Obscurin is a giant sarcomeric protein expressed in striated muscles known to establish several interactions with other proteins of the sarcomere, but also with proteins of the sarcoplasmic reticulum and costameres. Here, we report experiments aiming to better understand the contribution of obscurin to skeletal muscle fibers, starting with a detailed characterization of the diaphragm muscle function, which we previously reported to be the most affected muscle in obscurin (Obscn) KO mice. Twitch and tetanus tension were not significantly different in the diaphragm of WT and Obscn KO mice, while the time to peak (TTP) and half relaxation time (HRT) were prolonged. Differences in force-frequency and force-velocity relationships and an enhanced fatigability are observed in an Obscn KO diaphragm with respect to WT controls. Voltage clamp experiments show that a sarcoplasmic reticulum’s Ca²⁺ release and SERCA reuptake rates were decreased in muscle fibers from Obscn KO mice, suggesting that an impairment in intracellular Ca²⁺ dynamics could explain the observed differences in the TTP and HRT in the diaphragm. In partial contrast with previous observations, Obscn KO mice show a normal exercise tolerance, but fiber damage, the altered sarcomere ultrastructure and M-band disarray are still observed after intense exercise.

Calsequestrin 1 (CASQ1) and Ryanodine receptor 1 (RYR1) are two of the main players in excitation–contraction (EC) coupling. CASQ1-knockout mice and mice carrying a mutation in RYR1 (Y522S) linked to human malignant hyperthermia susceptibility (MHS) both suffer lethal hypermetabolic episodes when exposed to halothane (MHS crises) and to environmental heat (heat stroke, HS). The phenotype of Y522S is more severe than that of CASQ1-null mice. As MHS and HS are hypermetabolic responses, we studied the metabolism of adult CASQ1-null and Y522S mice using wild-type (WT) mice as controls. We found that CASQ1-null and Y522S mice have increased food consumption and higher core temperature at rest. By indirect calorimetry, we then verified that CASQ1-null and Y522S mice show an increased oxygen consumption and a lower respiratory quotient (RQ). The accelerated metabolism of CASQ1-null and Y522S mice was also accompanied with a reduction in body fat. Moreover, both mouse models displayed increased oxygen consumption and a higher core temperature during heat stress. The results collected suggest that metabolic rate, oxygen consumption, and body temperature at rest, all more elevated in Y522S than in CASQ1-null mice, could possibly be used as predictors of the level of susceptibility to hyperthermic crises of mice (and possibly humans).

Store-operated Ca²⁺ entry (SOCE) is a mechanism that allows muscle fibers to recover external Ca²⁺, which first enters the cytoplasm and then, via SERCA pump, also refills the depleted intracellular stores (i.e., the sarcoplasmic reticulum, SR). We recently discovered that SOCE is mediated by Calcium Entry Units (CEUs), intracellular junctions formed by: (i) SR stacks containing STIM1; and (ii) I-band extensions of the transverse tubule (TT) containing Orai1. The number and size of CEUs increase during prolonged muscle activity, though the mechanisms underlying exercise-dependent formation of new CEUs remain to be elucidated. Here, we first subjected isolated extensor digitorum longus (EDL) muscles from wild type mice to an ex vivo exercise protocol and verified that functional CEUs can assemble also in the absence of blood supply and innervation. Then, we evaluated whether parameters that are influenced by exercise, such as temperature and pH, may influence the assembly of CEUs. Results collected indicate that higher temperature (36 °C vs. 25 °C) and lower pH (7.2 vs. 7.4) increase the percentage of fibers containing SR stacks, the n. of SR stacks/area, and the elongation of TTs at the I band. Functionally, assembly of CEUs at higher temperature (36 °C) or at lower pH (7.2) correlates with increased fatigue resistance of EDL muscles in the presence of extracellular Ca²⁺. Taken together, these results indicate that CEUs can assemble in isolated EDL muscles and that temperature and pH are two of the possible regulators of CEU formation.

Environmental heat-stroke (HS) is a life-threatening response often triggered by hot and humid weather. Several lines of evidence indicate that HS is caused by excessive heat production in skeletal muscle, which in turn is the result of abnormal Ca²⁺ leak from the sarcoplasmic reticulum (SR) and excessive production of oxidative species of oxygen and nitrogen. As a high fat diet is known to increase oxidative stress, the objective of the present study was to investigate the effects of 3 months of high-fat diet (HFD) on the HS susceptibility of wild type (WT) mice. HS susceptibility was tested in an environmental chamber where 4 months old WT mice were exposed to heat stress (41 °C for 1 h). In comparison with mice fed with a regular diet, mice fed with HFD showed: (a) increased body weight and accumulation of adipose tissue; (b) elevated oxidative stress in skeletal muscles; (c) increased heat generation and oxygen consumption during exposure to heat stress; and finally, (d) enhanced sensitivity to both temperature and caffeine of isolated muscles during in-vitro contracture test. These data (a) suggest that HFD predisposes WT mice to heat stress and (b) could have implications for guidelines regarding food intake during periods of intense environmental heat.

The precise arrangement and peculiar interaction of transverse tubule (T-tubule) and sarcoplasmic reticulum (SR) membranes efficiently guarantee adequate contractile properties of skeletal muscle fibers. Fast muscle fibers from mice lacking calsequestrin 1 (CASQ1) are characterized by the profound ultrastructural remodeling of T-tubule/SR junctions. This study investigates the role of CASQ1, an essential component of calcium release units (CRUs), in the postnatal development of muscle fibers. By using CASQ1-knockout mice, we examined the maturation of CRUs and the involvement of different junctional proteins in the juxtaposition of the membrane system. Our morphological investigation of both wild-type (WT) and CASQ1-null extensor digitorum longus (EDL) fibers, from 1 week to 4 months of age, yielded noteworthy findings. Firstly, we observed that the absence of CASQ1 hindered the full maturation of CRUs, despite the correct localization of key junctional components (ryanodine receptor, dihydropyridine receptor, and triadin) to the junctional SR in adult animals. Furthermore, analysis of protein expression profiles related to T-tubule biogenesis and organization (junctophilin 1, amphiphysin 2, caveolin 3, and mitsugumin 29) demonstrated delayed progression in their expression during postnatal development in the absence of CASQ1, suggesting the impaired maturation of CRUs. The absence of CASQ1 directly impacts the proper assembly of CRUs during development and influences the expression and coordination of other proteins involved in T-tubule biogenesis and organization.

Ozone (O ₃ ) and polystyrene nanoparticles (PNPs) display diffusive behavior that leads to toxicity in many tissues of the adult organism. In this study, we evaluated the interactions between atmospheric pollutants and human muscle, using human myogenic progenitor cells (huMPCs) derived from vastus lateralis skeletal muscle. To achieve this goal, O ₃ and PNPs were first tested individually to understand the impact of the single pollutant on huMPCs. Subsequently, pollutants were tested in combination to examine their potential synergistic effects, given the simultaneous presence of multiple pollutants in the atmosphere. Cell viability was assessed after treatment with O ₃ and PNPs, and it seems to be significantly affected in huMPCs exposed to the pollutants, tested both alone and in combination. Similarly, the differentiation capability of treated huMPCs was evaluated, and it was found to be significantly reduced compared to controls, especially when O ₃ and PNPs are tested in combination. Furthermore, an alteration in the expression of microRNAs involved in myogenic cells’ proliferation and differentiation pathways was found. In light of the correlation between pollutants and increased oxidant levels, and O ₃ ’s ability to produce the superoxide anion, superoxide anion levels in huMPCs exposed to pollutants were also assessed, and an increase in this oxidant was recorded. Thus, this preliminary study suggests that exposure to O ₃ and PNPs affects human muscle, as it alters all the analyzed parameters in huMPCs, filling a gap in the current literature.