Conserved and species-specific transcriptional responses to daily programmed resistance exercise in rat and mouse.

Mice are often used in gain or loss of function studies to understand how genes regulate metabolism and adaptation to exercise in skeletal muscle. Once-daily resistance training with electrical nerve stimulation produces hypertrophy of the dorsiflexors in rat, but not in mouse. Using implantable pulse generators, we assessed the acute transcriptional response (1-h post-exercise) after 2, 10, and 20 days of training in free-living mice and rats using identical nerve stimulation paradigms. RNA sequencing revealed strong concordance in the timecourse of many transcriptional responses in the tibialis anterior muscles of both species including responses related to "stress responses/immediate-early genes, and "collagen homeostasis," "ribosomal subunits," "autophagy," and "focal adhesion." However, pathways associated with energy metabolism including "carbon metabolism," "oxidative phosphorylation," "mitochondrial translation," "propanoate metabolism," and "valine, leucine, and isoleucine degradation" were oppositely regulated between species. These pathways were suppressed in the rat but upregulated in the mouse. Our transcriptional analysis suggests that although many pathways associated with growth show remarkable similarities between species, the absence of an actual growth response in the mouse may be because the mouse prioritizes energy metabolism, specifically the replenishment of fuel stores and intermediate metabolites.

Abaloparatide prevents immobilization-induced cortical but not trabecular bone loss after spinal cord injury.

Spinal cord injury (SCI) causes severe and resistant sublesional disuse bone loss. Abaloparatide, a modified parathyroid hormone related peptide, is an FDA approved drug for treatment of severe osteoporosis with potent anabolic activity. The effects of abaloparatide on SCI-induced bone loss remain undefined. Thus, female mice underwent sham or severe contusion thoracic SCI causing hindlimb paralysis. Mice then received subcutaneous injection of vehicle or 20 µg/kg/day abaloparatide for 35 days. Micro-computed tomography (micro-CT) analysis of the distal and midshaft femoral regions of the SCI-vehicle mice revealed reduced trabecular fractional bone volume (56%), thickness (75%), and cortical thickness (80%) compared to sham-vehicle controls. Treatment with abaloparatide did not prevent SCI-induced changes in trabecular or cortical bone. However, histomorphometry evaluation of the SCI-abaloparatide mice demonstrated that abaloparatide treatment increased osteoblast (241%) and osteoclast (247%) numbers and the mineral apposition rate (131%) compared to SCI-vehicle animals. In another independent experiment, treatment with 80 µg/kg/day abaloparatide significantly attenuated SCI-induced loss in cortical bone thickness (93%) when compared to SCI-vehicle mice (79%) but did not prevent SCI-induced trabecular bone loss or elevation in cortical porosity. Biochemical analysis of the bone marrow supernatants of the femurs showed that SCI-abaloparatide animals had 2.3-fold increase in procollagen type I N-terminal propeptide, a bone formation marker than SCI-vehicle animals. SCI groups had 70% higher levels of cross-linked C-telopeptide of type I collagen, a bone resorption marker, than sham-vehicle mice. These findings suggest that abaloparatide protects the cortical bone against the deleterious effects of SCI by promoting bone formation.

Neuroprotective macromolecular methylprednisolone prodrug nanomedicine prevents glucocorticoid-induced muscle atrophy and osteoporosis in a rat model of spinal cord injury.

To address the adverse side effects associated with systemic high-dose methylprednisolone (MP) therapy for acute spinal cord injury (SCI), we have developed a N-2-hydroxypropyl methacrylamide copolymer-based MP prodrug nanomedicine (Nano-MP). Intravenous Nano-MP selectively targeted to the inflamed SCI lesion and significantly improved neuroprotection and functional recovery after acute SCI. In the present study, we comprehensively assessed the potential adverse side effects associated with the treatment in the SCI rat models, including reduced body weight and food intake, impaired glucose metabolism, and reduced musculoskeletal mass and integrity. In contrast to free MP treatment, intravenous Nano-MP after acute SCI not only offered superior neuroprotection and functional recovery but also significantly mitigated or even eliminated the aforementioned adverse side effects. The superior safety features of Nano-MP observed in this study further confirmed the clinical translational potential of Nano-MP as a highly promising drug candidate for better clinical management of patients with acute SCI.

Targeted-delivery of nanomedicine-enabled methylprednisolone to injured spinal cord promotes neuroprotection and functional recovery after acute spinal cord injury in rats.

To date, no therapy has been proven to be efficacious in fully restoring neurological functions after spinal cord injury (SCI). Systemic high-dose methylprednisolone (MP) improves neurological recovery after acute SCI in both animal and human. MP therapy remains controversial due to its modest effect on functional recovery and significant adverse effects. To overcome the limitation of MP therapy, we have developed a N-(2-hydroxypropyl) methacrylamide copolymer-based MP prodrug nanomedicine (Nano-MP) that can selectively deliver MP to the SCI lesion when administered systemically in a rat model of acute SCI. Our in vivo data reveal that Nano-MP is significantly more effective than free MP in attenuating secondary injuries and neuronal apoptosis. Nano-MP is superior to free MP in improving functional recovery after acute SCI in rats. These data support Nano-MP as a promising neurotherapeutic candidate, which may provide potent neuroprotection and accelerate functional recovery with improved safety for patients with acute SCI.

Assessing the impact of boldine on the gastrocnemius using multiomics profiling at 7 and 28 days post-complete spinal cord injury in young male mice.

Spinal cord injury (SCI) results in rapid muscle loss. Exogenous molecular interventions to slow muscle atrophy after SCI have been relatively ineffective and require the search for novel therapeutic targets. Connexin hemichannels (CxHCs) allow nonselective passage of small molecules into and out of the cell. Boldine, a CxHC-inhibiting aporphine found in the boldo tree (Peumus boldus), has shown promising preclinical results in slowing atrophy during sepsis and restoring muscle function in dysferlinopathy. We administered 50 mg/kg/day of boldine to spinal cord transected mice beginning 3 days post-injury. Tissue was collected 7 and 28 days post-SCI and the gastrocnemius was used for multiomics profiling. Boldine did not prevent body or muscle mass loss but attenuated SCI-induced changes in the abundance of the amino acids proline, phenylalanine, leucine and isoleucine, as well as glucose, 7 days post-SCI. SCI resulted in the differential expression of ∼7,700 and ∼2,000 genes at 7 and 28 days, respectively, compared with Sham controls. Pathway enrichment of these genes highlighted ribosome biogenesis at 7 days and translation and oxidative phosphorylation at both timepoints. Boldine altered the expression of ∼150 genes at 7 days and ∼110 genes at 28 days post-SCI. Pathway enrichment of these genes indicated a potential role for boldine in suppressing protein ubiquitination and degradation at the 7-day timepoint. Methylation analyses showed minimal differences between groups. Taken together, boldine is not an efficacious therapy to preserve body and muscle mass after complete SCI, though it attenuated some SCI-induced changes across the metabolome and transcriptome.NEW & NOTEWORTHY This is the first study to describe the multiome of skeletal muscle paralyzed by a spinal cord injury (SCI) in mice across the acute and subacute timeframe after injury. We show large-scale changes in the metabolome and transcriptome at 7 days post-injury compared with 28 days. Furthermore, we show that the alkaloid boldine was able to prevent SCI-induced changes in muscle glucose and free amino acid levels at 7 days, but not 28 days, after SCI.

Denervation Atrophy of Skeletal Muscle is Not Influenced by Numb Levels in Mice.

Skeletal muscle undergoes rapid and extensive atrophy following nerve transection though the underlying mechanisms remain incompletely understood. We previously showed transiently elevated Notch 1 signaling in denervated skeletal muscle that was abrogated by administration of nandrolone (an anabolic steroid) combined with replacement doses of testosterone. Numb is an adaptor molecule present in myogenic precursors and skeletal muscle fibers that is vital for normal tissue repair after muscle injury and for skeletal muscle contractile function. It is unclear whether the increase in Notch signaling observed in denervated muscle contributes to denervation and whether expression of Numb in myofibers slows denervation atrophy. To address these questions, the degree of denervation atrophy, Notch signaling, and Numb expression was studied over time after denervation in C57B6J mice treated with nandrolone, nandrolone plus testosterone or vehicle. Nandrolone increased Numb expression and reduced Notch signaling. Neither nandrolone alone nor nandrolone plus testosterone changed the rate of denervation atrophy. We next compared rates of denervation atrophy between mice with conditional, tamoxifen-inducible knockout of Numb in myofibers and genetically identical mice treated with vehicle. Numb cKO had no effect on denervation atrophy in this model. Taken together, the data indicate that loss of Numb in myofibers does not alter the course of denervation atrophy and that upregulation of Numb and blunting of the denervation-atrophy induced activation of Notch do not change the course of denervation atrophy.

Predictors of muscle hypertrophy responsiveness to electrically evoked resistance training after spinal cord injury.

The purpose of the study was to identify potential predictors of muscle hypertrophy responsiveness following neuromuscular electrical stimulation resistance training (NMES-RT) in persons with chronic spinal cord injury (SCI). Data for twenty individuals with motor complete SCI who completed twice weekly NMES-RT lasting 12-16 weeks as part of their participation in one of two separate clinical trials were pooled and retrospectively analyzed. Magnetic resonance imaging (MRI) was used to measure muscle cross-sectional area (CSA) of the whole thigh and knee extensor muscle before and after NMES-RT. Muscle biopsies and fasting biomarkers were also measured. Following the completion of the respective NMES-RT trials, participants were classified into either high-responders (n = 8; muscle CSA > 20%) or low-responders (n = 12; muscle CSA < 20%) based on whole thigh muscle CSA hypertrophy. Whole thigh muscle and knee extensors CSAs were significantly greater (P < 0.0001) in high-responders (29 ± 7% and 47 ± 15%, respectively) compared to low-responders (12 ± 3% and 19 ± 6%, respectively). There were no differences in total caloric intake or macronutrient intake between groups. Extensor spasticity was lower in the high-responders compared to the low-responders as was the dosage of baclofen. Prior to the intervention, the high-responders had greater body mass compared to the low-responders with SCI (87.8 ± 13.7 vs. 70.4 ± 15.8 kg; P = 0.012), body mass index (BMI: 27.6 ± 2.7 vs. 22.9 ± 6.0 kg/m2; P = 0.04), as well as greater percentage in whole body and regional fat mass (P < 0.05). Furthermore, high-responders had a 69% greater increase (P = 0.086) in total Akt protein expression than low-responders. High-responders also exhibited reduced circulating IGF-1 with a concomitant increase in IGFBP-3. Exploratory analyses revealed upregulation of mRNAs for muscle hypertrophy markers [IRS-1, Akt, mTOR] and downregulation of protein degradation markers [myostatin, MurF-1, and PDK4] in the high-responders compared to low-responders. The findings indicate that body composition, spasticity, baclofen usage, and multiple signaling pathways (anabolic and catabolic) are involved in the differential muscle hypertrophy response to NMES-RT in persons with chronic SCI.

Notch, Numb and Numb-like responses to exercise-induced muscle damage in human skeletal muscle.

NEW FINDINGS: What is the central question of this study? Do Notch, Numb and Numb-like expression change in human skeletal muscle after exercise-induced muscle damage? What are the main finding and its importance? Notch gene expression trends toward an increase in response to an acute bout of exercise-induced muscle damage, while Numb and Numb-like expression does not change. These results suggest that human skeletal muscle response to exercise-induced muscle damage is dynamic and may differ from Drosophila and rodent models. Furthermore, the timing of muscle biopsies, training status and muscle damage protocols should be considered. ABSTRACT: This investigation examined changes in the gene and protein expression of Notch, Numb and Numb-like (Numbl) in human skeletal muscle after an acute bout of eccentric exercise-induced muscle damage. Twelve recreationally active male subjects participated in this study. These individuals completed seven sets of 10 repetitions of eccentric leg extension at 120% of one-repetition max with 2 min of rest period between sets. Four muscle biopsies of the vastus lateralis were collected: before exercise (Pre), and 3 h, 2 days and 5 days post-muscle damage. Biopsy samples were used to probe Notch, Numb and Numbl utilizing western blot and RT-qPCR techniques. The results were analysed using a one-way repeated-measures ANOVA. Notch1 mRNA expression trended toward a significant increase from Pre to 2 days post-muscle damage from baseline measures (P = 0.087), while Numb (P = 0.804) and Numbl (P = 0.480) expression was unaltered post-muscle damage. There were no significant differences in protein expression post-muscle damage for any of the proteins. These results suggest that exercise-induced muscle damage, via eccentric exercise, slightly elevates Notch1 mRNA expression.

Locomotor training with adjuvant testosterone preserves cancellous bone and promotes muscle plasticity in male rats after severe spinal cord injury.

Loading and testosterone may influence musculoskeletal recovery after spinal cord injury (SCI). Our objectives were to determine (a) the acute effects of bodyweight-supported treadmill training (TM) on hindlimb cancellous bone microstructure and muscle mass in adult rats after severe contusion SCI and (b) whether longer-term TM with adjuvant testosterone enanthate (TE) delivers musculoskeletal benefit. In Study 1, TM (40 min/day, 5 days/week, beginning 1 week postsurgery) did not prevent SCI-induced hindlimb cancellous bone loss after 3 weeks. In Study 2, TM did not attenuate SCI-induced plantar flexor muscles atrophy nor improve locomotor recovery after 4 weeks. In our main study, SCI produced extensive distal femur and proximal tibia cancellous bone deficits, a deleterious slow-to-fast fiber-type transition in soleus, lower muscle fiber cross-sectional area (fCSA), impaired muscle force production, and levator ani/bulbocavernosus (LABC) muscle atrophy after 8 weeks. TE alone (7.0 mg/week) suppressed bone resorption, attenuated cancellous bone loss, constrained the soleus fiber-type transition, and prevented LABC atrophy. In comparison, TE+TM concomitantly suppressed bone resorption and stimulated bone formation after SCI, produced near-complete cancellous bone preservation, prevented the soleus fiber-type transition, attenuated soleus fCSA atrophy, maintained soleus force production, and increased LABC mass. 75% of SCI+TE+TM animals recovered voluntary over-ground hindlimb stepping, while no SCI and only 20% of SCI+TE animals regained stepping ability. Positive associations between testosterone and locomotor function suggest that TE influenced locomotor recovery. In conclusion, short-term TM alone did not improve bone, muscle, or locomotor recovery in adult rats after severe SCI, while longer-term TE+TM provided more comprehensive musculoskeletal benefit than TE alone.

Mitochondrial health and muscle plasticity after spinal cord injury.

Mitochondria are responsible for aerobic respiration and large-scale ATP production in almost all cells of the body. Their function is decreased in many neurodegenerative and cardiovascular disease states, in metabolic disorders such as type II diabetes and obesity, and as a normal component of aging. Disuse of skeletal muscle from immobilization or unloading triggers alterations of mitochondrial density and activity. Resultant mitochondrial dysfunction after paralysis, which precedes muscle atrophy, may augment subsequent release of reactive oxygen species leading to protein ubiquitination and degradation. Spinal cord injury is a unique form of disuse atrophy as there is a complete or partial disruption in tonic communication between the central nervous system (CNS) and skeletal muscle. Paralysis, unloading and disruption of CNS communication result in a rapid decline in skeletal muscle function and metabolic status with disruption in activity of peroxisome-proliferator-activated receptor-gamma co-activator 1 alpha and calcineurin, key regulators of mitochondrial health and function. External interventions, both acute and chronical with training using body-weight-assisted treadmill training or electrical stimulation have consistently demonstrated adaptations in skeletal muscle mitochondria, and expression of the genes and proteins required for mitochondrial oxidation of fats and carbohydrates to ATP, water, and carbon dioxide. The purpose of this mini-review is to highlight our current understanding as to how paralysis mechanistically triggers downstream regulation in mitochondrial density and activity and to discuss how mitochondrial dysfunction may contribute to skeletal muscle atrophy.

Myostatin inhibits osteoblastic differentiation by suppressing osteocyte-derived exosomal microRNA-218: A novel mechanism in muscle-bone communication.

Muscle and bone are closely associated in both anatomy and function, but the mechanisms that coordinate their synergistic action remain poorly defined. Myostatin, a myokine secreted by muscles, has been shown to inhibit muscle growth, and the disruption of the myostatin gene has been reported to cause muscle hypertrophy and increase bone mass. Extracellular vesicle-exosomes that carry microRNA (miRNA), mRNA, and proteins are known to perform an important role in cell-cell communication. We hypothesized that myostatin may play a crucial role in muscle-bone interactions and may promote direct effects on osteocytes and on osteocyte-derived exosomal miRNAs, thereby indirectly influencing the function of other bone cells. We report herein that myostatin promotes expression of several bone regulators such as sclerostin (SOST), DKK1, and RANKL in cultured osteocytic (Ocy454) cells, concomitant with the suppression of miR-218 in both parent Ocy454 cells and derived exosomes. Exosomes produced by Ocy454 cells that had been pretreated with myostatin could be taken up by osteoblastic MC3T3 cells, resulting in a marked reduction of Runx2, a key regulator of osteoblastic differentiation, and in decreased osteoblastic differentiation via the down-regulation of the Wnt signaling pathway. Importantly, the inhibitory effect of myostatin-modified osteocytic exosomes on osteoblast differentiation is completely reversed by expression of exogenous miR-218, through a mechanism involving miR-218-mediated inhibition of SOST. Together, our findings indicate that myostatin directly influences osteocyte function and thereby inhibits osteoblastic differentiation, at least in part, through the suppression of osteocyte-derived exosomal miR-218, suggesting a novel mechanism in muscle-bone communication.

Sclerostin Antibody Reverses the Severe Sublesional Bone Loss in Rats After Chronic Spinal Cord Injury.

To date, no efficacious therapy exists that will prevent or treat the severe osteoporosis in individuals with neurologically motor-complete spinal cord injury (SCI). Recent preclinical studies have demonstrated that sclerostin antibody (Scl-Ab) can prevent sublesional bone loss after acute SCI in rats. However, it remains unknown whether sclerostin inhibition reverses substantial bone loss in the vast majority of the SCI population who have been injured for several years. This preclinical study tested the efficacy of Scl-Ab to reverse the bone loss that has occurred in a rodent model after chronic motor-complete SCI. Male Wistar rats underwent either complete spinal cord transection or only laminectomy. Twelve weeks after SCI, the rats were treated with Scl-Ab at 25 mg/kg/week or vehicle for 8 weeks. In the SCI group that did not receive Scl-Ab, 20 weeks of SCI resulted in a significant reduction of bone mineral density (BMD) and estimated bone strength, and deterioration of bone structure at the distal femoral metaphysis. Treatment with Scl-Ab largely restored BMD, bone structure, and bone mechanical strength. Histomorphometric analysis showed that Scl-Ab increased bone formation in animals with chronic SCI. In ex vivo cultures of bone marrow cells, Scl-Ab inhibited osteoclastogenesis, and promoted osteoblastogenesis accompanied by increased Tcf7, ENC1, and the OPG/RANKL ratio expression, and decreased SOST expression. Our findings demonstrate for the first time that Scl-Ab reverses the sublesional bone loss when therapy is begun after relatively prolonged spinal cord transection. The study suggests that, in addition to being a treatment option to prevent bone loss after acute SCI, sclerostin antagonism may be a valid clinical approach to reverse the severe bone loss that invariably occurs in patients with chronic SCI.

Alterations in mitochondrial fission, fusion, and mitophagic protein expression in the gastrocnemius of mice after a sciatic nerve transection.

INTRODUCTION: Paralysis and unloading of skeletal muscle leads to a rapid loss in muscle size, function and oxidative capacity. The reduction in metabolic capability after disuse leads to dysregulation and increased breakdown of mitochondria by mitophagy. METHODS: Eight-week-old C57BL/6 male mice were given a sham surgery or sciatic nerve transection. Animals were euthanized at 7, 14, 21, or 35 days postsurgery. Whole gastrocnemius muscles were isolated from the animal, weighed and used for Western blotting. RESULTS: Markers of mitochondrial fusion were reduced while fission proteins were elevated following a sciatic nerve transection. There were elevations in phosphorylated unc-51-like kinase 1 (ULK1S555 ) and total expression of Beclin1, and of the mitophagy markers PINK1, p62, and microtubule-associated proteins 1A/1B light chain 3b (LC3-II). CONCLUSIONS: Paralysis of the gastrocnemius leads to a progressive elevation in expression of mitochondrial fission and mitophagic proteins. Rehabilitative or pharmaceutical interventions to limit excess mitophagy may be effective therapies to protect paralyzed muscle mass and function. Muscle Nerve 58: 592-599, 2018.

Plasma adiponectin levels are correlated with body composition, metabolic profiles, and mitochondrial markers in individuals with chronic spinal cord injury.

STUDY DESIGN: Cross-sectional design. OBJECTIVES: This study examined the relationships between circulating adiponectin levels, body composition, metabolic profile, and measures of skeletal muscle mitochondrial enzyme activity and biogenesis. SETTINGS: Clinical Research in a Medical Center. METHODS: Plasma adiponectin was quantified in 19 individuals with chronic spinal cord injury (SCI). Body composition was evaluated by dual x-ray absorptiometry and magnetic resonance imaging. Metabolic profile was assessed by basal metabolic rate (BMR), oxygen uptake (VO2), and intravenous glucose tolerance testing. Mitochondrial enzyme activity of skeletal muscle was obtained by spectrophotometric assays and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and 5' AMP-activated protein kinase (AMPK) protein expression was assessed by Western blots. RESULTS: Adiponectin was negatively related to both total and regional fat mass and positively related to lean mass and muscle mass. Furthermore, there were positive relationships between adiponectin and BMR (r = 0.52, P = 0.02) and VO2 (r = 0.73, P = 0.01). Furthermore, adiponectin was positively related to citrate synthase (r = 0.68, P = 0.002) and complex III activity (r = 0.57, P = 0.02). The relationships between adiponectin and body composition remained significant after accounting for age. The relationships between adiponectin, metabolic profile, and markers of mitochondria mass and activity were influenced by age. CONCLUSIONS: The study demonstrated that adiponectin is closely related to body composition and metabolic profile in persons with SCI and further supports mechanistic studies suggesting that adiponectin may stimulate mitochondrial biogenesis.

Phospholipid dysregulation contributes to ApoE4-associated cognitive deficits in Alzheimer's disease pathogenesis.

The apolipoprotein E4 (ApoE4) allele is the strongest genetic risk factor for developing sporadic Alzheimer's disease (AD). However, the mechanisms underlying the pathogenic nature of ApoE4 are not well understood. In this study, we have found that ApoE proteins are critical determinants of brain phospholipid homeostasis and that the ApoE4 isoform is dysfunctional in this process. We have found that the levels of phosphoinositol biphosphate (PIP2) are reduced in postmortem human brain tissues of ApoE4 carriers, in the brains of ApoE4 knock-in (KI) mice, and in primary neurons expressing ApoE4 alleles compared with those levels in ApoE3 counterparts. These changes are secondary to increased expression of a PIP2-degrading enzyme, the phosphoinositol phosphatase synaptojanin 1 (synj1), in ApoE4 carriers. Genetic reduction of synj1 in ApoE4 KI mouse models restores PIP2 levels and, more important, rescues AD-related cognitive deficits in these mice. Further studies indicate that ApoE4 behaves similar to ApoE null conditions, which fails to degrade synj1 mRNA efficiently, unlike ApoE3 does. These data suggest a loss of function of ApoE4 genotype. Together, our data uncover a previously unidentified mechanism that links ApoE4-induced phospholipid changes to the pathogenic nature of ApoE4 in AD.

Regulation of pannexin and connexin channels and their functional role in skeletal muscles.

Myogenic precursor cells express connexins (Cx) and pannexins (Panx), proteins that form different membrane channels involved in cell-cell communication. Cx channels connect either the cytoplasm of adjacent cells, called gap junction channels (GJC), or link the cytoplasm with the extracellular space, termed hemichannels (HC), while Panx channels only support the latter. In myoblasts, Panx1 HCs play a critical role in myogenic differentiation, and Cx GJCs and possibly Cx HCs coordinate metabolic responses during later steps of myogenesis. After innervation, myofibers do not express Cxs, but still express Panx1. In myotubes and innervated myofibers, Panx1 HCs allow release of adenosine triphosphate and thus they might be involved in skeletal muscle plasticity. In addition, Panx1 HCs present in adult myofibers mediate adenosine triphosphate release and glucose uptake required for potentiation of muscle contraction. Under pathological conditions, such as upon denervation and spinal cord injury, levels of Panx1 are upregulated. However, Panx1(-/-) mice show similar degree of atrophy as denervated wild-type muscles. Skeletal muscles also express Cx HCs in the sarcolemma after denervation or spinal cord injury, plus other non-selective membrane channels, including purinergic P2X7 receptors and transient receptor potential type V2 channels. The absence of Cx43 and Cx45 is sufficient to drastically reduce denervation atrophy. Moreover, inflammatory cytokines also induce the expression of Cxs in myofibers, suggesting the expression of these Cxs as a common factor for myofiber degeneration under diverse pathological conditions. Inhibitors of skeletal muscle Cx HCs could be promising tools to prevent muscle wasting induced by conditions associated with synaptic dysfunction and inflammation.

De novo expression of connexin hemichannels in denervated fast skeletal muscles leads to atrophy.

Denervation of skeletal muscles induces atrophy, preceded by changes in sarcolemma permeability of causes not yet completely understood. Here, we show that denervation-induced Evans blue dye uptake in vivo of fast, but not slow, myofibers was acutely inhibited by connexin (Cx) hemichannel/pannexin1 (Panx1) channel and purinergic ionotropic P2X7 receptor (P2X7R) blockers. Denervated myofibers showed up-regulation of Panx1 and de novo expression of Cx39, Cx43, and Cx45 hemichannels as well as P2X7Rs and transient receptor potential subfamily V, member 2, channels, all of which are permeable to small molecules. The sarcolemma of freshly isolated WT myofibers from denervated muscles also showed high hemichannel-mediated permeability that was slightly reduced by blockade of Panx1 channels or the lack of Panx1 expression, but was completely inhibited by Cx hemichannel or P2X7R blockers, as well as by degradation of extracellular ATP. However, inhibition of transient receptor potential subfamily V, member 2, channels had no significant effect on membrane permeability. Moreover, activation of the transcription factor NFκB and higher mRNA levels of proinflammatory cytokines (TNF-α and IL-1ß) were found in denervated WT but not Cx43/Cx45-deficient muscles. The atrophy observed after 7 d of denervation was drastically reduced in Cx43/Cx45-deficient but not Panx1-deficient muscles. Therefore, expression of Cx hemichannels and P2X7R promotes a feed-forward mechanism activated by extracellular ATP, most likely released through hemichannels, that activates the inflammasome. Consequently, Cx hemichannels are potential targets for new therapeutic agents to prevent or reduce muscle atrophy induced by denervation of diverse etiologies.

ANKRD1 modulates inflammatory responses in C2C12 myoblasts through feedback inhibition of NF-κB signaling activity.

Transcription factors of the nuclear factor-kappa B (NF-κB) family play a pivotal role in inflammation, immunity and cell survival responses. Recent studies revealed that NF-κB also regulates the processes of muscle atrophy. NF-κB activity is regulated by various factors, including ankyrin repeat domain 2 (AnkrD2), which belongs to the muscle ankyrin repeat protein family. Another member of this family, AnkrD1 is also a transcriptional effector. The expression levels of AnkrD1 are highly upregulated in denervated skeletal muscle, suggesting an involvement of AnkrD1 in NF-κB mediated cellular responses to paralysis. However, the molecular mechanism underlying the interactive role of AnkrD1 in NF-κB mediated cellular responses is not well understood. In the current study, we examined the effect of AnkrD1 on NF-κB activity and determined the interactions between AnkrD1 expression and NF-κB signaling induced by TNFα in differentiating C2C12 myoblasts. TNFα upregulated AnkrD1 mRNA and protein levels. AnkrD1-siRNA significantly increased TNFα-induced transcriptional activation of NF-κB, whereas overexpression of AnkrD1 inhibited TNFα-induced NF-κB activity. Co-immunoprecipitation studies demonstrated that AnkrD1 was able to bind p50 subunit of NF-κB and vice versa. Finally, CHIP assays revealed that AnkrD1 bound chromatin at a NF-κB binding site in the AnrkD2 promoter and required NF-κB to do so. These results provide evidence of signaling integration between AnkrD1 and NF-κB pathways, and suggest a novel anti-inflammatory role of AnkrD1 through feedback inhibition of NF-κB transcriptional activity by which AnkrD1 modulates the balance between physiological and pathological inflammatory responses in skeletal muscle.

Focal adhesion kinase and its role in skeletal muscle.

Skeletal muscle has a remarkable ability to respond to different physical stresses. Loading muscle through exercise, either anaerobic or aerobic, can lead to increases in muscle size and function while, conversely, the absence of muscle loading stimulates rapid decreases in size and function. A principal mediator of this load-induced change is focal adhesion kinase (FAK), a downstream non-receptor tyrosine kinase that translates the cytoskeletal stress and strain signals transmitted across the cytoplasmic membrane by integrins to activate multiple anti-apoptotic and cell growth pathways. Changes in FAK expression and phosphorylation have been found to correlate to specific developmental states in myoblast differentiation, muscle fiber formation and muscle size in response to loading and unloading. With the capability to regulate costamere formation, hypertrophy and glucose metabolism, FAK is a molecule with diverse functions that are important in regulating muscle cell health.