On the molecular mechanism of excitation–transcription coupling in skeletal muscle

An important question in neuromuscular biology is how skeletal muscle cells decipher the stimulation pattern coming from motoneurons to define their phenotype-activating transcriptional changes in a process named excitation–transcription coupling. We have shown in adult muscle fibers that 20 Hz electrical stimulation (ES) activates a signaling cascade that starts with Cav1.1 activation, ATP release trough pannexin-1 channel, activation of purinergic receptors, and IP3-dependent Ca2+ signals inducing transcriptional changes related to muscle plasticity from fast to slow phenotype. Extracellular addition of 30 µM ATP mimics transcriptional changes induced by ES at 20 Hz. ATP release occurs in two peaks, the first around 15 s after ES and a second around 300 s after ES. In the present work, we used apyrase to hydrolyze ATP 60 s after ES, maintaining the first peak and eliminating the second peak. In this condition, transcriptional changes were abolished, indicating that the second peak is the one crucial to activate transcription. Additionally, we observed a small depolarization of fibers after ES. The addition of 30 to 100 µM external ATP also induced depolarization of muscle fibers. This depolarization was unable to activate contraction but was able to induce transcriptional changes induced by 20 Hz ES. These changes were completely inhibited by the IP3R blocker xestospongin B, suggesting that IP3-dependent events are triggered at these membrane depolarization values. Moreover, transcriptional changes induced by addition of 30 µM extracellular ATP was blocked by incubation of fibers with 25 µM Nifedipine. These results suggest that the second ATP peak observed after 20 Hz ES is responsible for transcriptional activation by inducing small depolarizations of fiber membranes that are also sensed by Cav1.1. Finally, we show evidence that downstream of purinergic receptors, PKC is activated, likely causing phosphorylation of ClC-1 chloride channels, possibly responsible for depolarization after 20 Hz.

Comparison of Shifts in Skeletal Muscle Plasticity Parameters in Horses in Three Different Muscles, in Answer to 8 Weeks of Harness Training

Training-induced follow-up of multiple muscle plasticity parameters in postural stability vs. locomotion muscles provides an integrative physiological view on shifts in the muscular metabolic machinery. It can be expected that not all muscle plasticity parameters show the same expression time profile across muscles. This knowledge is important to underpin results of metabolomic studies. Twelve non-competing Standardbred mares were subjected to standardized harness training. Muscle biopsies were taken on a non-training day before and after 8 weeks. Shifts in muscle fiber type composition and muscle fiber cross-sectional area (CSA) were compared in the m. pectoralis, the m. vastus lateralis, and the m. semitendinosus. In the m. vastus lateralis, which showed most pronounced training-induced plasticity, two additional muscle plasticity parameters (capillarization and mitochondrial density) were assessed. In the m. semitendinosus, additionally the mean minimum Feret's diameter was assessed. There was a significant difference in baseline profiles. The m. semitendinosus contained less type I and more type IIX fibers compatible with the most pronounced anaerobic profile. Though no baseline fiber type-specific and overall mean CSA differences could be detected, there was a clear post-training decrease in fiber type specific CSA, most pronounced for the m. vastus lateralis, and this was accompanied by a clear increase in capillary supply. No shifts in mitochondrial density were detected. The m. semitendinosus showed a decrease in fiber type specific CSA of type IIAX fibers and a decrease of type I fiber Feret's diameter as well as mean minimum Feret's diameter. The training-induced increased capillary supply in conjunction with a significant decrease in muscle fiber CSA suggests that the muscular machinery models itself toward an optimal smaller individual muscle fiber structure to receive and process fuels that can be swiftly delivered by the circulatory system. These results are interesting in view of the recently identified important fuel candidates such as branched-chain amino acids, aromatic amino acids, and gut microbiome-related xenobiotics, which need a rapid gut–muscle gateway to reach these fibers and are less challenging for the mitochondrial system. More research is needed with that respect. Results also show important differences between muscle groups with respect to baseline and training-specific modulation.

Cannabinoid receptor 1 expression is higher in muscle of old vs. young males, and increases upon resistance exercise in older adults

Aged skeletal muscle undergoes metabolic and structural alterations eventually resulting in a loss of muscle strength and mass, i.e. age-related sarcopenia. Therefore, novel targets for muscle growth purposes in elderly are needed. Here, we explored the role of the cannabinoid system in muscle plasticity through the expression of muscle cannabinoid receptors (CBs) in young and old humans. The CB1 expression was higher (+ 25%; p = 0.04) in muscle of old (≥ 65 years) vs. young adults (20–27 years), whereas CB2 was not differently expressed. Furthermore, resistance exercise tended to increase the CB1 (+ 11%; p = 0.055) and CB2 (+ 37%; p = 0.066) expression in muscle of older adults. Interestingly, increases in the expression of CB2 following resistance exercise positively correlated with changes in key mechanisms of muscle homeostasis, such as catabolism (FOXO3a) and regenerative capacity (Pax7, MyoD). This study for the first time shows that CB1 is differentially expressed with aging and that changes in CB2 expression upon resistance exercise training correlate with changes in mediators that play a central role in muscle plasticity. These data confirm earlier work in cells and mice showing that the cannabinoid system might orchestrate muscle growth, which is an incentive to further explore CB-based strategies that might counteract sarcopenia.

Nuclear factor E2-related factor 2 (NRF2) deficiency accelerates fast fibre type transition in soleus muscle during space flight

Microgravity induces skeletal muscle atrophy, particularly in the soleus muscle, which is predominantly composed of slow-twitch myofibre (type I) and is sensitive to disuse. Muscle atrophy is commonly known to be associated with increased production of reactive oxygen species. However, the role of NRF2, a master regulator of antioxidative response, in skeletal muscle plasticity during microgravity-induced atrophy, is not known. To investigate the role of NRF2 in skeletal muscle within a microgravity environment, wild-type and Nrf2-knockout (KO) mice were housed in the International Space Station for 31 days. Gene expression and histological analyses demonstrated that, under microgravity conditions, the transition of type I (oxidative) muscle fibres to type IIa (glycolytic) was accelerated in Nrf2-KO mice without affecting skeletal muscle mass. Therefore, our results suggest that NRF2 affects myofibre type transition during space flight.

Dose–Response Effect of Vibratory Stimulus on Synaptic and Muscle Plasticity in a Middle-Aged Murine Model

Whole body vibration plays a central role in many work categories and can represent a health risk to the musculoskeletal system and peripheral nervous system. However, studies in animal and human models have shown that vibratory training, experimentally and/or therapeutically induced, can exert beneficial effects on the whole body, as well as improve brain functioning and reduce cognitive decline related to the aging process. Since the effects of vibratory training depend on several factors, such as vibration frequency and vibration exposure time, in this work, we investigated whether the application of three different vibratory protocols could modulate synaptic and muscle plasticity in a middle-aged murine model, counteracting the onset of early symptoms linked to the aging process. To this end, we performed in vitro electrophysiological recordings of the field potential in the CA1 region of mouse hippocampal slices, as well as histomorphometric and ultrastructural analysis of muscle tissue by optic and transmission electron microscopy, respectively. Our results showed that protocols characterized by a low vibration frequency and/or a longer recovery time exert positive effects at both hippocampal and muscular level, and that these effects improve significantly by varying both parameters, with an action comparable with a dose–response effect. Thus, we suggested that vibratory training may be an effective strategy to counteract cognitive impairment, which is already present in the early stages of the aging process, and the onset of sarcopenia, which is closely related to a sedentary lifestyle. Future studies are needed to understand the underlying molecular mechanisms and to determine an optimal vibratory training protocol.

Nuclear Mechanotransduction in Skeletal Muscle

Skeletal muscle is composed of multinucleated, mature muscle cells (myofibers) responsible for contraction, and a resident pool of mononucleated muscle cell precursors (MCPs), that are maintained in a quiescent state in homeostatic conditions. Skeletal muscle is remarkable in its ability to adapt to mechanical constraints, a property referred as muscle plasticity and mediated by both MCPs and myofibers. An emerging body of literature supports the notion that muscle plasticity is critically dependent upon nuclear mechanotransduction, which is transduction of exterior physical forces into the nucleus to generate a biological response. Mechanical loading induces nuclear deformation, changes in the nuclear lamina organization, chromatin condensation state, and cell signaling, which ultimately impacts myogenic cell fate decisions. This review summarizes contemporary insights into the mechanisms underlying nuclear force transmission in MCPs and myofibers. We discuss how the cytoskeleton and nuclear reorganizations during myogenic differentiation may affect force transmission and nuclear mechanotransduction. We also discuss how to apply these findings in the context of muscular disorders. Finally, we highlight current gaps in knowledge and opportunities for further research in the field.

Changes in Gene Expression of the MCU Complex Are Induced by Electrical Stimulation in Adult Skeletal Muscle

The slow calcium transient triggered by low-frequency electrical stimulation (ES) in adult muscle fibers and regulated by the extracellular ATP/IP3/IP3R pathway has been related to muscle plasticity. A regulation of muscular tropism associated with the MCU has also been described. However, the role of transient cytosolic calcium signals and signaling pathways related to muscle plasticity over the regulation of gene expression of the MCU complex (MCU, MICU1, MICU2, and EMRE) in adult skeletal muscle is completely unknown. In the present work, we show that 270 0.3-ms-long pulses at 20-Hz ES (and not at 90 Hz) transiently decreased the mRNA levels of the MCU complex in mice flexor digitorum brevis isolated muscle fibers. Importantly, when ATP released after 20-Hz ES is hydrolyzed by the enzyme apyrase, the repressor effect of 20 Hz on mRNA levels of the MCU complex is lost. Accordingly, the exposure of muscle fibers to 30 μM exogenous ATP produces the same effect as 20-Hz ES. Moreover, the use of apyrase in resting conditions (without ES) increased mRNA levels of MCU, pointing out the importance of extracellular ATP concentration over MCU mRNA levels. The use of xestospongin B (inhibitor of IP3 receptors) also prevented the decrease of mRNA levels of MCU, MICU1, MICU2, and EMRE mediated by a low-frequency ES. Our results show that the MCU complex can be regulated by electrical stimuli in a frequency-dependent manner. The changes observed in mRNA levels may be related to changes in the mitochondria, associated with the phenotypic transition from a fast- to a slow-type muscle, according to the described effect of this stimulation frequency on muscle phenotype. The decrease in mRNA levels of the MCU complex by exogenous ATP and the increase in MCU levels when basal ATP is reduced with the enzyme apyrase indicate that extracellular ATP may be a regulator of the MCU complex. Moreover, our results suggest that this regulation is part of the axes linking low-frequency stimulation with ATP/IP3/IP3R.

β2-Adrenergic Signaling Modulates Mitochondrial Function and Morphology in Skeletal Muscle in Response to Aerobic Exercise

The molecular mechanisms underlying skeletal muscle mitochondrial adaptations induced by aerobic exercise (AE) are not fully understood. We have previously shown that AE induces mitochondrial adaptations in cardiac muscle, mediated by sympathetic stimulation. Since direct sympathetic innervation of neuromuscular junctions influences skeletal muscle homeostasis, we tested the hypothesis that β2-adrenergic receptor (β2-AR)-mediated sympathetic activation induces mitochondrial adaptations to AE in skeletal muscle. Male FVB mice were subjected to a single bout of AE on a treadmill (80% Vmax, 60 min) under β2-AR blockade with ICI 118,551 (ICI) or vehicle, and parameters of mitochondrial function and morphology/dynamics were evaluated. An acute bout of AE significantly increased maximal mitochondrial respiration in tibialis anterior (TA) isolated fiber bundles, which was prevented by β2-AR blockade. This increased mitochondrial function after AE was accompanied by a change in mitochondrial morphology towards fusion, associated with increased Mfn1 protein expression and activity. β2-AR blockade fully prevented the increase in Mfn1 activity and reduced mitochondrial elongation. To determine the mechanisms involved in mitochondrial modulation by β2-AR activation in skeletal muscle during AE, we used C2C12 myotubes, treated with the non-selective β-AR agonist isoproterenol (ISO) in the presence of the specific β2-AR antagonist ICI or during protein kinase A (PKA) and Gαi protein blockade. Our in vitro data show that β-AR activation significantly increases mitochondrial respiration in myotubes, and this response was dependent on β2-AR activation through a Gαs-PKA signaling cascade. In conclusion, we provide evidence for AE-induced β2-AR activation as a major mechanism leading to alterations in mitochondria function and morphology/dynamics. β2-AR signaling is thus a key-signaling pathway that contributes to skeletal muscle plasticity in response to exercise.