Sergey A. Tyganov

The depolarization of the sarcolemma is one of the first effects of unloading on skeletal muscle. We hypothesized that unloading-induced activation of the dihydropyridine receptor (DHPR), a voltage-sensitive L-type Ca²⁺ channel, and depolarization of the sarcolemma trigger intracellular Ca²⁺ release from the sarcoplasmic reticulum and activation of Ca²⁺-dependent signaling pathways, resulting in muscle atrophy. Nifedipine, a DHPR calcium channel blocker, was used to study the role of DHPR in the regulation of signaling pathways during three days of rat soleus muscle unloading/hindlimb suspension. Inhibition of the DHPR during unloading attenuates the decrease in soleus muscle contractile properties, prevents the accumulation of ATP, ROS, and Ca²⁺ content in the sarcoplasm and the mitochondria, and blocks the decrease in PGC1alpha mRNA expression and Junctophilin-1 (JP1) proteolysis. In nifedipine-treated rats, the improvement of the unloaded soleus muscle contractile properties could be mediated by blocking the calpain-mediated degradation of the cytoskeletal proteins. DHPR blocking could be one of the future directions for the preservation of contractile properties of inactive/unloaded muscle.

Dihydropyridine receptors (DHPR) participate in changes of the membrane potential during muscle unloading. We used nifedipine, a dihydropyridine derivative and Ca²⁺ antagonist, to study the role of L‑type Ca²⁺ channels (Cav1.1, DHPR) in regulation of energy and Ca²⁺ metabolism and impact on functional properties of m. soleus during unloading. Male rats were divided into 3 groups (n = 16 in each group): vivarium control with placebo (C), 3-day hindlimb suspension with placebo (HS), and 3-day hindlimb suspension with intraperitoneal administration of the nifedipine (N). It was found that nifedipine administration during hindlimb suspension produced the following effects: (1) it decreased the ATP accumulation; (2) prevented the reduction of the maximum force of a single contraction and the time of contraction, and (3) prevented the increase in the content of intramitochondrial and myoplasmic calcium. Thus, DHPR participates in energy and Ca²⁺ metabolism and affects the functional properties of muscles.

The elimination of ground reaction force (support withdrawal) vastly affects slow postural muscles in terms of their regulation and structure. One of the effects of support withdrawal in this study was an immediate postural muscle inactivation, followed by the daily gradual development of spontaneous activity of the slow postural soleus muscle in response to rat hindlimb suspension to mimic space flight. The origin of this activity is somewhat akin to muscle spasticity after spinal cord injuries and is the result of KCC2 content decline in the spinal cord’s motor neurons. However, the physiological consequences of unloading-induced spontaneous activity remain unexplored. We have conducted an experiment with the administration of a highly specific KCC2 activator during 7-day unloading. For this experiment, 32 male Wistar rats were divided into 4 groups: C+placebo, C+CLP-290 (100 mg/kg b w), 7HS+placebo, and 7HS+CLP—hindlimb-suspended group with CLP-290 administration (100 mg/kg b w). The soleus muscles of the animals were dissected and analyzed for several proteostasis- and metabolism-related parameters. CLP-290 administration to the unloaded animals led to the upregulation of AMPK downstream (p-ACC) and mTOR targets (p-p70S6k and p-4E-BP) and an enhanced PGC1alpha decrease vs. the 7HS group, but neither prevented nor enhanced atrophy of the soleus muscle or myofiber CSA.

It is known that activation of protein synthesis and hypertrophy of muscle fibers in response to mechanical stress is realized through an anabolic mTORC1-dependent signaling pathway. However, mechanosensors through which a mechanical signal can be perceived and further transmitted to the mTORC1-dependent signaling pathway (mechanotransduction) are poorly identified. Mechanically activated (MA) ion channels are candidates for the role of such sarcolemmal mechanosensors. In this regard, the aim of this study was to investigate the potential role of MA channels (Piezo1) in the activation of the mTORC1-dependent pathway in the isolated rat soleus muscle in response to mechanical stress. Wistar rats were divided into 3 groups: (1) “Control” (animal muscles were not exposed to MA channel inhibitor or Piezo1 channel activator); (2) “Gadolinium” (animal muscles were incubated with gadolinium chloride, MA channel inhibitor), and (3) “Yoda” (animal muscles were incubated with Yoda1, Piezo1 MA channel activator). In rats from each group, m. soleus was isolated from the left limb and incubated in the appropriate solution without mechanical stress in the form of a series of stretching (“Rest”); m. soleus from the right limb was subjected to a series of stretching (“Stretch”) and then incubated in the appropriate solution. Phosphorylation of mTORC1 targets (p70S6K, rpS6, and 4E-BP1) in rat m. soleus was determined by PAAG electrophoresis and immunoblotting. A series of passive stretches of isolated m. soleus led to an increase in the phosphorylation of p70S6K, its substrate rpS6, and 4E-BP1 by 38.5, 168 and 112%, respectively, compared to the muscle that was not subjected to mechanical stress. Incubation of the muscles with gadolinium completely prevented the activation of mTORC1 markers caused by a series of stretches. Incubation of m. soleus in a solution with Yoda1 resulted in a decrease in the mechano-dependent phosphorylation of p70S6K, rpS6 and 4E-BP1 compared to the muscle that was not exposed to Yoda1. Thus, the methodological approach used in this work did not reveal the participation of Piezo1 in mechano-anabolic coupling in rat m. soleus.

Skeletal muscle abnormalities and atrophy during unloading are accompanied by the accumulation of excess calcium in the sarcoplasm. We hypothesized that calcium accumulation may occur, among other mechanisms, due to the inhibition of sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) activity. Consequently, the use of the SERCA activator will reduce the level of calcium in the sarcoplasm and prevent the negative consequences of muscle unloading. Wistar rats were randomly assigned into one of three groups (eight rats per group): control rats with placebo (C), 7 days of unloading/hindlimb suspension with placebo (7HS), and 7 days of unloading treated with SERCA activator CDN1163 (7HSC). After seven days of unloading the soleus muscle, the 7HS group displayed increased fatigue in the ex vivo test, a significant increase in the level of calcium-dependent CaMK II phosphorylation and the level of tropomyosin oxidation, as well as a decrease in the content of mitochondrial DNA and protein, slow-type myosin mRNA, and the percentage of slow-type muscle fibers. All of these changes were prevented in the 7HSC group. Moreover, treatment with CDN1163 blocked a decrease in the phosphorylation of p70S6k, an increase in eEF2 phosphorylation, and an increase in MuRF-1 mRNA expression. Nevertheless, there were no differences in the degree of fast and slow muscle fiber atrophy between the 7HS and 7HSC groups. Conclusion: SERCA activation during 7 days of unloading prevented an increase in soleus fatigue, the decrease of slow-type myosin, mitochondrial markers, and markers of calcium homeostasis but had no effect on muscle atrophy.

Dysfunction of skeletal muscles and their atrophy under unloading are accompanied by excess calcium accumulation in the myoplasm of muscle fibers. We hypothesized that calcium accumulation may occur, among other reasons, due to inhibition of SERCA activity under muscle unloading. In this case, the use of a sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) activator would reduce the calcium level in the myoplasm and prevent the consequences of unloading. Male Wistar rats were divided into 3 groups: vivarium control with placebo administration (C, n = 8), 7-day suspension group with placebo administration (7HS, n = 8), and 7-day suspension group with intraperitoneal administration of the SERCA activator CDN1163 (7HS + CDN, 50 mg/kg, n = 8). One m. soleus of each rat was frozen in liquid nitrogen, the second was tested for functional properties. 7HS rats demonstrated an increased m. soleus fatigue in the ex vivo test, a significant increase in mRNA and the number of fast-twitch muscle fibers, an increase in the level of calcium-dependent CaMK II phosphorylation and tropomyosin oxidation, as well as a decrease in mitochondrial DNA and protein levels. All these changes were prevented in the 7HS + CDN group administered with SERCA activator. Conclusion: 7-day SERCA activator administration does not delay m. soleus atrophy but prevents the development of its fatigue, probably due to prevention of a reduction in the number of type I fibers and mitochondrial biogenesis markers.

Skeletal muscle disuse leads to pathological muscle activity as well as to slow-to-fast fiber-type transformation. Fast-type fibers are more fatigable than slow-type, so this transformation leads to a decline in muscle function. Prochlorperazine injections previously were shown to attenuate autonomous rat soleus muscle electrical activity under unloading conditions. In this study, we found that prochlorperazine blocks slow-to-fast fiber-type transformation in disused skeletal muscles of rats, possibly through affecting calcium and ROS-related signaling.

Currently, no ideal treatment exists to combat skeletal muscle disuse-induced atrophy and loss of strength. Because the activity of AMP-activated protein kinase (AMPK) in rat soleus muscle is suppressed at the early stages of disuse, we hypothesized that pre-treatment of rats with metformin (an AMPK activator) would exert beneficial effects on skeletal muscle during disuse. Muscle disuse was performed via hindlimb suspension (HS). Wistar rats were divided into four groups: (1) control (C), (2) control + metformin for 10 days (C+Met), (3) HS for 7 days (HS), (4) metformin treatment for 7 days before HS and during the first 3 days of 1-week HS (HS+Met). Anabolic and catabolic markers were assessed using WB and RT-PCR. Treatment with metformin partly prevented an HS-induced decrease in rat soleus weight and size of slow-twitch fibers. Metformin prevented HS-related slow-to-fast fiber transformation. Absolute soleus muscle force in the HS+Met group was increased vs. the HS group. GSK-3β (Ser9) phosphorylation was significantly increased in the HS+Met group vs. the HS group. Metformin pre-treatment partly prevented HS-induced decrease in 18S+28S rRNA content and attenuated upregulation of calpain-1 and ubiquitin. Thus, pre-treatment of rats with metformin can ameliorate disuse-induced reductions in soleus muscle weight, the diameter of slow-type fibers, and absolute muscle strength.

Disuse muscle atrophy is usually accompanied by changes in skeletal muscle structure, signaling, and contractile potential. Different models of muscle unloading can provide valuable information, but the protocols of experiments with complete immobilization are not physiologically representative of a sedentary lifestyle, which is highly prevalent among humans now. In the current study, we investigated the potential effects of restricted activity on the mechanical characteristics of rat postural (soleus) and locomotor (extensor digitorum longus, EDL) muscles. The restricted-activity rats were kept in small Plexiglas cages (17.0 × 9.6 × 13.0 cm) for 7 and 21 days. After this, soleus and EDL muscles were collected for ex vivo mechanical measurements and biochemical analysis. We demonstrated that while a 21-day movement restriction affected the weight of both muscles, in soleus muscle we observed a greater decrease. The maximum isometric force and passive tension in both muscles also significantly changed after 21 days of movement restriction, along with a decrease in the level of collagen 1 and 3 mRNA expression. Furthermore, the collagen content itself changed only in soleus after 7 and 21 days of movement restriction. With regard to cytoskeletal proteins, in our experiment we observed a significant decrease in telethonin in soleus, and a similar decrease in desmin and telethonin in EDL. We also observed a shift towards fast-type myosin heavy chain expression in soleus, but not in EDL. In summary, in this study we showed that movement restriction leads to profound specific changes in the mechanical properties of fast and slow skeletal muscles. Future studies may include evaluation of signaling mechanisms regulating the synthesis, degradation, and mRNA expression of the extracellular matrix and scaffold proteins of myofibers.