Exertional heat stroke causes long-term skeletal muscle epigenetic reprogramming, altered gene expression, and impaired satellite cell function in mice.

The effect of exertional heat stroke (EHS) exposure on skeletal muscles is incompletely understood. Muscle weakness is an early symptom of EHS but is not considered a major target of multiorgan injury. Previously, in a preclinical mouse model of EHS, we observed the vulnerability of limb muscles to a second EHS exposure, suggesting hidden processes contributing to declines in muscle resilience. Here, we evaluated the possible molecular origins of EHS-induced declines in muscle resilience. Female C57BL/6 mice [total n = 56; 28/condition, i.e., EHS and exercise control (EXC)] underwent forced wheel running at 37.5°C/40% relative humidity until symptom limitation (unconsciousness). EXC mice exercised identically at room temperature (22-23°C). After 1 mo of recovery, the following were assessed: 1) specific force and caffeine-induced contracture in soleus (SOL) and extensor digitorum longus (EDL) muscles; 2) transcriptome and DNA methylome responses in gastrocnemius (GAST); and 3) primary satellite cell function (proliferation and differentiation). There were no differences in specific force in either SOL or EDL from EXC. Only EHS solei exhibited lower caffeine sensitivity. EHS GAST exhibited higher RNA expression of genes encoding structural proteins of slow fibers, heat shock proteins, and myogenesis. A total of ∼2,500 differentially methylated regions of DNA that could potentially affect many cell functions were identified. Primary satellite cells exhibited suppressed proliferation rates but normal differentiation responses. Results demonstrate long-term changes in skeletal muscles 1 mo after EHS that could contribute to declines in muscle resilience. Skeletal muscle may join other, more recognized tissues considered vulnerable to long-term effects of EHS.NEW & NOTEWORTHY Exertional heat stroke (EHS) in mice induces long-term molecular and functional changes in limb muscle that could reflect a loss of "resilience" to further stress. The phenotype was characterized by altered caffeine sensitivity and suppressed satellite cell proliferative potential. This was accompanied by changes in gene expression and DNA methylation consistent with ongoing muscle remodeling and stress adaptation. We propose that EHS may induce a prolonged vulnerability of skeletal muscle to further stress or injury.

The impact of hindlimb disuse on sepsis-induced myopathy in mice.

Sepsis induces a myopathy characterized by loss of muscle mass and weakness. Septic patients undergo prolonged periods of limb muscle disuse due to bed rest. The contribution of limb muscle disuse to the myopathy phenotype remains poorly described. To characterize sepsis-induced myopathy with hindlimb disuse, we combined the classic sepsis model via cecal ligation and puncture (CLP) with the disuse model of hindlimb suspension (HLS) in mice. Male C57bl/6j mice underwent CLP or SHAM surgeries. Four days after surgeries, mice underwent HLS or normal ambulation (NA) for 7 days. Soleus (SOL) and extensor digitorum longus (EDL) were dissected for in vitro muscle mechanics, morphological, and histological assessments. In SOL muscles, both CLP+NA and SHAM+HLS conditions elicited ~20% reduction in specific force (p < 0.05). When combined, CLP+HLS elicited ~35% decrease in specific force (p < 0.05). Loss of maximal specific force (~8%) was evident in EDL muscles only in CLP+HLS mice (p < 0.05). CLP+HLS reduced muscle fiber cross-sectional area (CSA) and mass in SOL (p < 0.05). In EDL muscles, CLP+HLS decreased absolute mass to a smaller extent (p < 0.05) with no changes in CSA. Immunohistochemistry revealed substantial myeloid cell infiltration (CD68+) in SOL, but not in EDL muscles, of CLP+HLS mice (p < 0.05). Combining CLP with HLS is a feasible model to study sepsis-induced myopathy in mice. Hindlimb disuse combined with sepsis induced muscle dysfunction and immune cell infiltration in a muscle dependent manner. These findings highlight the importance of rehabilitative interventions in septic hosts to prevent muscle disuse and help attenuate the myopathy.

Deletion of muscle Igf1 exacerbates disuse atrophy weakness in mice.

Muscle atrophy occurs as a result of prolonged periods of reduced mechanical stimulation associated with injury or disease. The growth hormone/insulin-like growth factor-1 (GH/IGF-1) axis and load sensing pathways can both aid in recovery from disuse through their shared downstream signaling, but their relative contributions to these processes are not fully understood. The goal of this study was to determine whether reduced muscle IGF-1 altered the response to disuse and reloading. Adult male mice with inducible muscle-specific IGF-1 deletion (MID) induced 1 wk before suspension and age-matched controls (CON) were subjected to hindlimb suspension and reloading. Analysis of muscle force, morphology, gene expression, signaling, and tissue weights was performed in nonsuspended (NS) mice, and those suspended for 7 days or reloaded following suspension for 3, 7, and 14 days. MID mice displayed diminished IGF-1 protein levels and muscle atrophy before suspension. Muscles from suspended CON mice displayed a similar extent of atrophy and depletion of IGF-1, yet combined loss of load and IGF-1 was not additive with respect to muscle mass. In contrast, soleus force generation capacity was diminished to the greatest extent when both suspension and IGF-1 deletion occurred. Recovery of mass, force, and gene expression patterns following suspension were similar in CON and MID mice, even though IGF-1 levels increased only in muscles from CON mice. Diminished strength in disuse atrophy is exacerbated with the loss of muscle IGF-1 production, whereas recovery of mass and strength upon reloading can occur even IGF-1 is low.NEW & NOTEWORTHY A mouse model with skeletal muscle-specific inducible deletion of Igf1 was used to address the importance of this growth factor for the consequences of disuse atrophy. Rapid and equivalent loss of IGF-I and mass occurred with deletion or disuse. Decrements in strength were most severe with combined loss of load and IGF-1. Return of mass and strength upon reloading was independent of IGF-1.

Matrix Metalloproteinase 13 from Satellite Cells is Required for Efficient Muscle Growth and Regeneration.

BACKGROUND/AIMS: Cell migration and extracellular matrix remodeling underlie normal mammalian development and growth as well as pathologic tumor invasion. Skeletal muscle is no exception, where satellite cell migration replenishes nuclear content in damaged tissue and extracellular matrix reforms during regeneration. A key set of enzymes that regulate these processes are matrix metalloproteinases (MMP)s. The collagenase MMP-13 is transiently upregulated during muscle regeneration, but its contribution to damage resolution is unknown. The purpose of this work was to examine the importance of MMP-13 in muscle regeneration and growth in vivo and to delineate a satellite cell specific role for this collagenase. METHODS: Mice with total and satellite cell specific Mmp13 deletion were utilized to determine the importance of MMP-13 for postnatal growth, regeneration after acute injury, and in chronic injury from a genetic cross with dystrophic (mdx) mice. We also evaluated insulin-like growth factor 1 (IGF-1) mediated hypertrophy in the presence and absence of MMP-13. We employed live-cell imaging and 3D migration measurements on primary myoblasts obtained from these animals. Outcome measures included muscle morphology and function. RESULTS: Under basal conditions, Mmp13-/- mice did not exhibit histological or functional deficits in muscle. However, following acute injury, regeneration was impaired at 11 and 14 days post injury. Muscle hypertrophy caused by increased IGF-1 was blunted with minimal satellite cell incorporation in the absence of MMP-13. Mmp13-/- primary myoblasts displayed reduced migratory capacity in 2D and 3D, while maintaining normal proliferation and differentiation. Satellite cell specific deletion of MMP-13 recapitulated the effects of global MMP-13 ablation on muscle regeneration, growth and myoblast movement. CONCLUSION: These results show that satellite cells provide an essential autocrine source of MMP-13, which not only regulates their migration, but also supports postnatal growth and resolution of acute damage.

Contrast-Enhanced Near-Infrared Optical Imaging Detects Exacerbation and Amelioration of Murine Muscular Dystrophy.

Assessment of muscle pathology is a key outcome measure to measure the success of clinical trials studying muscular dystrophies; however, few robust minimally invasive measures exist. Indocyanine green (ICG)-enhanced near-infrared (NIR) optical imaging offers an objective, minimally invasive, and longitudinal modality that can quantify pathology within muscle by imaging uptake of ICG into the damaged muscles. Dystrophic mice lacking dystrophin (mdx) or gamma-sarcoglycan (Sgcg-/-) were compared to control mice by NIR optical imaging and magnetic resonance imaging (MRI). We determined that optical imaging could be used to differentiate control and dystrophic mice, visualize eccentric muscle induced by downhill treadmill running, and restore the membrane integrity in Sgcg-/- mice following adeno-associated virus (AAV) delivery of recombinant human SGCG (desAAV8hSGCG). We conclude that NIR optical imaging is comparable to MRI and can be used to detect muscle damage in dystrophic muscle as compared to unaffected controls, monitor worsening of muscle pathology in muscular dystrophy, and assess regression of pathology following therapeutic intervention in muscular dystrophies.