Myofiber Cross-sectional Area Research Articles

Cystine/glutamate antiporter xCT controls skeletal muscle glutathione redox, bioenergetics and differentiation

Cysteine, the rate-controlling amino acid in cellular glutathione synthesis is imported as cystine, by the cystine/glutamate antiporter, xCT, and subsequently reduced to cysteine. As glutathione redox is important in muscle regeneration in aging, we hypothesized that xCT exerts upstream control over skeletal muscle glutathione redox, metabolism and regeneration. Bioinformatic analyses of publicly available datasets revealed that expression levels of xCT and GSH-related genes are inversely correlated with myogenic differentiation genes. Muscle satellite cells (MuSCs) isolated from Slc7a11sut/sut mice, which harbour a mutation in the Slc7a11 gene encoding xCT, required media supplementation with 2-mercaptoethanol to support cell proliferation but not myotube differentiation, despite persistently lower GSH. Slc7a11sut/sut primary myotubes were larger compared to WT myotubes, and also exhibited higher glucose uptake and cellular oxidative capacities. Immunostaining of myogenic markers (Pax7, MyoD, and myogenin) in cardiotoxin-damaged tibialis anterior muscle fibres revealed greater MuSC activation and commitment to differentiation in Slc7a11sut/sut muscle compared to WT mice, culminating in larger myofiber cross-sectional areas at 21 days post-injury. Slc7a11sut/sut mice subjected to a 5-week exercise training protocol demonstrated enhanced insulin tolerance compared to WT mice, but blunted muscle mitochondrial biogenesis and respiration in response to exercise training. Our results demonstrate that the absence of xCT inhibits cell proliferation but promotes myotube differentiation by regulating cellular metabolism and glutathione redox. Altogether, these results support the notion that myogenesis is a redox-regulated process and may help inform novel therapeutic approaches for muscle wasting and dysfunction in aging and disease.

Single-Nuclei RNA-Sequencing Uncovers Alterations in the Transcriptional Profile of Skeletal Muscle in Peripheral Artery Disease

BACKGROUND: Lower extremity peripheral artery disease (PAD) is caused by atherosclerotic occlusion of lower limb blood vessels, impacting >200 million people worldwide. PAD causes life-altering symptomatology such as claudication, ischemic rest pain, and gangrene, with limited effective treatment options. Herein, we performed single-nuclei RNA-sequencing (snRNAseq) to facilitate a better understanding of the complex pathobiological environment within the PAD limb and intercellular communication. METHODS: Gastrocnemius muscle specimens were collected from 20 patients with PAD and 12 non-PAD controls. Patients with PAD were classified as Rutherford category 3 (severe claudication). Non-PAD participants were of similar age, body mass index and medication usage as patients with PAD. Nuclei were isolated and snRNAseq was performed. Bioinformatic analysis was conducted to determine differences in gene expression between PAD and non-PAD nuclei, as well as analysis of intercellular signaling networks. Histological analysis of muscle specimens assessed the following: myosin heavy chain fiber distributions, myofiber cross-sectional area and myofiber denervation. RESULTS: 57,364 high-quality nuclei were captured (21,417 PAD; 35,947 non-PAD) and unsupervised clustering revealed 12 distinct nuclei populations based on their transcriptional profile. Quantification of the relative abundance of nuclei populations identified several differences, including PAD muscles having a lower percentage of type I myonuclei, muscle stem cells, and endothelial cells but a higher proportion of type II and regenerating myonuclei. Differentially expressed gene analysis of myonuclei populations uncovered upregulation of genes involved in stress response, autophagy, and atrophy in patients with PAD. Ligand-receptor interactions were used to determine how PAD alters the communication between identified nuclei populations. The inferred strength of interactions was greater in non-PAD compared to PAD patients. Further, intercellular communication network analysis revealed fibro-adipogenic progenitors as a primary signaling hub in PAD muscle. CONCLUSION: snRNAseq uncovered novel transcriptional differences and intercellular communication pathways unique to PAD muscle. This study was supported by National Institutes of Health (NIH) grant R01-HL149704 (T. Ryan). K. Kim supported by the American Heart Association grant POST903198. T. Thome supported by NIH grant F31-DK128920. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

TRIM Enhances Angiogenesis in Dystrophic Muscle 140 Days Post Treatment

Background: Duchenne Muscular Dystrophy (DMD) causes limb muscle dysfunction and impaired mobility while promoting ischemia in effected muscles. For treatment, we developed a novel biomaterial, Time Release Ion Matrix (TRIM), that promotes angiogenesis and enhances muscle structure and function in dystrophic (D2. mdx) mice at 14- and 70-days post injection (dpi). Whether TRIM remains effective at 140 dpi is unknown. Hypothesis: At 140 dpi in D2. mdx mice TRIM will: 1) enhance isometric force and myofiber cross-sectional area (CSA) of the tibialis anterior (TA) muscle; and 2) stimulate angiogenic growth factors and increase vascularity. Methods: The right and left TA of adult male D2. mdx mice (male, n=7-8, age 7 months) were injected with saline alone (control) or 250 μg of TRIM suspended in saline. At 140 dpi, the left TA muscle was isolated for in situ muscle force measurements and histological analysis; the right TA was frozen for biochemical analysis. Muscle cross-sections were immunostained for laminin (myofiber borders), DAPI (nuclei), and CD31 (endothelial cells). Vascular endothelial growth factor (VEGF) ELISA and immunoblots were performed on muscle homogenates. Results: No difference in peak isometric force was recorded between groups at 140 dpi (means ± SE: TRIM, 65.4 ± 2.7g; Saline, 62.7 ± 1.5g; P= 0.34). TRIM enhanced the relative frequency of myofibers >1000 μm2 by 27% vs. saline (TRIM, 56 ± 2%; Saline, 44 ± 3%; P= 0.006). DAPI staining revealed a 36% increase in central nuclei indicating myofiber regeneration (TRIM, 30.0 ± 1.7%; Saline, 22.0% ± 2.5; P=0.02). Microvessel area (CD31 staining, μm2) was 2.5-fold greater in treated TA’s (TRIM, 131 ± 12 μm2; Saline, 53 ± 6 μm2, P=0.0001). In muscle homogenates, TRIM had no effect on [VEGF] (TRIM, 102 ± 5%; Saline, 100 ± 4%; P= 0.79), while STAT3 activation was greater (TRIM, 129 ± 11%; Saline, 100 ± 7%; P= 0.04). Conclusion: At 140 days following injection of TRIM into the TA muscle of dystrophic mice, peak isometric force was unchanged while myofiber CSA was enhanced. Furthermore, TRIM promotes angiogenic growth factors and muscle vascularity, which should promote the ability to sustain physical activity. University of Missouri Coulter Biomedical Accelerator (SSS), American Physiological Society SURF (AGN), Texas A&M Department of Kinesiology and Sport Management (ABM). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

TRIM Enhances Dystrophic Muscle Function 70 Days Post Treatment

Background: Duchenne Muscular Dystrophy (DMD) is associated with limb muscle dysfunction caused by progressive weakness and degeneration of skeletal muscle. Current FDA-approved therapeutic strategies are less effective than originally hoped. Our alternative approach was to develop a novel biomaterial, Time Release Ion Matrix (TRIM), shown to release angiogenic ions and enhance muscle structure and function at 14 days post injection (dpi) in dystrophic (D2. mdx) mice. However, the long-term effects of TRIM are unknown. Hypothesis: At 70 days post injection in D2. mdx mice TRIM will: 1) enhance isometric force and myofiber cross-sectional area (CSA) of the tibialis anterior (TA) muscle; and 2) promote vascular endothelial growth factor (VEGF) availability and muscle vascularity. Methods: The right and left TA of adult male D2. mdx mice (n=6-7, age 5 months) were injected with saline alone (control) or 250 μg of TRIM suspended in saline. At 70 dpi, the left TA was isolated for in situ muscle force measurements and histological analysis; the right TA was frozen for biochemical analysis. Muscle cross-sections were immunostained for laminin (myofiber borders), DAPI (nuclei), and CD31 (endothelial cells). An ELISA for VEGF was performed on homogenates of frozen muscles. Results: TA’s injected with TRIM increased peak isometric force (means ± SE: TRIM 70.9 ± 1.8 g; Saline, 65.9 ± 1.1 g; P= 0.04). TRIM-treated TA’s had a 2.2-fold increase in relative frequency of myofibers >1000 μm2 (TRIM, 31.8 ± 3.7%; Saline, 14.4 ± 1.9%; P= 0.001), and reduced the frequency of myofibers <400 μm2 by 32% (TRIM, 38.6% ± 2.8; Saline, 56.5% ± 3.5; P= 0.001). DAPI staining revealed a 50% increase in central myonuclei indicative of regenerated myofibers (TRIM, 30.2 ± 1.7%; Saline, 20.2 ± 1.3%; P=0.0006). Microvessel area (CD31 staining; μm2) was 2.1-fold greater in treated TA’s (TRIM, 135.6 ± 8.7 μm2; Saline, 63.6 ± 4.5 μm2; P=0.0001). In muscle homogenates, TRIM increased [VEGF] (TRIM, 139 ± 10%; Saline, 100 ± 6%; P= 0.009) Conclusion: Results support the hypothesis that 70 days following injection of TRIM into the TA of dystrophic mice, muscle force and myofiber CSA are enhanced. Moreover, TRIM enhances VEGF with increased vascularity, suggesting that the angiogenic effects of TRIM complement those on myogenesis and contractile function towards restoring muscle structure and function in DMD. University of Missouri Coulter Biomedical Accelerator (SSS), Texas A&M Department of Kinesiology and Sport Management (ABM). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Deletion of the Aryl Hydrocarbon Receptor in Endothelial Cells Improves Ischemic Angiogenesis in Chronic Kidney Disease

Background: Chronic kidney disease (CKD) is a strong yet underappreciated risk factor for peripheral arterial disease (PAD) that exacerbates disease progression and increases risk for limb amputation and cardiovascular mortality. An estimated 25-35% of CKD patients develop PAD, but mechanistic data linking these two pathologies is limited. Several tryptophan-derived uremic metabolites are retained in the blood and tissues as a consequence of CKD and are endogenous ligands of the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor that has been shown to regulate angiogenesis. Hypothesis: We tested the hypothesis that endothelial cell-specific conditional deletion of the AHR would enhance ischemic limb outcomes in mice with CKD and femoral artery ligation (FAL). Methods: Twelve-week-old male and female endothelium-specific AHR knockout mice (AHRecKO) and AHR floxed mice (AHRfl/fl, Cre-negative control) were fed 0.2% adenine to induce CKD or casein control diet (n=10/group/sex) prior to undergoing FAL to induce limb ischemia. Laser Doppler flowmetry was used to measure limb perfusion. Isometric contraction of the tibialis anterior in-situ via nerve stimulus was performed to measure muscle function. Skeletal muscle mitochondrial respiratory function and H2O2 production were evaluated. Primary endothelial cells were isolated from male and female wildtype mice and treated with known AHR ligand indoxyl sulfate (IS) under hypoxic conditions to uncover sex-dependent differences in AHR activating potential. Results: In male mice with CKD, endothelium-specific ablation of the AHR resulted in significant increases in percentage of perfused capillaries within the ischemic limb (AHRecKO 73.41% ± 4.80% vs AHRfl/fl 64.75% ± 8.325%; p= 0.0074), improved laser Doppler perfusion recovery in the paw and gastrocnemius muscle, and larger myofiber cross-sectional area ( p=0.0258) compared to AHRfl/fl mice. However, these improvements did not translate to improved muscle contractile function. In contrast, deletion of the AHR in female mice with CKD had no significant impact on major outcome measures. Primary cells from male mice treated with IS had significantly higher expression of the AHR controlled gene Cyp1a1 ( p=0.026) compared to cells from female mice. Conclusion: Endothelium-specific deletion of the AHR improved ischemic angiogenesis in male, but not female, mice with CKD. These data reveal sex-dependent differences in AHR activating potential within endothelial cells that exist in the absence of sex hormones. This study was supported by National Institutes of Health (NIH) grant R01-HL149704 (T.E.R.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Single Cell Profiling Reveals Unique Contributions of Senescent Macrophages across Musculoskeletal Tissues in Posttraumatic Osteoarthritis

Injury to the acute cruciate ligament (ACL) disrupts musculoskeletal tissue architecture and function, leading to atrophy and weakness associated with the development of posttraumatic osteoarthritis (PTOA). The objective of this study was to profile senescent cell burden across skeletal muscle and knee joint capsule compartments and to ascertain the effectiveness of senolytic treatment in mitigation of PTOA. Given the unique features of these tissues, we hypothesized that composition and physiological consequence of senescent cell burden would differ among muscle and joint capsule, and senolytic treatment would prevent cartilage degradation and improve muscle function. We employed a surgical ACL transection model (ACLT) and performed scRNAseq analysis on the quadriceps and joint capsule 14-days post-injury and discerned senescent status by SPiDER-βGal. We also examined the influence of senescent cells on neighboring populations in vitro. We investigated whether senolytic treatment (dasatinib and quercetin; D+Q) improves muscle and joint health 28-d post-injury. scRNAseq revealed that macrophages (MΦ) accounted for 83% of senescent cells in the quadriceps and 48% in the joint capsule. Senescent MΦ exhibited an alternatively activated phenotype (M2) . Gene ontology enrichment analysis in the quadriceps showed downregulation of positive regulation of muscle cell differentiation, and upregulation of secretory granule lumen; in the joint capsule, it revealed upregulation of glycosaminoglycan catabolic process and regulation of ossification. In vitro, we found that mesenchymal progenitors incubated with senescent MΦ conditioned media exhibited upregulation of senescence associated secretory phenotype (SASP) genes and extracellular matrix organization-related genes ( i.e.Col1a1 and Lox). We found upregulation of protein glycosylation and downregulation of chondroitin sulfate proteoglycan biosynthetic process pathways. PTOA mice exhibited significant reductions in quadriceps myofiber cross-sectional area, increased accumulation of M2-like Cd163+ MΦ, and increased fibrosis, which were blunted in D+Q-treated mice. D+Q-treated PTOA mice exhibited a trend of improved peak torque compared to vehicle-treated PTOA mice. Histochemical assessment of whole knee joints revealed compromised articular cartilage and moderate to severe PTOA in vehicle-treated mice, which was blunted in D+Q-treated mice. These findings reveal distinct transcriptional profiles of senescent macrophages isolated from unique musculoskeletal tissues, providing critical insight into the heterogeneity of senescent cell programming following traumatic injury. Further, we show that accumulation of senescent alternatively-activated MΦ likely contributes to musculoskeletal dysfunction and development of PTOA, and that senolytics may improve functional recovery. AR072061. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Shift from Pro- to Anti-Inflammatory Phase in Pelvic Floor Muscles at Postpartum Matches Histological Signs of Regeneration in Multiparous Rabbits.

Background and Objectives: Pelvic floor muscles (PFM) play a core role in defecation and micturition. Weakening of PFM underlies urogynecological disorders such as pelvic organ prolapse and stress urinary incontinence. Vaginal delivery damages PFM. Muscle trauma implies an inflammatory response mediated by myeloid cells, essential for subsequent recovery. Molecular signaling characterizing the pro-inflammatory phase shifts M1 macrophages to M2 macrophages, which modulate muscle repair. The present study aimed to evaluate histological characteristics and the presence of M1 and M2 macrophages in bulbospongiosus (Bsm) and pubococcygeus muscles (Pcm). Materials and Methods: Muscles from young nulliparous (N) and multiparous rabbits on postpartum days three (M3) and twenty (M20) were excised and histologically processed to measure the myofiber cross-sectional area (CSA) and count the centralized myonuclei in hematoxylin-eosinstained sections. Using immunohistochemistry, M1 and M2 macrophages were estimated in muscle sections. Kruskal-Wallis or one-way ANOVA testing, followed by post hoc tests, were conducted to identify significant differences (p < 0.05). Results: The myofiber CSA of both the Bsm and Pcm of the M3 group were more extensive than those of the N and M20 groups. Centralized myonuclei estimated in sections from both muscles of M20 rabbits were higher than those of N rabbits. Such histological outcomes matched significant increases in HLA-DR immunostaining in M3 rabbits with the CD206 immunostaining in muscle sections from M20 rabbits. Conclusions: A shift from the pro- to anti-inflammatory phase in the bulbospongiosus and pubococcygeus muscles of multiparous rabbits matches with centralized myonuclei, suggesting the ongoing regeneration of muscles.

N-Acetylcysteine Attenuates Sepsis-Induced Muscle Atrophy by Downregulating Endoplasmic Reticulum Stress.

(1) Background: Sepsis-induced muscle atrophy is characterized by a loss of muscle mass and function which leads to decreased quality of life and worsens the long-term prognosis of patients. N-acetylcysteine (NAC) has powerful antioxidant and anti-inflammatory properties, and it relieves muscle wasting caused by several diseases, whereas its effect on sepsis-induced muscle atrophy has not been reported. The present study investigated the effect of NAC on sepsis-induced muscle atrophy and its possible mechanisms. (2) Methods: The effect of NAC on sepsis-induced muscle atrophy was assessed in vivo and in vitro using cecal ligation and puncture-operated (CLP) C57BL/6 mice and LPS-treated C2C12 myotubes. We used immunofluorescence staining to analyze changes in the cross-sectional area (CSA) of myofibers in mice and the myotube diameter of C2C12. Protein expressions were analyzed by Western blotting. (3) Results: In the septic mice, the atrophic response manifested as a reduction in skeletal muscle weight and myofiber cross-sectional area, which is mediated by muscle-specific ubiquitin ligases-muscle atrophy F-box (MAFbx)/Atrogin-1 and muscle ring finger 1 (MuRF1). NAC alleviated sepsis-induced skeletal muscle wasting and LPS-induced C2C12 myotube atrophy. Meanwhile, NAC inhibited the sepsis-induced activation of the endoplasmic reticulum (ER) stress signaling pathway. Furthermore, using 4-Phenylbutyric acid (4-PBA) to inhibit ER stress in LPS-treated C2C12 myotubes could partly abrogate the anti-muscle-atrophy effect of NAC. Finally, NAC alleviated myotube atrophy induced by the ER stress agonist Thapsigargin (Thap). (4) Conclusions: NAC can attenuate sepsis-induced muscle atrophy, which may be related to downregulating ER stress.

Stem cell secretome treatment improves whole-body metabolism, reduces adiposity, and promotes skeletal muscle function in aged mice.

Aging coincides with the progressive loss of muscle mass and strength, increased adiposity, and diminished physical function. Accordingly, interventions aimed at improving muscle, metabolic, and/or physical health are of interest to mitigate the adverse effects of aging. In this study, we tested a stem cell secretome product, which contains extracellular vesicles and growth, cytoskeletal remodeling, and immunomodulatory factors. We examined the effects of 4 weeks of 2×/week unilateral intramuscular secretome injections (quadriceps) in ambulatory aged male C57BL/6 mice (22-24 months) compared to saline-injected aged-matched controls. Secretome delivery substantially increased whole-body lean mass and decreased fat mass, corresponding to higher myofiber cross-sectional area and smaller adipocyte size, respectively. Secretome-treated mice also had greater whole-body physical function (grip strength and rotarod performance) and had higher energy expenditure and physical activity levels compared to control mice. Furthermore, secretome-treated mice had greater skeletal muscle Pax7+ cell abundance, capillary density, collagen IV turnover, reduced intramuscular lipids, and greater Akt and hormone sensitive lipase phosphorylation in adipose tissue. Finally, secretome treatment invitro directly enhanced muscle cell growth and IL-6 production, and in adipocytes, it reduced lipid content and improved insulin sensitivity. Moreover, indirect treatment with secretome-treated myotube culture media also enhanced muscle cell growth and adipocyte size reduction. Together, these data suggest that intramuscular treatment with a stem cell secretome improves whole-body metabolism, physical function, and remodels skeletal muscle and adipose tissue in aged mice.

Marked irregular myofiber shape is a hallmark of human skeletal muscle ageing and is reversed by heavy resistance training.

Age-related loss of strength is disproportionally greater than the loss of mass, suggesting maladaptations in the neuro-myo-tendinous system. Myofibers are often misshaped in aged and diseased muscle, but systematic analyses of large sample sets are lacking. Our aim was to investigate myofiber shape in relation to age, exercise, myofiber type, species and sex. Vastus lateralis muscle biopsies (n=265) from 197 males and females, covering an age span of 20-97years, were examined. The gastrocnemius and soleus muscles of 11+22-month-old male C57BL/6 mice were also examined. Immunofluorescence and ATPase stainings of muscle cross-sections were used to measure myofiber cross-sectional area (CSA) and perimeter. From these, a shape factor index (SFI) was calculated in a fibre-type-specific manner (type I/II in humans; type I/IIa/IIx/IIb in mice), with higher values indicating increased deformity. Heavy resistance training (RT) was performed three times per week for 3-4months by a subgroup (n=59). Correlation analyses were performed comparing SFI and CSA with age, muscle mass, maximal voluntary contraction (MVC), rate of force development and specific force (MVC/muscle mass). In human muscle, SFI was positively correlated with age for both type I (R2 =0.20) and II (R2 =0.38) myofibers. When subjects were separated into age cohorts, SFI was lower for type I (4%, P<0.001) and II (6%, P<0.001) myofibers in young (20-36) compared with old (60-80) and higher for type I (5%, P<0.05) and II (14%, P<0.001) myofibers in the oldest old (>80) compared with old. The increased SFI in old muscle was observed in myofibers of all sizes. Within all three age cohorts, type II myofiber SFI was higher than that for type I myofiber (4-13%, P<0.001), which was also the case in mice muscles (8-9%, P<0.001). Across age cohorts, there was no difference between males and females in SFI for either type I (P=0.496/0.734) or II (P=0.176/0.585) myofibers. Multiple linear regression revealed that SFI, after adjusting for age and myofiber CSA, has independent explanatory power for 8/10 indices of muscle mass and function. RT reduced SFI of type II myofibers in both young and old (3-4%, P<0.001). Here, we identify type I and II myofiber shape in humans as a hallmark of muscle ageing that independently predicts volumetric and functional assessments of muscle health. RT reverts the shape of type II myofibers, suggesting that a lack of myofiber recruitment might lead to myofiber deformity.

Dietary bile acids improve breast muscle growth in chickens through FXR/IGF2 pathway

It is a common practice to provide fast-growing broilers with high-fat diets in the context of integrated farms in Northeast China. Therefore, fat digestion, absorption, and utilization efficiency are critical for broiler meat production. Bile acids (BA) promote fat digestion and absorption, but whether and how BA affects muscle growth in broilers remains unclear. In this study, 1-day-old broilers were fed diets containing varying levels of crude fat (low, medium, and high) with or without BA supplementation for 42 d. Chickens fed a high-fat diet supplemented with BA exhibited significantly (P < 0.05) higher body weight (BW) at 21 d and average daily gain (ADG) during the first 21 d compared to the other groups. Throughout the entire experiment, feed conversion rate (FCR) was significantly (P < 0.05) lower in the high-fat group without the addition of BA, which was further decreased (P < 0.05) with BA supplementation. The improved growth performance in the BA-supplemented high-fat group was associated with significantly (P < 0.05) higher lipase activity in the small intestine chyme, a decreased trend (P = 0.06) in abdominal fat ratio, and significantly (P < 0.05) higher breast muscle mass. Histological analysis revealed significant (P < 0.05) increases in myofiber diameter, cross-sectional area, and RNA and DNA concentrations in the breast muscle of BA-supplemented broilers on the high-fat diet. Additional histological analysis further revealed significant (P < 0.05) enhancements in myofiber diameter, cross-sectional area, and RNA and DNA concentrations within the breast muscles of broilers supplemented with BA and a high-fat diet. The increased insulin-like growth factor 2 (IGF2) in the breast muscle of broilers fed a BA-supplemented high-fat diet correlated with significantly (P < 0.05) increased farnesoid X factor (FXR) protein expression and binding to the IGF2 promoter. These results suggest that dietary BA supplementation improves FCR and breast muscle growth in broilers fed a high-fat diet, potentially through the FXR-mediated IGF2 pathway.

Effects of sclerostin injection on soleus and extensor digitorum longus muscle tissue in male mice.

Sclerostin, a potent inhibitor of the Wnt signaling pathway, plays a critical role in bone homeostasis. Evidence suggests that sclerostin may also be involved in crosstalk between other tissues, including muscle. This pilot study attempted to examine the effects of sclerostin on soleus and extensor digitorum longus (EDL) muscle tissue from male mice that were given continuous recombinant sclerostin injections for 4 weeks. A total of 48 10-week-old male C57BL/6J mice were assigned to be sedentary or perform 1 h treadmill running per day for 4 weeks and administered subcutaneous injections of either saline or recombinant sclerostin 5 days/week. Sclerostin injection led to a reduction in the soleus myosin heavy chain (MHC) I, MHC I/IIA, MHC IIA/X, and MHC IIB cross-sectional area (p< 0.05) with no exercise effects on these reductions. In contrast, there were no effects of sclerostin injections or exercise on the fast-twitch EDL muscle in terms of size, MHC protein, or markers of Wnt signaling. These findings provide preliminary evidence of sclerostin's endocrine role in muscle via decreases in myofiber cross-sectional area, which seems to be independent of fiber type but muscle type-specific. More studies, however, are needed to confirm these preliminary results.

Abstract 18748: Prevention of Skeletal Muscle Atrophy and Fibrosis in Congestive Heart Failure by Skeletal Myoblast-Targeted Overexpression of Angiotensin II Type 2 Receptor

Introduction: We have previously shown that angiotensin II (Ang II) type 2 receptor (AT2R) positively regulates skeletal myoblast differentiation. Systemic angiotensin II (Ang II) increase or congestive heart failure suppress AT2R expression, likely contributing to the development of skeletal muscle atrophy and fibrosis associated with these conditions. Hypothesis: We hypothesized that AT2R overexpression in skeletal myoblasts could prevent skeletal muscle atrophy and fibrosis associated with congestive heart failure (CHF). Approach: Two mouse models, systemic Ang II infusion and left anterior descending coronary artery (LAD) ligation were used in this study. Myoblast differentiation, muscle atrophy and muscle fibrosis were assessed by analyzing gene expressions (e.g. myogenin and atrogin-1), myofiber cross sectional area, and the collagen deposition (picrosirius red staining). Primary myoblasts were isolated from mouse hindlimb muscles. Myoblast-specific AT2R-overexpressing transgenic mice were generated by crossing Pax7 CreER/+ mice (tamoxifen-inducible cre recombinase is expressed under Pax7 promoter) and CAG-stop flox -AT2R mice (AT2R is expressed under CMV early enhancer element, chicken β-actin gene promoter upon cre-mediated excision of floxed stop codon). Results: AT2R expression was robustly increased during myoblast differentiation both in vitro and in vivo . siRNA-mediated AT2R knockdown significantly suppressed myoblast differentiation in vitro and skeletal muscle regeneration in vivo . In both Ang II infusion and LAD ligation models, AT2R induction during myoblast differentiation was significantly suppressed, associated with reduced muscle regeneration and increased atrophy and fibrosis. Myoblast-specific AT2R overexpression in vivo restored muscle regenerative capacity, and prevented muscle atrophy and fibrosis. Conclusions: AT2R induction in skeletal myoblast is suppressed in CHF, possibly via increased Ang II. Decreased AT2R expression and its downstream signaling in myoblast lead to lowered muscle regenerative capacity and increased muscle atrophy and fibrosis. Restoration of myoblast AT2R expression could lead to a novel therapy to prevent muscle atrophy and fibrosis in CHF condition.

PSV-5 The Effect of a Prenatal Immune Challenge on Offspring Skeletal Muscle Characteristics in the Longissimus Muscle of Weaned Pigs

Abstract An insult to the health status of an animal is associated with decreased skeletal muscle growth. Furthermore, an immune challenge to a gestating dam may inhibit pre- and postnatal muscle growth of the offspring. The objective of this study was to determine if the administration of low-dose lipopolysaccharide (LPS) to gestating sows would alter muscle characteristics of the longissimus muscle (LM) in weaned pigs exposed to LPS. Pregnant Camborough sows were randomly assigned to receive LPS (LPS; n = 7) at a dose of 2.5 μg/kg BW or saline (CON; n= 7) on 78 ± 1.8 d of gestation. At weaning (21 ± 1.3 d of age), barrows (CON n = 17; LPS n = 17) from each sow received a secondary LPS challenge. Following the postnatal challenge, barrows were euthanized (31 ± 1.3 d of age), and both LM were collected. The left LM was weighed, measured for length, and measured for circumference at the cranial, middle, and caudal portion of the muscle. Two samples from the right LM were preserved for histological analyses. Morphometric, fiber type count, primary myofiber clustering, and satellite cell counts were analyzed using the MIXED procedure of SAS 9.4 with a main effect of prenatal treatment and covariate of body weight. There were no differences (P &amp;gt; 0.248) between any morphometric measures. The average diameter for myosin heavy chain (MHC) type I and IIB/X fibers was increased (P &amp;lt; 0.048) 8.6 and 5.9% respectively in LPS offspring compared with CON offspring. Average cross-sectional area (CSA) was increased 10.6% (P = 0.030) in MHC IIB/X fibers and tended to be increased 14.9% (P = 0.080) in MHC 1 fibers of LPS offspring. Number of myofibers within the LM was increased 16.1% (P = 0.032) in CON compared with LPS offspring. Diameter, CSA, and total CSA of oxidative myofibers (MHC type I and IIA) clusters was increased (P &amp;lt; 0.001) 13.8, 27.6, and 22.3% respectively in LPS compared with CON offspring. There were no differences (P &amp;gt; 0.186) between treatment groups for total nuclei or nuclei positive for MYF5, PAX7, or MYF5 and PAX7 nuclei. Based on the timing of the maternal challenge, it can be theorized that LPS hinders hyperplasia during secondary myogenesis following an immune challenge to the dam, with a rescue response of increased hypertrophy of MHC I and IIB/X myofibers post-weaning and glycolytic myofiber profile. However, due to the reduction in total myofibers, the impact of LPS on parameters such as average daily gain, feed efficiency, and lean skeletal muscle accretion should be studied further.

Glycine and N-Acetylcysteine (GlyNAC) Combined with Body Weight Support Treadmill Training Improved Spinal Cord and Skeletal Muscle Structure and Function in Rats with Spinal Cord Injury.

Skeletal muscle atrophy is a frequent complication after spinal cord injury (SCI) and can influence the recovery of motor function and metabolism in affected patients. Delaying skeletal muscle atrophy can promote functional recovery in SCI rats. In the present study, we investigated whether a combination of body weight support treadmill training (BWSTT) and glycine and N-acetylcysteine (GlyNAC) could exert neuroprotective effects, promote motor function recovery, and delay skeletal muscle atrophy in rats with SCI, and we assessed the therapeutic effects of the double intervention from both a structural and functional viewpoint. We found that, after SCI, rats given GlyNAC alone showed an improvement in Basso-Beattie-Bresnahan (BBB) scores, gait symmetry, and results in the open field test, indicative of improved motor function, while GlyNAC combined with BWSTT was more effective than either treatment alone at ameliorating voluntary motor function in injured rats. Meanwhile, the results of the skeletal muscle myofiber cross-sectional area (CSA), hindlimb grip strength, and acetylcholinesterase (AChE) immunostaining analysis demonstrated that GlyNAC improved the structure and function of the skeletal muscle in rats with SCI and delayed the atrophication of skeletal muscle.

Parathyroid Hormone Inhibits Fatty Infiltration and Muscle Atrophy After Rotator Cuff Tear by Browning of Fibroadipogenic Progenitors in a Rodent Model

Background: Progressive fatty infiltration and muscle atrophy after rotator cuff tears lead to tendon repair failure and poor outcomes. Fibro-adipogenic progenitors (FAPs) are involved in fatty infiltration and muscle homeostasis of skeletal muscle. Inducing FAP differentiation into brown adipocyte–like “beige adipocytes” suppresses fatty infiltration and muscle atrophy. Hypothesis: Parathyroid hormone (PTH) suppresses fatty infiltration and muscle atrophy after rotator cuff tears in a rat model by browning of FAPs. Study Design: Controlled laboratory study. Methods: PTH was administered subcutaneously for 4 or 8 weeks to a rotator cuff tear model in rats. After treatment, fatty infiltration of supraspinatus muscles was assessed using Oil Red O staining and muscle atrophy using wet muscle weight and muscle fiber cross-sectional area. Costaining of platelet-derived growth factor receptor α (FAP marker) and uncoupling protein 1 (browning marker) was performed to confirm FAP browning by PTH. Mouse-isolated FAPs were cultured with PTH and evaluated for browning-related gene expression and adipogenic differentiation using BODIPY staining. Myogenic differentiation of C2C12 myoblasts was evaluated using coculture of PTH-treated browning FAPs with C2C12. Results: PTH inhibited fatty infiltration after rotator cuff tear at 8 weeks. Rotator cuff wet muscle loss of PTH-treated rats was inhibited at 4 and 8 weeks. Furthermore, PTH-treated rats demonstrated larger myofiber cross-sectional area than did untreated rats at 4 and 8 weeks. Costaining indicated colocalization of platelet-derived growth factor receptor α and uncoupling protein 1 and promoted PTH-induced FAP browning. PTH increased the expression of browning-related genes in FAPs and suppressed fat droplet accumulation in vitro. Coculture with PTH-treated FAPs promoted C2C12 cell differentiation into myotubes. Conclusion: PTH induced FAP-derived beige adipocytes by upregulating browning-related gene expression, and the browning effect of PTH on FAPs inhibited fatty infiltration and muscle atrophy in the rat rotator cuff tear model. PTH might have potential as a therapeutic drug for fatty infiltration and muscle atrophy after rotator cuff tears. Clinical Relevance: PTH may expand treatment options for rotator cuff tears by reducing fatty infiltration and muscle atrophy after rotator cuff tears by browning of FAPs.

Rab44 deficiency accelerates recovery from muscle damage by regulating mTORC1 signaling and transport of fusogenic regulators.

The skeletal muscle is a tissue that shows remarkable plasticity to adapt to various stimuli. The development and regeneration of skeletal muscles are regulated by numerous molecules. Among these, we focused on Rab44, a large Rab GTPase, that has been recently identified in immune cells and osteoclasts. Recently, bioinformatics data has revealed that Rab44 is upregulated during the myogenic differentiation of myoblasts into myotubes in C2C12 cells. Thus, Rab44 may be involved in myogenesis. Here, we have investigated the effects of Rab44 deficiency on the development and regeneration of skeletal muscle in Rab44 knockout (KO) mice. Although KO mice exhibited body and muscle weights similar to those of wild-type (WT) mice, the histochemical analysis showed that the myofiber cross-sectional area (CSA) of KO mice was significantly smaller than that of WT mice. Importantly, the results of muscle regeneration experiments using cardiotoxin revealed that the CSA of KO mice was significantly larger than that of WT mice, suggesting that Rab44 deficiency promotes muscle regeneration. Consistent with the in vivo results, in vitro experiments indicated that satellite cells derived from KO mice displayed enhanced proliferation and differentiation. Mechanistically, KO satellite cells exhibited an increased mechanistic target of rapamycin complex 1 (mTORC1) signaling compared to WT cells. Additionally, enhanced cell surface transport of myomaker and myomixer, which are essential membrane proteins for myoblast fusion, was observed in KO satellite cells compared to WT cells. Therefore, Rab44 deficiency enhances muscle regeneration by modulating the mTORC1 signaling pathway and transport of fusogenic regulators.

Surface Electrical Impedance Myography Detects Skeletal Muscle Atrophy in Aged Wildtype Zebrafish and Aged gpr27 Knockout Zebrafish.

Throughout a vertebrate organism's lifespan, skeletal muscle mass and function progressively decline. This age-related condition is termed sarcopenia. In humans, sarcopenia is associated with risk of falling, cardiovascular disease, and all-cause mortality. As the world population ages, projected to reach 2 billion older adults worldwide in 2050, the economic burden on the healthcare system is also projected to increase considerably. Currently, there are no pharmacological treatments for sarcopenia, and given the long-term nature of aging studies, high-throughput chemical screens are impractical in mammalian models. Zebrafish is a promising, up-and-coming vertebrate model in the field of sarcopenia that could fill this gap. Here, we developed a surface electrical impedance myography (sEIM) platform to assess skeletal muscle health, quantitatively and noninvasively, in adult zebrafish (young, aged, and genetic mutant animals). In aged zebrafish (~85% lifespan) as compared to young zebrafish (~20% lifespan), sEIM parameters (2 kHz phase angle, 2 kHz reactance, and 2 kHz resistance) robustly detected muscle atrophy (p < 0.000001, q = 0.000002; p = 0.000004, q = 0.000006; p = 0.000867, q = 0.000683, respectively). Moreover, these same measurements exhibited strong correlations with an established morphometric parameter of muscle atrophy (myofiber cross-sectional area), as determined by histological-based morphometric analysis (r = 0.831, p = 2 × 10-12; r = 0.6959, p = 2 × 10-8; and r = 0.7220; p = 4 × 10-9, respectively). Finally, the genetic deletion of gpr27, an orphan G-protein coupled receptor (GPCR), exacerbated the atrophy of skeletal muscle in aged animals, as evidenced by both sEIM and histology. In conclusion, the data here show that surface EIM techniques can effectively discriminate between healthy young and sarcopenic aged muscle as well as the advanced atrophied muscle in the gpr27 KO animals. Moreover, these studies show how EIM values correlate with cell size across the animals, making it potentially possible to utilize sEIM as a "virtual biopsy" in zebrafish to noninvasively assess myofiber atrophy, a valuable measure for muscle and gerontology research.

Chronic repetitive cooling and caffeine-induced intracellular Ca2+ elevation differentially impact adaptations in slow- and fast-twitch rat skeletal muscles.

Intracellular Ca2+ concentration ([Ca2+]i) is considered important in the regulation of skeletal muscle mass. This study tested the hypothesis that chronic repeated cooling and/or caffeine ingestion would acutely increase [Ca2+]i and hypertrophy muscles potentially in a fiber-type-dependent manner. Control rats and those fed caffeine were subjected to repeated bidiurnal treatments of percutaneous icing, under anesthesia, to reduce the muscle temperature below ∼5°C. The predominantly fast-twitch tibialis anterior (TA) and slow-twitch soleus (SOL) muscles were evaluated after 28 days of intervention. The [Ca2+]i elevating response to icing was enhanced by caffeine loading only in the SOL muscle, with the response present across a significantly higher temperature range than in the TA muscle under caffeine-loading conditions. In both the TA and SOL muscles, myofiber cross-sectional area (CSA) was decreased by chronic caffeine treatment (mean reductions of 10.5% and 20.4%, respectively). However, in the TA, but not the SOL, CSA was restored by icing (+15.4 ± 4.3% vs. noniced, P < 0.01). In the SOL, but not TA, icing + caffeine increased myofiber number (20.5 ± 6.7%, P < 0.05) and satellite cell density (2.5 ± 0.3-fold) in cross sections. These contrasting muscle responses to cooling and caffeine may reflect fiber-type-specific [Ca2+]i responses and/or differential responses to elevated [Ca2+]i.

Exploring skeletal muscle tolerance and whole-body metabolic effects of FDA-approved drugs in a volumetric muscle loss model.

Volumetric muscle loss (VML) is associated with persistent functional impairment due to a lack of de novo muscle regeneration. As mechanisms driving the lack of regeneration continue to be established, adjunctive pharmaceuticals to address the pathophysiology of the remaining muscle may offer partial remediation. Studies were designed to evaluate the tolerance and efficacy of two FDA-approved pharmaceutical modalities to address the pathophysiology of the remaining muscle tissue after VML injury: (1) nintedanib (an anti-fibrotic) and (2) combined formoterol and leucine (myogenic promoters). Tolerance was first established by testing low- and high-dosage effects on uninjured skeletal muscle mass and myofiber cross-sectional area in adult male C57BL/6J mice. Next, tolerated doses of the two pharmaceutical modalities were tested in VML-injured adult male C57BL/6J mice after an 8-week treatment period for their ability to modulate muscle strength and whole-body metabolism. The most salient findings indicate that formoterol plus leucine mitigated the loss in muscle mass, myofiber number, whole-body lipid oxidation, and muscle strength, and resulted in a higher whole-body metabolic rate (p ≤ 0.016); nintedanib did not exacerbate or correct aspects of the muscle pathophysiology after VML. This supports ongoing optimization efforts, including scale-up evaluations of formoterol treatment in large animal models of VML.