular junction degeneration.

Neuromuscular junction degeneration is a prominent aspect of sarcopenia, the age-associated loss of skeletal muscle integrity. Previously, we showed that muscle stem cells activate and contribute to mouse neuromuscular junction regeneration in response to denervation (Liu et al., 2015). Here, we examined gene expression profiles and neuromuscular junction integrity in aged mouse muscles, and unexpectedly found limited denervation despite a high level of degenerated neuromuscular junctions. Instead, degenerated neuromuscular junctions were associated with reduced contribution from muscle stem cells. Indeed, muscle stem cell depletion was sufficient to induce neuromuscular junction degeneration at a younger age. Conversely, prevention of muscle stem cell and derived myonuclei loss was associated with attenuation of age-related neuromuscular junction degeneration, muscle atrophy, and the promotion of aged muscle force generation. Our observations demonstrate that deficiencies in muscle stem cell fate and post-synaptic myogenesis provide a cellular basis for age-related neuromuscular junction degeneration and associated skeletal muscle decline.

Smad4 restricts differentiation to promote expansion of satellite cell derived progenitors during skeletal muscle regeneration.

Skeletal muscle regenerative potential declines with age, in part due to deficiencies in resident stem cells (satellite cells, SCs) and derived myogenic progenitors (MPs); however, the factors responsible for this decline remain obscure. TGFß superfamily signaling is an inhibitor of myogenic differentiation, with elevated activity in aged skeletal muscle. Surprisingly, we find reduced expression of Smad4, the downstream cofactor for canonical TGFß superfamily signaling, and the target Id1 in aged SCs and MPs during regeneration. Specific deletion of Smad4 in adult mouse SCs led to increased propensity for terminal myogenic commitment connected to impaired proliferative potential. Furthermore, SC-specific Smad4 disruption compromised adult skeletal muscle regeneration. Finally, loss of Smad4 in aged SCs did not promote aged skeletal muscle regeneration. Therefore, SC-specific reduction of Smad4 is a feature of aged regenerating skeletal muscle and Smad4 is a critical regulator of SC and MP amplification during skeletal muscle regeneration.