Ablation of specific insulin-like growth factor I forms reveals the importance of cleavage for regenerative capacity and glycosylation for skeletal muscle storage.

Insulin-like growth factor-I (IGF-I) facilitates mitotic and anabolic actions in all tissues. In skeletal muscle, IGF-I can promote growth and resolution of damage by promoting satellite cell proliferation and differentiation, suppressing inflammation, and enhancing fiber formation. While the most well-characterized form of IGF-I is the mature protein, alternative splicing and post-translational modification complexity lead to several additional forms of IGF-I. Previous studies showed muscle efficiently stores glycosylated pro-IGF-I. However, non-glycosylated forms display more efficient IGF-I receptor activation in vitro, suggesting that the removal of the glycosylated C terminus is a necessary step to enable increased activity. We employed CRISPR-Cas9 gene editing to ablate IGF-I glycosylation sites (2ND) or its cleavage site (3RA) in mice to determine the necessity of glycosylation or cleavage for IGF-I function in postnatal growth and during muscle regeneration. 3RA mice had the highest circulating and muscle IGF-I content, whereas 2ND mice had the lowest levels compared to wild-type mice. After weaning, 4-week-old 2ND mice exhibited higher body and skeletal muscle mass than other strains. However, by 16 weeks of age, muscle and body size differences disappeared. Even though 3RA mice had more IGF-I stored in muscle in homeostatic conditions, regeneration was delayed after cardiotoxin-induced injury, with prolonged necrosis most evident at 5 days post injury (dpi). In contrast, 2ND displayed improved regeneration with reduced necrosis, and greater fiber size and muscle mass at 11 and 21 dpi. Overall, these results demonstrate that while IGF-I glycosylation may be important for storage, cleavage is needed to enable IGF-I to be used for efficient activity in postnatal growth and following acute injury.

Loss of calpain 3 dysregulates store-operated calcium entry and its exercise response in mice.

Limb-Girdle Muscular Dystrophy 2A (LGMD2A) is caused by mutations in the CAPN3 gene encoding Calpain 3, a skeletal-muscle specific, Ca2+-dependent protease. Localization of Calpain 3 within the triad suggests it contributes to Ca2+ homeostasis. Through live-cell Ca2+ measurements, muscle mechanics, immunofluorescence, and electron microscopy (EM) in Capn3 deficient (C3KO) and wildtype (WT) mice, we determined if loss of Calpain 3 altered Store-Operated Calcium Entry (SOCE) activity. Direct Ca2+ influx measurements revealed loss of Capn3 elicits elevated resting SOCE and increased resting cytosolic Ca2+, supported by high incidence of calcium entry units (CEUs) observed by EM. C3KO and WT mice were subjected to a single bout of treadmill running to elicit SOCE. Within 1HR post-treadmill running, C3KO mice exhibited diminished force production in extensor digitorum longus muscles and a greater decay of Ca2+ transients in flexor digitorum brevis muscle fibers during repetitive stimulation. Striking evidence for impaired exercise-induced SOCE activation in C3KO mice included poor colocalization of key SOCE proteins, stromal-interacting molecule 1 (STIM1) and ORAI1, combined with disappearance of CEUs in C3KO muscles. These results demonstrate that Calpain 3 is a key regulator of SOCE in skeletal muscle and identify SOCE dysregulation as a contributing factor to LGMD2A pathology.

S100A8 and S100A9 are elevated in chronically threatened ischemic limb muscle and induce ischemic mitochondrial pathology in mice.

Objective: The objective of the present study was to determine whether elevated levels of S100A8 and S100A9 (S100A8/A9) alarmins contribute to ischemic limb pathology. Methods: Gastrocnemius muscle was collected from control patients without peripheral arterial disease (PAD; n = 14) and patients with chronic limb threatening limb ischemia (CLTI; n = 14). Mitochondrial function was assessed in permeabilized muscle fibers, and RNA and protein analyses were used to quantify the S100A8/A9 levels. Additionally, a mouse model of hindlimb ischemia with and without exogenous delivery of S100A8/A9 was used. Results: Compared with the non-PAD control muscles, CLTI muscles displayed significant increases in the abundance of S100A8 and S100A9 at both mRNA and protein levels (P < .01). The CLTI muscles also displayed significant impairment in mitochondrial oxidative phosphorylation and increased mitochondrial hydrogen peroxide production compared with the non-PAD controls. The S100A8/A9 levels correlated significantly with the degree of muscle mitochondrial dysfunction (P < .05 for all). C57BL6J mice treated with recombinant S100A8/A9 displayed impaired perfusion recovery and muscle mitochondrial impairment compared with the placebo-treated mice after hindlimb ischemia surgery. These mitochondrial deficits observed after S100A8/A9 treatment were confirmed in the muscle cell culture system under normoxic conditions. Conclusions: The S100A8/A9 levels were increased in CLTI limb muscle specimens compared with the non-PAD control muscle specimens, and the level of accumulation was associated with muscle mitochondrial impairment. Elevated S100A8/A9 levels in mice subjected to hindlimb ischemia impaired perfusion recovery and mitochondrial function. Together, these findings suggest that the inflammatory mediators S100A8/A9 might be directly involved in ischemic limb pathology.

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.