Reciprocal Homer1a and Homer2 Isoform Expression Is a Key Mechanism for Muscle Soleus Atrophy in Spaceflown Mice

The molecular mechanisms of skeletal muscle atrophy under extended periods of either disuse or microgravity are not yet fully understood. The transition of Homer isoforms may play a key role during neuromuscular junction (NMJ) imbalance/plasticity in space. Here, we investigated the expression pattern of Homer short and long isoforms by gene array, qPCR, biochemistry, and laser confocal microscopy in skeletal muscles from male C57Bl/N6 mice (n = 5) housed for 30 days in space (Bion-flight = BF) compared to muscles from Bion biosatellite on the ground-housed animals (Bion ground = BG) and from standard cage housed animals (Flight control = FC). A comparison study was carried out with muscles of rats subjected to hindlimb unloading (HU). Gene array and qPCR results showed an increase in Homer1a transcripts, the short dominant negative isoform, in soleus (SOL) muscle after 30 days in microgravity, whereas it was only transiently increased after four days of HU. Conversely, Homer2 long-form was downregulated in SOL muscle in both models. Homer immunofluorescence intensity analysis at the NMJ of BF and HU animals showed comparable outcomes in SOL but not in the extensor digitorum longus (EDL) muscle. Reduced Homer crosslinking at the NMJ consequent to increased Homer1a and/or reduced Homer2 may contribute to muscle-type specific atrophy resulting from microgravity and HU disuse suggesting mutual mechanisms.

JNK activation in TA and EDL muscle is load-dependent in rats receiving identical excitation patterns

As the excitation–contraction coupling is inseparable during voluntary exercise, the relative contribution of the mechanical and neural input on hypertrophy-related molecular signalling is still poorly understood. Herein, we use a rat in-vivo strength exercise model with an electrically-induced standardized excitation pattern, previously shown to induce a load-dependent increase in myonuclear number and hypertrophy, to study acute effects of load on molecular signalling. We assessed protein abundance and specific phosphorylation of the four protein kinases FAK, mTOR, p70S6K and JNK after 2, 10 and 28 min of a low- or high-load contraction, in order to assess the effects of load, exercise duration and muscle-type on their response to exercise. Specific phosphorylation of mTOR, p70S6K and JNK was increased after 28 min of exercise under the low- and high-load protocol. Elevated phosphorylation of mTOR and JNK was detectable already after 2 and 10 min of exercise, respectively, but greatest after 28 min of exercise, and JNK phosphorylation was highly load-dependent. The abundance of all four kinases was higher in TA compared to EDL muscle, p70S6K abundance was increased after exercise in a load-independent manner, and FAK and JNK abundance was reduced after 28 min of exercise in both the exercised and control muscles. In conclusion, the current study shows that JNK activation after a single resistance exercise is load-specific, resembling the previously reported degree of myonuclear accrual and muscle hypertrophy with repetition of the exercise stimulus.

IL-6 Protein Expression is Increased in Fast Glycolytic Muscle Fibers Over Time in the SOD1G93A Mouse model.

The deregulation of the inflammatory cytokine interleukin (IL)-6 has been associated to a variety of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). The aim of this work was to analyze the variation of IL-6 levels in blood and damaged tissues during the course of the disease. We studied IL-6 protein expression in spinal cord, extensor digital longus (EDL) muscle and soleus (SOL) muscle of the SOD1G93A animal model at four stages of the disease by western blot. Concurrently, we analyzed IL-6 gene and protein expression in blood of ALS patients, healthy subjects and patients with other neuropathies through RTqPCR and ELISA. The results revealed different expression patterns depending on both the tissue analyzed and the stage of the disease, showing increasing IL-6 levels in EDL muscle over time. Moreover, lower IL-6 levels in blood were found in ALS patients. The decreased levels of IL-6 in blood from ALS patients could suggest that IL-6 might not be the main pro-inflammatory biomarker in the last stages in whole blood. In contrast, IL-6 may play a main role in fast glycolytic muscle fibers associated with muscle atrophy, suggesting that modulation of IL-6 in this tissue could be a potential target for anti-inflammatory therapies in ALS.

Fast skeletal myosin-binding protein-C regulates fast skeletal muscle contraction

Fast skeletal myosin-binding protein-C (fMyBP-C) is one of three MyBP-C paralogs and is predominantly expressed in fast skeletal muscle. Mutations in the gene that encodes fMyBP-C, MYBPC2, are associated with distal arthrogryposis, while loss of fMyBP-C protein is associated with diseased muscle. However, the functional and structural roles of fMyBP-C in skeletal muscle remain unclear. To address this gap, we generated a homozygous fMyBP-C knockout mouse (C2−/−) and characterized it both in vivo and in vitro compared to wild-type mice. Ablation of fMyBP-C was benign in terms of muscle weight, fiber type, cross-sectional area, and sarcomere ultrastructure. However, grip strength and plantar flexor muscle strength were significantly decreased in C2−/− mice. Peak isometric tetanic force and isotonic speed of contraction were significantly reduced in isolated extensor digitorum longus (EDL) from C2−/− mice. Small-angle X-ray diffraction of C2−/− EDL muscle showed significantly increased equatorial intensity ratio during contraction, indicating a greater shift of myosin heads toward actin, while MLL4 layer line intensity was decreased at rest, indicating less ordered myosin heads. Interfilament lattice spacing increased significantly in C2−/− EDL muscle. Consistent with these findings, we observed a significant reduction of steady-state isometric force during Ca2+-activation, decreased myofilament calcium sensitivity, and sinusoidal stiffness in skinned EDL muscle fibers from C2−/− mice. Finally, C2−/− muscles displayed disruption of inflammatory and regenerative pathways, along with increased muscle damage upon mechanical overload. Together, our data suggest that fMyBP-C is essential for maximal speed and force of contraction, sarcomere integrity, and calcium sensitivity in fast-twitch muscle.

JNK activation in TA and EDL muscle is load-dependent in rats receiving identical excitation patterns

ABSTACTAimAs the excitation-contraction coupling is inseparable during voluntary exercise, the relative contribution of the mechanical and neural input is poorly understood. Herein, we use a rat in-vivo strength training setup with an electrically induced standardized excitation pattern previously shown to lead to a load-dependent increase in myonuclear number and hypertrophy, to study acute effects of load per se on molecular signalling.MethodsAnaesthetized rats were subjected to unliteral identical electrically-paced contractions of the TA and EDL muscles under a high or low load for a duration of 2, 10 or 28-minutes. Muscle soluble proteins were extracted, and abundance and specific phosphorylations of FAK, mTOR, p70S6K and JNK were measured. Effects of exercise, load, muscle and exercise duration were assessed.ResultsSpecific phosphorylation of S2448-mTOR, T421/S424-p70S6K and T183/Y185-JNK was increased after 28-minutes of exercise under the high- and low-load protocol. Elevated phosphorylation of mTOR and JNK was detectable already after 2 and 10 minutes of exercise, respectively, but greatest after 28-minutes of exercise. T183/Y185-JNK and S2448-mTOR demonstrated a load-dependent increase in phosphorylation in the exercised muscles that for mTOR depended on muscle type. The abundance of all four kinases was higher in TA compared to EDL muscle. FAK and JNK abundance was reduced after 28 minutes of exercise in both the exercised and control muscle.ConclusionThe current study shows that JNK and mTOR activation is load-driven, and together with muscle-type specific mTOR and p70S6K effects it may drive muscle-type specific exercise and load-responses.

Different fuel regulation in two types of myofiber results in different antioxidant strategies in Daurian ground squirrels (Spermophilus dauricus) during hibernation

We previously showed that different skeletal muscles in Daurian ground squirrels (Spermophilus dauricus) possess different antioxidant strategies during hibernation; however, the reason for these varied strategies remains unclear. To clarify this issue, we studied REDD1, FOXO4, PGC-1α, FOXO1, and atrogin-1 proteins to determine the potential cause of the different antioxidant strategies in Daurian ground squirrels during hibernation, and to clarify whether different strategies affect atrophy-related signals. Results showed that the soleus (SOL) muscle experienced intracellular hypoxia during interbout arousal, but no oxidative stress. This may be due to increased PGC-1α expression enhancing antioxidant capacity in the SOL under hypoxic conditions. Extensor digitorum longus (EDL) muscle showed no change in oxidative stress, hypoxia, or antioxidant capacity during hibernation. The FOXO1 and PGC-1α results strongly suggested differentially regulated fuel metabolism in the SOL and EDL muscles during hibernation, i.e., enhanced lipid oxidation and maintained anaerobic glycolysis, respectively. Atrogin-1 expression did not increase during hibernation in either the SOL or EDL, indicating that protein synthesis was not inhibited by atrogin-1. Thus, our results suggest that different fuel regulation may be one mechanism related to antioxidant defense strategy formation in different kinds of skeletal muscle fibers of Daurian ground squirrels during hibernation.

Neuroprotective Fragment C of Tetanus Toxin Modulates IL-6 in an ALS Mouse Model

Neuroinflammation plays a significant role in amyotrophic lateral sclerosis (ALS) pathology, leading to the development of therapies targeting inflammation in recent years. Our group has studied the tetanus toxin C-terminal fragment (TTC) as a therapeutic molecule, showing neuroprotective properties in the SOD1G93A mouse model. However, it is unknown whether TTC could have some effect on inflammation. The objective of this study was to assess the effect of TTC on the regulation of inflammatory mediators to elucidate its potential role in modulating inflammation occurring in ALS. After TTC treatment in SOD1G93A mice, levels of eotaxin-1, interleukin (IL)-2, IL-6 and macrophage inflammatory protein (MIP)-1 alpha (α) and galectin-1 were analyzed by immunoassays in plasma samples, whilst protein expression of caspase-1, IL-1β, IL-6 and NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) was measured in the spinal cord, extensor digitorum longus (EDL) muscle and soleus (SOL) muscle. The results showed reduced levels of IL-6 in spinal cord, EDL and SOL in treated SOD1G93A mice. In addition, TTC showed a different role in the modulation of NLRP3 and caspase-1 depending on the tissue analyzed. In conclusion, our results suggest that TTC could have a potential anti-inflammatory effect by reducing IL-6 levels in tissues drastically affected by the disease. However, further research is needed to study more in depth the anti-inflammatory effect of TTC in ALS.

The role of group I p21-activated kinases in contraction-stimulated skeletal muscle glucose transport

AimMuscle contraction stimulates skeletal muscle glucose transport. Since it occurs independently of insulin, it is an important alternative pathway to increase glucose uptake in insulin-resistant states, but the intracellular signalling mechanisms are not fully understood. Muscle contraction activates group I p21-activated kinases (PAKs) in mouse and human skeletal muscle. PAK1 and PAK2 are downstream targets of Rac1, which is a key regulator of contraction-stimulated glucose transport. Thus, PAK1 and PAK2 could be downstream effectors of Rac1 in contraction-stimulated glucose transport. The current study aimed to test the hypothesis that PAK1 and/or PAK2 regulate contraction-induced glucose transport.MethodsGlucose transport was measured in isolated soleus and extensor digitorum longus (EDL) mouse skeletal muscle incubated either in the presence or absence of a pharmacological inhibitor (IPA-3) of group I PAKs or originating from whole-body PAK1 knockout (KO), muscle-specific PAK2 (m)KO or double whole-body PAK1 and muscle-specific PAK2 knockout mice.ResultsIPA-3 attenuated (−22%) the increase in muscle glucose transport in response to electrically-stimulated contraction. PAK1 was dispensable for contraction-stimulated glucose uptake in both soleus and EDL muscle. Lack of PAK2, either alone (−13%) or in combination with PAK1 (−14%), reduced contraction-stimulated glucose transport compared to control littermates in EDL, but not soleus muscle.ConclusionContraction-stimulated glucose transport in isolated glycolytic mouse EDL muscle is partly dependent on PAK2, but not PAK1.