Three-dimensional imaging studies in mice identify cellular dynamics of skeletal muscle regeneration.

The function of many organs, including skeletal muscle, depends on their three-dimensional structure. Muscle regeneration therefore requires not only reestablishment of myofibers but also restoration of tissue architecture. Resident muscle stem cells (SCs) are essential for regeneration, but how SCs regenerate muscle architecture is largely unknown. We address this problem using genetic labeling of mouse SCs and whole-mount imaging to reconstruct, in three dimensions, muscle regeneration. Unexpectedly, we found that myofibers form via two distinct phases of fusion and the residual basement membrane of necrotic myofibers is critical for promoting fusion and orienting regenerated myofibers. Furthermore, the centralized myonuclei characteristic of regenerated myofibers are associated with myofibrillogenesis and endure months post injury. Finally, we elucidate two cellular mechanisms for the formation of branched myofibers, a pathology characteristic of diseased muscle. We provide a synthesis of the cellular events of regeneration and show that these differ from those used during development.

Ablation of skeletal muscle estrogen receptor alpha impairs contractility in male mice.

Estradiol and estrogen receptor α (ERα) have been shown to be important for the maintenance of skeletal muscle strength in females; however, little is known about the roles of estradiol and ERα in male muscle. The purpose of this study was to determine if skeletal muscle ERα is required for optimal contractility in male mice. We hypothesize that reduced ERα in skeletal muscle impairs contractility in male mice. Skeletal muscle-specific knockout (skmERαKO) male mice exhibited reduced strength across multiple muscles and several contractile parameters related to force generation and kinetics compared with wild-type littermates (skmERαWT). Isolated EDL muscle-specific isometric tetanic force, peak twitch force, peak concentric and peak eccentric forces, as well as the maximal rates of force development and relaxation were 11%-21% lower in skmERαKO compared with skmERαWT mice. In contrast, isolated soleus muscles from skmERαKO mice were not affected. In vivo peak torque of the anterior crural muscles was 20% lower in skmERαKO compared with skmERαWT mice. Muscle masses, contractile protein contents, fiber types, phosphorylation of the myosin regulatory light chain, and caffeine-elicited force did not differ between muscles of skmERαKO and skmERαWT mice, suggesting that strength deficits were not due to size, composition, or calcium release components of muscle contraction. These results indicate that in male mice, reduced skeletal muscle ERα blunts contractility to a magnitude similar to that previously reported in females; however, the mechanism may be sexually dimorphic.NEW & NOTEWORTHY We comprehensively measured in vitro and in vivo contractility of leg muscles with reduced estrogen receptor α (ERα) in male mice and reported that force generation and contraction kinetics are impaired. In contrast to findings in females, phosphorylation of myosin regulatory light chain cannot account for low force production in male skeletal muscle ERα knockout mice. These results indicate that ERα is required for optimal contractility in males and females but via sexually dimorphic means.

Fibroblast-derived Hgf controls recruitment and expansion of muscle during morphogenesis of the mammalian diaphragm.

The diaphragm is a domed muscle between the thorax and abdomen essential for breathing in mammals. Diaphragm development requires the coordinated development of muscle, connective tissue, and nerve, which are derived from different embryonic sources. Defects in diaphragm development cause the common and often lethal birth defect, congenital diaphragmatic hernias (CDH). HGF/MET signaling is required for diaphragm muscularization, but the source of HGF and the specific functions of this pathway in muscle progenitors and effects on phrenic nerve have not been explicitly tested. Using conditional mutagenesis in mice and pharmacological inhibition of MET, we demonstrate that the pleuroperitoneal folds (PPFs), transient embryonic structures that give rise to the connective tissue in the diaphragm, are the source of HGF critical for diaphragm muscularization. PPF-derived HGF is directly required for recruitment of MET+ muscle progenitors to the diaphragm and indirectly (via its effect on muscle development) required for phrenic nerve primary branching. In addition, HGF is continuously required for maintenance and motility of the pool of progenitors to enable full muscularization. Localization of HGF at the diaphragm's leading edges directs dorsal and ventral expansion of muscle and regulates its overall size and shape. Surprisingly, large muscleless regions in HGF and Met mutants do not lead to hernias. While these regions are likely more susceptible to CDH, muscle loss is not sufficient to cause CDH.

It takes all kinds: heterogeneity among satellite cells and fibro-adipogenic progenitors during skeletal muscle regeneration.

Vertebrate skeletal muscle is composed of multinucleate myofibers that are surrounded by muscle connective tissue. Following injury, muscle is able to robustly regenerate because of tissue-resident muscle stem cells, called satellite cells. In addition, efficient and complete regeneration depends on other cells resident in muscle - including fibro-adipogenic progenitors (FAPs). Increasing evidence from single-cell analyses and genetic and transplantation experiments suggests that satellite cells and FAPs are heterogeneous cell populations. Here, we review our current understanding of the heterogeneity of satellite cells, their myogenic derivatives and FAPs in terms of gene expression, anatomical location, age and timing during the regenerative process - each of which have potentially important functional consequences.

Preservation of satellite cell number and regenerative potential with age reveals locomotory muscle bias.

BACKGROUND: Although muscle regenerative capacity declines with age, the extent to which this is due to satellite cell-intrinsic changes vs. environmental changes has been controversial. The majority of aging studies have investigated hindlimb locomotory muscles, principally the tibialis anterior, in caged sedentary mice, where those muscles are abnormally under-exercised. METHODS: We analyze satellite cell numbers in 8 muscle groups representing locomotory and non-locomotory muscles in young and 2-year-old mice and perform transplantation assays of low numbers of hind limb satellite cells from young and old mice. RESULTS: We find that satellite cell density does not decline significantly by 2 years of age in most muscles, and one muscle, the masseter, shows a modest but statistically significant increase in satellite cell density with age. The tibialis anterior and extensor digitorum longus were clear exceptions, showing significant declines. We quantify self-renewal using a transplantation assay. Dose dilution revealed significant non-linearity in self-renewal above a very low threshold, suggestive of competition between satellite cells for space within the pool. Assaying within the linear range, i.e., transplanting fewer than 1000 cells, revealed no evidence of decline in cell-autonomous self-renewal or regenerative potential of 2-year-old murine satellite cells. CONCLUSION: These data demonstrate the value of comparative muscle analysis as opposed to overreliance on locomotory muscles, which are not used physiologically in aging sedentary mice, and suggest that self-renewal impairment with age is precipitously acquired at the geriatric stage, rather than being gradual over time, as previously thought.

Estrogen Regulates the Satellite Cell Compartment in Females.

Skeletal muscle mass, strength, and regenerative capacity decline with age, with many measures showing a greater deterioration in females around the time estrogen levels decrease at menopause. Here, we show that estrogen deficiency severely compromises the maintenance of muscle stem cells (i.e., satellite cells) as well as impairs self-renewal and differentiation into muscle fibers. Mechanistically, by hormone replacement, use of a selective estrogen-receptor modulator (bazedoxifene), and conditional estrogen receptor knockout, we implicate 17ß-estradiol and satellite cell expression of estrogen receptor α and show that estrogen signaling through this receptor is necessary to prevent apoptosis of satellite cells. Early data from a biopsy study of women who transitioned from peri- to post-menopause are consistent with the loss of satellite cells coincident with the decline in estradiol in humans. Together, these results demonstrate an important role for estrogen in satellite cell maintenance and muscle regeneration in females.

Aging of the musculoskeletal system: How the loss of estrogen impacts muscle strength.

Skeletal muscle weakness occurs with aging and in females this is compounded by the loss of estrogen with ovarian failure. Estrogen deficiency mediates decrements in muscle strength from both inadequate preservation of skeletal muscle mass and decrements in the quality of the remaining skeletal muscle. Processes and components of skeletal muscle that are affected by estrogens are beginning to be identified. This review focuses on mechanisms that contribute to the loss of muscle force generation when estrogen is low in females, and conversely the maintenance of strength by estrogen. Evidence is accumulating that estrogen deficiency induces apoptosis in skeletal muscle contributing to loss of mass and thus strength. Estrogen sensitive processes that affect quality, i.e., force generating capacity of muscle, include myosin phosphorylation and satellite cell function. Further detailing these mechanisms and identifying additional mechanisms that underlie estrogenic effects on skeletal muscle is important foundation for the design of therapeutic strategies to minimize skeletal muscle pathologies, such as sarcopenia and dynapenia.

Effects of ovarian hormones and estrogen receptor α on physical activity and skeletal muscle fatigue in female mice.

Menopause is associated with declines in physical activity and skeletal muscle strength. Physical activity is also reduced in rodents after ovariectomy (OVX) and whole-body estrogen receptor α (ERα) knockout. However, it is unclear if the effects are estradiol (E2) specific. Thus, the overall purpose of this study was to investigate the effects of the ovarian hormones, E2 and progesterone (P4), and skeletal muscle ERα (skmERα) on physical activity and skeletal muscle contractility in female mice. METHODS: Study 1: Forty female C57Bl/6J mice were given free access to running wheels for 2 weeks to assess baseline running and randomized into 4 treatment groups: OVX, OVX + E2, OVX + P4, OVX + E2 + P4. All mice underwent OVX, returned to wheels for 2 weeks, received hormone pellet implants and returned to running wheels for 6 weeks, after which soleus muscle contractility testing was completed. Study 2: Thirty-two skeletal muscle specific ERα knock-out (skmERαKO) mice and wildtype (WT) littermates were randomized into 4 groups: skmERαKO-Run, skmERαWT-Run, skmERαKO-Sed, and skmERαWT-Sed. Run mice were given free access to wheels for 20 wk and sedentary (Sed) mice maintained normal cage activities. At the end point, muscle contractility was tested. RESULTS: Study 1: OVX + E2 + P4 group ran greater distances than both the OVX and OVX + P4 groups (p ≤ 0.009). After fatiguing contractions, soleus muscles of the OVX + E2 + P4 group maintained greater submaximal force than those of other groups (p = 0.023). Immediately after the fatiguing contractions, OVX + E2 + P4 muscles had greater maximal force production than the OVX + E2 group (p = 0.027). Study 2: There were no differences in running distance between skmERαWT and skmERαKO mice (p = 0.240). Soleus muscles of skmERαKO mice were more fatigable (p < 0.001) and did not recover force as well as skmERαWT mice (p < 0.001). In vivo isometric, concentric and eccentric torque was decreased in skmERαKO mice compared to skmERαWT mice (p ≤ 0.029). CONCLUSIONS: Combined treatment of E2 + P4 in OVX mice restored physical activity, predominantly driven by E2, and protected soleus muscles against fatigue. Muscle of skmERαKO mice was weak regardless of physical activity. Although 20 wk of wheel running partially prevented force loss during fatigue in skmERαKO mice, force production during recovery remained low, indicating that estradiol functions through ERα in skeletal muscle.

Won't You Be My Neighbor? Muscle Stem Cells Recruit Endothelial Cells to Their Niche.

The maintenance of a pool of quiescent satellite cells (muscle stem cells) is necessary for long-term muscle health. In this issue of Cell Stem Cell, Verma et al. (2018) show that satellite cells recruit endothelial cells to create a vascular niche and that cross-talk between endothelial and satellite cells is vital for replenishment and maintenance of quiescent satellite cells.

Deletion of estrogen receptor α in skeletal muscle results in impaired contractility in female mice.

Estradiol deficiency in females can result in skeletal muscle strength loss, and treatment with estradiol mitigates the loss. There are three primary estrogen receptors (ERs), and estradiol elicits effects through these receptors in various tissues. Ubiquitous ERα-knockout mice exhibit numerous biological disorders, but little is known regarding the specific role of ERα in skeletal muscle contractile function. The purpose of this study was to determine the impact of skeletal muscle-specific ERα deletion on contractile function, hypothesizing that ERα is a main receptor through which estradiol affects muscle strength in females. Deletion of ERα specifically in skeletal muscle (skmERαKO) did not affect body mass compared with wild-type littermates (skmERαWT) until 26 wk of age, at which time body mass of skmERαKO mice began to increase disproportionally. Overall, skmERαKO mice had low strength demonstrated in multiple muscles and by several contractile parameters. Isolated extensor digitorum longus muscles from skmERαKO mice produced 16% less eccentric and 16-26% less submaximal and maximal isometric force, and isolated soleus muscles were more fatigable, with impaired force recovery relative to skmERαWT mice. In vivo maximal torque productions by plantarflexors and dorsiflexors were 16% and 12% lower in skmERαKO than skmERαWT mice, and skmERαKO muscles had low phosphorylation of myosin regulatory light chain. Plantarflexors also generated 21-32% less power, submaximal isometric and peak concentric torques. Data support the hypothesis that ablation of ERα in skeletal muscle results in muscle weakness, suggesting that the beneficial effects of estradiol on muscle strength are receptor mediated through ERα. NEW & NOTEWORTHY We comprehensively measured in vitro and in vivo skeletal muscle contractility in female estrogen receptor α (ERα) skeletal muscle-specific knockout mice and report that force generation is impaired across multiple parameters. These results support the hypothesis that a primary mechanism through which estradiol elicits its effects on strength is mediated by ERα. Evidence is presented that estradiol signaling through ERα appears to modulate force at the molecular level via posttranslational modifications of myosin regulatory light chain.

Estradiol modulates myosin regulatory light chain phosphorylation and contractility in skeletal muscle of female mice.

Impairment of skeletal muscle function has been associated with changes in ovarian hormones, especially estradiol. To elucidate mechanisms of estradiol on skeletal muscle strength, the hormone's effects on phosphorylation of the myosin regulatory light chain (pRLC) and muscle contractility were investigated, hypothesizing an estradiol-specific beneficial impact. In a skeletal muscle cell line, C2C12, pRLC was increased by 17ß-estradiol (E2) in a concentration-dependent manner. In skeletal muscles of C57BL/6 mice that were E2 deficient via ovariectomy (OVX), pRLC was lower than that from ovary-intact, sham-operated mice (Sham). The reduced pRLC in OVX muscle was reversed by in vivo E2 treatment. Posttetanic potentiation (PTP) of muscle from OVX mice was low compared with that from Sham mice, and this decrement was reversed by acute E2 treatment, demonstrating physiological consequence. Western blot of those muscles revealed that low PTP corresponded with low pRLC and higher PTP with greater pRLC. We aimed to elucidate signaling pathways affecting E2-mediated pRLC using a kinase inhibitor library and C2C12 cells as well as a specific myosin light chain kinase inhibitor in muscles. PI3K/Akt, MAPK, and CamKII were identified as candidate kinases sensitive to E2 in terms of phosphorylating RLC. Applying siRNA strategy in C2C12 cells, pRLC triggered by E2 was found to be mediated by estrogen receptor-ß and the G protein-coupled estrogen receptor. Together, these results provide evidence that E2 modulates myosin pRLC in skeletal muscle and is one mechanism by which this hormone can affect muscle contractility in females.

The myosin super-relaxed state is disrupted by estradiol deficiency.

We have used quantitative epifluorescence microscopy of fluorescent ATP to measure single-nucleotide turnover in skinned skeletal muscle fibers from mouse models of female aging and hormone treatment. Aging causes declines in muscle strength, often leading to frailty, disability, and loss of independence for the elderly. Female muscle is additionally affected by age due to reduction of ovarian hormone production with menopause. Estradiol (E2) is the key hormonal signal to skeletal muscle in females, and strength loss is attenuated by E2 treatment. To investigate E2 mechanisms on skeletal muscle, single fibers were isolated from sham-operated or ovariectomized (OVX) mice, with or without E2 treatment, and were incubated with 2'-(or-3')-O-(N-methylanthraniloyl) adenosine 5'-triphosphate (mantATP). We measured decay of mantATP fluorescence in an ATP-chase experiment, as pioneered by Cooke and coworkers, who unveiled a novel regulated state of muscle myosin characterized by slow nucleotide turnover on the order of minutes, termed the super-relaxed state (SRX). We detected a slow phase of nucleotide turnover in a portion of the myosin heads from sham fibers, consistent with SRX. Turnover was substantially faster in OVX fibers, with a turnover time constant for the slow phase of 65 ± 8s as compared to 102 ± 7s for sham fibers. 60-days E2 treatment in OVX mice substantially reversed this effect on SRX, while acute exposure of isolated muscles from OVX mice to E2 had no effect. We conclude that E2-mediated signaling reversibly regulates slow ATP turnover by myosin. Age- and hormone-related muscle functional losses may be targetable at the level of myosin structure/function for strategies to offset weakness and metabolic changes that occur with age.