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Spinal cord injury (SCI) results in rapid muscle loss. The mechanisms of muscle atrophy have been well-described but there is limited information specific to SCI. Exogenous molecular interventions to slow muscle atrophy in severe-to-complete SCI have been relatively ineffective and the wide-ranging physiologic response to SCI requires the search for novel therapeutic targets. Connexin hemichannels (CxHC) allow non-selective 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 pre-clinical results in slowing atrophy during sepsis and dysferlinopathy. We administered 50 mg/kg/d of boldine to spinal cord transected mice beginning 3 d post-injury. Tissue was collected 7 and 28 d post-SCI and the gastrocnemius was used for multiomic profiling. Boldine did not prevent body or muscle mass loss but attenuated SCI-induced changes in the abundance of proline, phenylalanine, leucine and isoleucine, as well as glucose, 7 d post-SCI. SCI resulted in the differential expression of ~7,700 and ~2,000 genes at 7 and 28 d, respectively, compared to sham animals, with enrichment for pathways associated with ribosome biogenesis, translation and oxidative phosphorylation. Boldine altered the expression of ~150 genes at 7 d and ~110 genes at 28 d post-SCI. Methylation analyses highlighted distinct patterns at both 7 and 28 d following SCI both with and without boldine. Taken together, boldine is not an efficacious therapy to preserve body and muscle mass after complete SCI, though it preserved or attenuated SCI-induced changes across the metabolome, transcriptome and methylome.

BackgroundThe role of Numb, a protein that is important for cell fate and development was investigated in adult skeletal muscle in mice using a conditional, inducible knockout (cKO) model.MethodsNumb expression was evaluated by Western blot. Numb localization was determined by confocal microscopy. The effects of cKO of Numb and the closely-related gene Numb-like in skeletal muscle fibers was evaluated by in-situ physiology; transmission and focused ion beam scanning electron microscopy; 3-dimensional reconstruction of mitochondrial; lipidomics; and bulk RNA-sequencing. Additional studies using primary mouse myotubes investigated the effects the effects of Numb knockdown on cell fusion, mitochondrial function and calcium transients.ResultsNumb protein expression was reduced by ∼70% (p < 0.01) at 24 as compared to 3 months of age. Numb was localized within muscle fibers as bands traversing fibers at regularly spaced intervals in close proximity to dihydropyridine receptors.The cKO of Numb and Numb-like reduced specific tetanic force by 36%, p < 0.01), altered mitochondrial spatial relationships to sarcomeric structures, increased Z-line spacing by 30% (p < 0.0001), perturbed sarcoplasmic reticulum organization and reduced mitochondrial volume by over 80% (p < 0.01). Only six genes were differentially expressed in cKO mice: Itga4, Sema7a, Irgm2, Vezf1, Mib1 and Tmem132a. Several lipid mediators derived from polyunsaturated fatty acid (PUFAs) through lipoxygenases were upregulated in Numb cKO skeletal muscle; 12-HEPE was increased by ∼250% (p < 0.05) and 17,18-EpETE by ∼240% (p < 0.05). In mouse primary myotubes, Numb knock-down reduced cell fusion (∼20%, p < 0.01) and mitochondrial function and delayed the caffeine-induced rise in cytosolic calcium concentrations by more than 100% (p < 0.01).ConclusionsThese findings implicate Numb as a critical factor in skeletal muscle structure and function which appear to be critical for calcium release; we therefore speculate Numb plays critical roles in excitation-contraction coupling, one of the putative targets of aged skeletal muscles. These findings provide new insights into the molecular underpinnings of the loss of muscle function observed with sarcopenia.

Spinal cord injury (SCI) is associated with obesity and is a risk factor for type 2 diabetes mellitus (T2DM). Immobilization, muscle atrophy, obesity, and loss of sympathetic innervation to the liver are believed to contribute to risks of these abnormalities. Systematic study of the mechanisms underlying SCI-induced metabolic disorders has been limited by a lack of animal models of insulin resistance following SCI. Therefore, the effects of a high-fat diet (HFD), which causes weight gain and glucose intolerance in neurologically intact mice, was tested in mice that had undergone a spinal cord transection at thoracic vertebra 10 (T10) or a sham-transection. At 84 days after surgery, Sham-HFD and SCI-HFD mice showed impaired intraperitoneal glucose tolerance when compared with Sham control (Sham-Con) or SCI control (SCI-Con) mice fed a standard control chow. Glucose tolerance in SCI-Con mice was comparable to that of Sham-Con mice. The mass of paralyzed skeletal muscle, liver, and epididymal, inguinal, and omental fat deposits were lower in SCI versus Sham groups, with lower liver mass present in SCI-HFD versus SCI-Con animals. SCI also produced sublesional bone loss, with no differences between SCI-Con and SCI-HFD groups. The results suggest that administration of a HFD to mice after SCI may provide a model to better understand mechanisms leading to insulin resistance post-SCI, as well as an approach to study pathogenesis of glucose intolerance that is independent of obesity.

This study aimed to characterize the effects of laparotomy on post-operative physical function and skeletal muscle gene expression in C57BL/6N mice at 3, 20 and 24 months of age to investigate late-life vulnerability and resiliency to acute surgical stress. Pre- and post-operative physical functioning were assessed by forelimb grip strength and motor coordination. Laparotomy induced an age-associated post-operative decline in forelimb grip strength that was greatest in the oldest mice. In contrast, while motor coordination declined with increasing age at baseline, it was unaffected by laparotomy. Moreover, baseline physical function as stratified by motor coordination performance (low vs. high functioning) in 24-month-old mice did not differentially affect post-laparotomy reduction in grip strength. RNA sequencing of soleus muscles showed that laparotomy induced age-associated differential gene expression and canonical pathway activation with the greatest effects in the youngest mice. Examples of such age-associated, metabolically important pathways that were only activated in the youngest mice after laparotomy included oxidative phosphorylation and NRF2-mediated oxidative stress response. Analysis of lipid mediators in serum and gastrocnemius muscle showed alterations in profiles of these mediators during aging and confirmed an association between such changes and functional status in gastrocnemius muscle. These findings demonstrate a mouse model of laparotomy which recapitulated some features of post-operative skeletal muscle decline in older adults following surgery, and identified age-associated, laparotomy-induced molecular signatures in skeletal muscles. Future research can build upon this mouse model to study molecular mechanisms of late-life vulnerability to acute surgical stress and resiliency to counter surgery-induced physical decline.

Spinal cord injury (SCI) causes adverse changes in body composition and metabolic profile due to dramatic loss of skeletal muscle mass and an extremely sedentary lifestyle. These abrupt and permanent changes increase the risk for cardiovascular disease, liver disorders, and type 2 diabetes compared to the general population. We have reported that metabolic dysregulation following SCI due to transection is associated with impaired fibroblast growth factor 21 (FGF21) and adiponectin (ADPN) signaling [1], two important regulators of metabolism [2, 3]. JTV519, which stabilizes ryanodine receptors and blocks calcium uptake via SERCA [4], has been reported to have a strong cardioprotective effect via stabilization of ryanodine receptor 2. We examined the effects of SCI caused by contusion (a more common cause of SCI), with or without JTV519 administration, on FGF21 and ADPN signaling in tissues collected from mice at 56 days after SCI. SCI reduced serum and hepatic mRNA levels of FGF21 by 75% and 45%, respectively, compared to sham mice. SCI also significantly diminished FGF21 receptor (FGF-R1) and coreceptor (beta-klotho, KLB) mRNA expression in adipose tissue. In addition, SCI decreased serum ADPN levels, particularly its high molecular weight (HMW) isoform which is the most potent form of the protein. These inhibitory effects were associated with decreased hepatic mRNA expression for PPARalpha (55%), type-2 adiponectin receptor (AdipoR2; 70%), and Glut4 (75%). In addition, SCI reduced mRNA expression of leptin (by 85%) and UCP1 (by 95%) in adipose tissue. In skeletal muscle (gastrocnemius), SCI lowered mRNA expression of PGC1alpha (by 38%), ADPN (by 80%), FGF-R1 (by 35%) and AdipoR1 (by 20%) compared to sham mice. Treating sham-mice with JTV519 upregulated FGF21 and ADPN serum and mRNA levels as well as the expression of the associated metabolic genes of interest (mentioned above). Likewise, SCI-mice treated with JTV519 also demonstrated upregulation of serum levels of FGF21, the HMW isoform of ADPN, and hepatic FGF21 mRNA expression compared to vehicle-treated SCI-mice. Decreased hepatic mRNA expression of PPARalpha, AdipoR2, Glut4 and FGF-R1 and KLB mRNA in adipose tissue were also reversed after JTV519 administration, with levels approximately equivalent to those observed in sham-vehicle treated mice but lower than sham-JTV519 treated mice. Moreover, JTV519 treatment protected against SCI-induced decreases of PGC1alpha, ADPN and FGF-R1 mRNA expression in the gastrocnemius. These data imply that JTV-519 is able to alleviate SCI-induced impairments of FGF21/ADPN signaling. Therefore, JTV519 could be a potential pharmacological agent to treat metabolic dysfunction after SCI.References: (1) Liu XH et al. Front Endocrinol. 12: 668984, 2021. (2) Staiger H et al. Endocrine Rev. 38: 468, 2017. (3) Hui X et al. J. Mol. Cell Biol. 8: 110, 2016. (4) Kaneko N et al. Curr Clin Pharmacol. 4: 126, 2009. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Spinal cord injury (SCI) causes deleterious changes in the metabolic profile due to muscle atrophy and sedentary lifestyle. SCI is associated with increased risk for cardiovascular disease, liver disorders and type 2 diabetes mellitus (T2DM). Understanding of the mechanisms for these disorders in those with SCI is poor. Fibroblast growth factor 21 (FGF-21), a hepatic hormone, is reported to have beneficial effects on glucose/lipid metabolism in animal models and human subjects with T2DM. However, the expression levels and the potential effect of FGF-21 on SCI related metabolic dysfunction have not been studied. To address this issue, we compared FGF-21 serum and hepatic mRNA levels in sham and SCI mice fed control or high fat diet (HFD) at 84d after SCI. SCI reduced serum FGF-21 levels by 60% and hepatic FGF-21 mRNA levels by 50% compared to sham mice; levels were further suppressed by HFD. These changes were associated with elevated serum levels of alanine aminotransferase, a liver damage marker. In adipose tissue (AT), SCI decreased mRNA expression of FGF-21 receptor (FGFR1), β-Klotho, adiponectin, PPARγ, and leptin. In addition, SCI reduced IRS1 and Glut4 mRNA levels in AT, indicating impaired insulin signaling. Moreover, elevated levels of TNFα in AT and CD11b (a macrophage marker) in the liver were observed, implying a proinflammatory state after SCI. Finally, post-SCI mice fed a HFD had elevated free fatty acid (FFA) levels in serum and liver compared to sham-mice fed a HFD, suggesting defective FFA uptake and metabolism. These data suggest that reduced FGF-21 signaling after SCI may, at least in part, be contributing to the metabolic dysfunction after SCI, and that FGF-21 may be a candidate for the treatment of SCI-related metabolic dysfunction. Disclosure X. Liu: None. L. Harlow: None. Z. Graham: None. J.F. Yarrow: None. K. Cusi: Consultant; Self; Allergan plc., AstraZeneca, Bristol-Myers Squibb, Genentech, Inc., Gilead Sciences, Inc., Merck & Co., Inc. Research Support; Self; Cirius Therapeutics, Echosens, Eli Lilly and Company, Inventiva Pharma, Janssen Pharmaceuticals, Inc., Novartis Pharmaceuticals Corporation, Novo Nordisk Inc., Poxel SA, Zydus Pharmaceuticals, Inc. W.A. Bauman: None. C.P. Cardozo: None. Funding U.S. Department of Veterans Affairs (B9212C, B2020C)

In older adults, pre-operative physical function predicts post-operative outcomes. The biological mechanisms underlying vulnerability to physical decline remain poorly understood. Using a mouse model of laparotomy, we sought to identify biological correlates of post-operative function. 24-month-old male C57BL/6N mice were categorized as high functioning (HF) or low functioning (LF) based on pre-operative performance on the accelerating rotarod. On post-operative days (POD) 2 and 4, LF mice had lower rotarod latency to fall times than HF mice did. Forelimb grip strength was reduced after laparotomy in both HF and LF groups on POD 1 and 3 and did not differ significantly between these groups. Whole transcriptome sequencing analysis (RNAseq) of soleus muscles collected on POD 5 showed 224 and 228 differentially expressed genes (DEGs) for HF and LF, respectively, compared to their respective controls. Only 21 DEGs were observed in both groups, including Pparα, Fst and Pla2g15. Such changes may be hallmarks of the post-surgical response in aging. Pathway analysis of DEGs using Ingenuity Pathway Analysis software (Qiagen) revealed one pathway common to HF and LF (osteoarthritis) whereas activation of GP6 signaling and apoptosis signaling was observed in HF and inhibition of PPARα/RXR activation and PPARα signaling was noted in LF. We conclude that pre-operative performance on the accelerating rotarod correlates with differences in skeletal muscle gene expression, which may contribute to the differences in functional outcomes post-operatively in HF and LF mice. Further studies are needed to delineate the roles of these signaling pathways in physical resilience to surgery.

Spinal cord injury (SCI) results in rapid muscle atrophy and an oxidative-to-glycolytic fiber-type shift. Those with chronic SCI are more at risk for developing insulin resistance and reductions in glucose clearance than able-bodied individuals, but how glucose metabolism is affected after SCI is not well known. An untargeted metabolomics approach was utilized to investigate changes in whole-muscle metabolites at an acute (7-day) and subacute (28-day) time frame after a complete T9 spinal cord transection in 20-week-old female C57BL/6 mice. Two hundred one metabolites were detected in all samples, and 83 had BinBase IDs. A principal components analysis showed the 7-day group as a unique cluster. Further, 36 metabolites were altered after 7- and/or 28-day post-SCI (p values

Unloading, neural lesions, and hormonal disorders after acute motor-complete spinal cord injury (SCI) cause one of the most severe forms of bone loss, a condition that has been refractory to available interventions tested to date. Thus, these features related to acute SCI provide a unique opportunity to study complex bone problems, potential efficacious interventions, and mechanisms of action that are associated with these dramatic pathological changes. This study was designed to explore the therapeutic potential of sclerostin antibody (Scl-Ab) in a rat model of bone loss after motor-complete SCI, and to investigate mechanisms underlying bone loss and Scl-Ab action. SCI rats were administered Scl-Ab (25 mg/kg/week) or vehicle beginning 7 days after injury then weekly for 7 weeks. SCI resulted in significant decreases in bone mineral density (-25%) and trabecular bone volume (-67%) at the distal femur; Scl-Ab completely prevented these deteriorations of bone in SCI rats, concurrent with markedly increased bone formation. Scanning electron microscopy revealed that SCI reduced numbers of osteocytes and dendrites concomitant with a morphology change from a spindle to round shape; Scl-Ab corrected these abnormalities in osteocytes. In ex vivo cultures of bone marrow cells, Scl-Ab inhibited osteoclastogenesis, and promoted osteoblastogenesis accompanied by increases in mRNA levels of LRP5, osteoprotegerin (OPG), and the OPG/RANKL ratio, and a decrease in DKK1 mRNA. Our findings provide the first evidence that robust bone loss after acute motor-complete SCI can be blocked by Scl-Ab, at least in part, through the preservation of osteocyte morphology and structure and related bone remodeling. Our findings support the inhibition of sclerostin as a promising approach to mitigate the striking bone loss that ensues after acute motor-complete SCI, and perhaps other conditions associated with disuse osteoporosis as a consequence of neurological disorders.

Exosomes are vesicles released by many eukaryotic cells; their cargo includes proteins, mRNA and microRNA (miR) that can be transferred to recipient cells and regulate cellular processes in an autocrine or paracrine manner. While cells of the myoblast lineage secrete exosomes, it is not known whether skeletal muscle fibers (myofibers) release exosomes. In this study, we found that cultured myofibers release nanovesicles that have bilamellar membranes and an average size of 60–130 nm, contain typical exosomal proteins and miRNAs and are taken up by C2C12 cells. miR-133a was found to be the most abundant myomiR in these vesicles while miR-720 was most enriched in exosomes compared to parent myofibers. Treatment of NIH 3T3 cells with myofiber-derived exosomes downregulated the miR-133a targets proteins Smarcd1 and Runx2, confirming that these exosomes have biologically relevant effects on recipient cells. Denervation resulted in a marked increase in miR-206 and reduced expression of miRs 1, 133a, and 133b in myofiber-derived exosomes. These findings demonstrate that skeletal muscle fibers release exosomes which can exert biologically significant effects on recipient cells, and that pathological muscle conditions such as denervation induce alterations in exosomal miR profile which could influence responses to disease states through autocrine or paracrine mechanisms.

Spinal cord injury (SCI) results in marked atrophy and dysfunction of skeletal muscle. There are currently no effective treatments for SCI-induced muscle atrophy or the dysfunction of the remaining muscle tissue. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-4 (Nox4) produces reactive oxygen species (ROS) in sarcoplasmic reticulum (SR) and has been identified as an important O2 sensor in skeletal muscle. Ryanodine receptors (RyRs) are calcium (Ca2+) channels that are responsible for Ca2+ release from SR. In skeletal muscle, type1 RyR (RyR1) is predominantly functional. RyR1 is regulated by multiple proteins, including calstabin1, which assures that they close appropriately once contraction has ceased. RyR1 function is also regulated by oxidation and redox-dependent cysteine nitrosylation. Excessive oxidation/nitrosylation of RyR1 is associated with dissociation of calstabin1 and reduced muscle force generation. However, whether Nox4 levels in skeletal muscle are elevated or whether RyR1 is oxidized or nitrosylated after SCI has not been determined. In this study, we examined Nox4 expression, oxidation/nitrolysation status, and association of calstabin1 with RyR1 in skeletal muscle derived from rats that were subjected to T4 complete transection (SCI), and observed elevated expression of Nox4 messenger RNA and protein in muscle after SCI associated with enhanced binding of Nox4 to RyR1, increased oxidation and nitrosylation of RyR1, and dissociation of calstabin1 from RyR1 in SCI rat muscle. Our data suggest that RyR1 dysfunction resulting from excessive oxidation/nitrosylation may contribute to reduced specific force after SCI and suggest that Nox4 may be the source of ROS responsible for increased oxidation and nitrosylation of RyR1.

Spinal cord injury (SCI) results in muscle atrophy, reduced force generation and an oxidative-to-glycolytic fiber type shift. The mechanisms responsible for these alterations remain incompletely understood. To gain new insights regarding mechanisms involved in deterioration of muscle after SCI, global expression profiles of miRs in paralyzed gastrocnemius muscle were compared between sham-operated (Sham) and spinal cord-transected (SCI) rats. Ingenuity Pathways Analysis of the altered miRs identified signaling via insulin, IGF-1, integrins and TGF-β as being significantly enriched for target genes. By qPCR, miRs 23a, 23b, 27b, 145, and 206, were downregulated in skeletal muscle 56 days after SCI. Using FISH, miR-145, a miR not previously implicated in the function of skeletal muscle, was found to be localized to skeletal muscle fibers. One predicted target of miR-145 was Cited2, a transcriptional regulator that modulates signaling through NF-κB, Smad3 and other transcription factors. The 3' UTR of Cited2 mRNA contained a highly conserved miR-145 seed sequence. Luciferase reporter assays confirmed that miR-145 interacts with this seed sequence. However, Cited2 protein levels were similar between Sham and SCI groups, indicating a biochemical interaction that was not involved in the context of adaptations after SCI. Taken together, the findings indicate dysregulation of several highly expressed miRs in skeletal muscle after SCI and suggest that reduced expression of miR-23a, 145 and 206 may have roles in alteration in skeletal muscle mass and insulin responsiveness in muscle paralyzed by upper motor neuron injuries.

Immobilization, as a result of motor‐complete spinal cord injury (SCI), is associated with severe osteoporosis. Whether parathyroid hormone (PTH) administration would reduce bone loss after SCI remains unclear. Thus, female mice underwent sham or surgery to produce complete spinal cord transection. PTH (80 μg/kg) or vehicle was injected subcutaneously (SC) daily starting on the day of surgery and continued for 35 days. Isolated tibias and femurs were examined by microcomputed tomography scanning (micro‐CT) and histology and serum markers of bone turnover were measured. Micro‐CT analysis of tibial metaphysis revealed that the SCI‐vehicle animals exhibited 49% reduction in fractional trabecular bone volume and 18% in trabecular thickness compared to sham‐vehicle controls. SCI‐vehicle animals also had 15% lower femoral cortical thickness and 16% higher cortical porosity than sham‐vehicle counterparts. Interestingly, PTH administration to SCI animals restored 78% of bone volume, increased connectivity to 366%, and lowered structure model index by 10% compared to sham‐vehicle animals. PTH further favorably attenuated femoral cortical bone loss to 5% and prevented the SCI‐associated cortical porosity. Histomorphometry evaluation of femurs of SCI‐vehicle animals demonstrated a marked 49% and 38% decline in osteoblast and osteoclast number, respectively, and 35% reduction in bone formation rate. In contrast, SCI‐PTH animals showed preserved osteoblast and osteoclast numbers and enhanced bone formation rate. Furthermore, SCI‐PTH animals had higher levels of bone formation and resorption markers than either SCI‐ or sham‐vehicle groups. Collectively, these findings suggest that intermittent PTH receptor activation is an effective therapeutic strategy to preserve bone integrity after severe immobilization.