Myofibre Cross-sectional Area Research Articles

RBP4 promotes denervation-induced muscle atrophy through STRA6-dependent pathway.

Fat infiltration of skeletal muscle has been recognized as a common feature of many degenerative muscle disorders. Retinol binding protein 4 (RBP4) is an adipokine that has been demonstrated to be correlated with the presence and severity of sarcopenia in the elderly. However, the exact role and the underlying mechanism of RBP4 in muscle atrophy remains unclear. Denervation-induced muscle atrophy model was constructed in wild-type and RBP4 knockout mice. To modify the expression of RBP4, mice were received intramuscular injection of retinol-free RBP4 (apo-RBP4), retinol-bound RBP4 (holo-RBP4) or oral gavage of RBP4 inhibitor A1120. Holo-RBP4-stimulated C2C12 myotubes were treated with siRNAs or specific inhibitors targeting signalling receptor and transporter of retinol 6 (STRA6)/Janus kinase 2 (JAK2)/Signal transducer and activator of transcription 3 (STAT3) pathway. Fat accumulation, myofibre cross-sectional area, myotube diameter and the expression of muscle atrophy markers and myogenesis markers were analysed. The expression levels of RBP4 in skeletal muscles were significantly up-regulated more than 2-fold from 7days and sustained for 28days after denervation. Immunofluorescence analysis indicated that increased RBP4 was localized in the infiltrated fatty region in denervated skeletal muscles. Knockout of RBP4 alleviated denervation-induced fatty infiltration and muscle atrophy together with decreased expression of atrophy marker Atrogin-1 and MuRF1 as well as increased expression of myogenesis regulators MyoD and MyoG. By contrast, injection of retinol-bound holo-RBP4 aggregated denervation-induced ectopic fat accumulation and muscle atrophy. Consistently, holo-RBP4 stimulation also had a dose-dependent effect on the reduction of C2C12 myotube diameter and myofibre cross-sectional area, as well as on the increase of Atrogin-1and MuRF1 expression and decrease of MyoD and MyoG expression. Mechanistically, holo-RBP4 treatment increased the expression of its membrane receptor STRA6 (>3-fold) and promoted the phosphorylation of downstream JAK2 and STAT3. Inhibition of STRA6/JAK2/STAT3 pathway either by specific siRNAs or inhibitors could decrease the expression of Atrogin-1 and MuRF1 (>50%) and decrease the expression of MyoD and MyoG (>3-fold) in holo-RBP4-treated C2C12 myotube. RBP4 specific pharmacological antagonist A1120 significantly inhibited the activation of STRA6/JAK2/STAT3 pathway, ameliorated ectopic fat infiltration and protected against denervation-induced muscle atrophy (30% increased myofibre cross-sectional area) in mice. In conclusion, our data reveal that RBP4 promotes fat infiltration and muscle atrophy through a STRA6-dependent and JAK2/STAT3 pathway-mediated mechanism in denervated skeletal muscle. Our results suggest that lowering RBP4 levels might serve as a promising therapeutic approach for prevention and treatment of muscle atrophy.

Lack of vitamin D signalling in mesenchymal progenitors causes fatty infiltration in muscle.

Recent studies have indicated the importance of muscle quality in addition to muscle quantity in sarcopenia pathophysiology. Intramuscular adipose tissue (IMAT), which originates from mesenchymal progenitors (MPs) in adult skeletal muscle, is a key factor affecting muscle quality in older adults, suggesting that controlling IMAT formation is a promising therapeutic strategy for sarcopenia. However, the molecular mechanism underlying IMAT formation in older adults has not been clarified. We recently found that the vitamin D receptor (VDR) is highly expressed in MPs in comparison to myotubes (P=0.028, N=3), indicating a potential role of vitamin D signalling in MPs. In this study, we aimed to clarify the role of vitamin D signalling in MP kinetics, with a focus on adipogenesis. MPs isolated from mouse skeletal muscles were subjected to adipogenic differentiation conditions with or without vitamin D (1α,25(OH)2D3, 100nM) for 7days, and adipogenicity was evaluated based on adipogenic marker expression. For in vivo analysis, tamoxifen-inducible MP-specific VDR-deficient (VdrMPcKO) mice were newly developed to investigate whether lack of vitamin D signalling in MPs is involved in IMAT formation. To induce muscle atrophy, VdrMPcKO male mice were subjected to tenotomy of the gastrocnemius muscle, and then muscle weight, myofibre cross-sectional area, adipogenic marker expression, and fatty infiltration into the muscle were evaluated at 3weeks after operation (N=3-4). In addition, a vitamin D-deficient diet was provided to wild-type male mice (3 and 20months of age, N=5) for 3months to investigate whether vitamin D deficiency causes IMAT formation. Vitamin D treatment nearly completely inhibited adipogenesis of MPs through Runx1-mediated transcriptional modifications of early adipogenic factors such as PPARγ (P=0.0031) and C/EBPα (P=0.0027), whereas VDR-deficient MPs derived from VdrMPcKO mice differentiated into adipocytes even in the presence of vitamin D (P=0.0044, Oil-Red O+ area). In consistency with in-vitro findings, VdrMPcKO mice and mice fed a vitamin D-deficient diet exhibited fat deposition in atrophied (P=0.0311) and aged (P=0.0216) skeletal muscle, respectively. Vitamin D signalling is important to prevent fate decision of MPs towards the adipogenic lineage. As vitamin D levels decline with age, our data indicate that decreased vitamin D levels may be one of the causes of IMAT formation in older adults, and vitamin D signalling may be a novel therapeutic target for sarcopenia.

Radiation induces long-term muscle fibrosis and promotes a fibrotic phenotype in fibro-adipogenic progenitors.

Radiation-induced muscle pathology, characterized by muscle atrophy and fibrotic tissue accumulation, is the most common debilitating late effect of therapeutic radiation exposure particularly in juvenile cancer survivors. In healthy muscle, fibro/adipogenic progenitors (FAPs) are required for muscle maintenance and regeneration, while in muscle pathology FAPs are precursors for exacerbated extracellular matrix deposition. However, the role of FAPs in radiation-induced muscle pathology has not previously been explored. Four-week-old Male CBA or C57Bl/6J mice received a single dose (16Gy) of irradiation (IR) to a single hindlimb with the shielded contralateral limb (CLTR) serving as a non-IR control. Mice were sacrificed 3, 7, 14 (acute IR response), and 56days post-IR (long-term IR response). Changes in skeletal muscle morphology, myofibre composition, muscle niche cellular dynamics, DNA damage, proliferation, mitochondrial respiration, and metabolism and changes in progenitor cell fate where assessed. Juvenile radiation exposure resulted in smaller myofibre cross-sectional area, particularly in type I and IIA myofibres (P<0.05) and reduced the proportion of type I myofibres (P<0.05). Skeletal muscle fibrosis (P<0.05) was evident at 56days post-IR. The IR-limb had fewer endothelial cells (P<0.05) and fibro-adipogenic progenitors (FAPs) (P<0.05) at 56days post-IR. Fewer muscle satellite (stem) cells were detected at 3 and 56days in the IR-limb (P<0.05). IR induced FAP senescence (P<0.05), increased their fibrogenic differentiation (P<0.01), and promoted their glycolytic metabolism. Further, IR altered the FAP secretome in a manner that impaired muscle satellite (stem) cell differentiation (P<0.05) and fusion (P<0.05). Our study suggests that following juvenile radiation exposure, FAPs contribute to long-term skeletal muscle atrophy and fibrosis. These findings provide rationale for investigating FAP-targeted therapies to ameliorate the negative late effects of radiation exposure in skeletal muscle.

Impact of short-term, pharmacological CARM1 inhibition on skeletal muscle mass, function, and atrophy in mice.

Coactivator-associated arginine methyltransferase 1 (CARM1) catalyzes the methylation of arginine residues on target proteins critical for health and disease. The purpose of this study was to characterize the effects of short-term, pharmacological CARM1 inhibition on skeletal muscle size, function, and atrophy. Adult mice (n = 10 or 11/sex) were treated with either a CARM1 inhibitor (150 mg/kg EZM2302; EZM) or vehicle (Veh) via oral gavage for 11-13 days and muscle mass, function, and exercise capacity were assessed. In addition, we investigated the effect of CARM1 suppression on unilateral hindlimb denervation (DEN)-induced muscle atrophy (n = 8/sex). We report that CARM1 inhibition caused significant reductions in the asymmetric dimethylation of known CARM1 substrates but no change in CARM1 protein or mRNA content in skeletal muscle. Reduced CARM1 activity did not affect body or muscle mass, however, we observed a decrease in exercise capacity and muscular endurance in male mice. CARM1 methyltransferase activity increased in the muscle of Veh-treated mice following 7 days of DEN, and this response was blunted in EZM-dosed mice. Skeletal muscle mass and myofiber cross-sectional area were significantly reduced in DEN compared with contralateral, non-DEN limbs to a similar degree in both treatment groups. Furthermore, skeletal muscle atrophy and autophagy gene expression programs were elevated in response to DEN independent of CARM1 suppression. Collectively, these results suggest that short-term, pharmacological CARM1 inhibition in adult animals affects muscle performance in a sex-specific manner but does not impact the maintenance and remodeling of skeletal muscle mass during conditions of neurogenic muscle atrophy.NEW & NOTEWORTHY Short-term pharmacological inhibition of coactivator-associated arginine methyltransferase 1 (CARM1) was effective at significantly reducing CARM1 methyltransferase function in skeletal muscle. CARM1 inhibition did not impact muscle mass, but exercise capacity was impaired, particularly in male mice, whereas morphological and molecular signatures of denervation-induced muscle atrophy were largely maintained in animals administered the inhibitor. Altogether, the role of CARM1 in neuromuscular biology remains complex and requires further investigation of its therapeutic potential in muscle-wasting conditions.

Resistance andendurance exercisetraining improves muscle mass and the inflammatory/fibrotic transcriptome in a rhabdomyosarcoma model.

Rhabdomyosarcoma (RMS) is an aggressive soft tissue sarcoma that most often develops in children. Chemoradiation therapy is a standard treatment modality; however, the detrimental long-term skeletal muscle consequences of this therapy in juvenile cancer survivors include muscle atrophy and fibrosis resulting in decreased physical performance. Using a novel model of murine resistanceand endurance exercisetraining, we investigate its role in preventing the long-term effects of juvenile RMS plus therapy. Four-week-old male (n=10) and female (n=10) C57Bl/6J mice were injected with M3-9-M RMS cell into the left gastrocnemius with the right limb serving as an internal control (CON). Mice received a systemic vincristine injection and then five doses of 4.8Gy of gamma radiation localized to the left hindlimb (RMS+Tx). Mice were then randomly divided into either sedentary (SED) or resistanceand endurance exercisetraining (RET) groups. Changes in exercise performance, body composition, myocellular adaptations and the inflammatory/fibrotic transcriptome were assessed. RET improved endurance performance (P<0.0001) and body composition (P=0.0004) compared to SED. RMS+Tx resulted in significantly lower muscle weight (P=0.015) and significantly smaller myofibre cross-sectional area (CSA) (P=0.014). Conversely, RET resulted in significantly higher muscle weight (P=0.030) and significantly larger Type IIA (P=0.014) and IIB (P=0.015) fibre CSA. RMS+Tx resulted in significantly more muscle fibrosis (P=0.028), which was not prevented by RET. RMS+Tx resulted in significantly fewer mononuclear cells (P<0.05) and muscle satellite (stem) cells (MuSCs) (P<0.05) and significantly more immune cells (P<0.05) than CON. RET resulted in significantly more fibro-adipogenic progenitors (P<0.05), a trend for more MuSCs (P=0.076) than SED and significantly more endothelial cells specifically in the RMS+Tx limb. Transcriptomic changes revealed significantly higher expression of inflammatory and fibrotic genes in RMS+Tx, which was prevented by RET. In the RMS+Tx model, RET also significantly altered expression of genes involved in extracellular matrix turnover. Our study suggests that RET preserves muscle mass and performance in a model of juvenile RMS survivorship while partially restoring cellular dynamics and the inflammatory and fibrotic transcriptome.

A novel rat model of sarcopenic obesity based on aging and high-fat diet consumption.

Sarcopenic obesity (SO) is defined as a combination of obesity and sarcopenia, leading to serious health consequences. However, a lack of suitable animal models has hampered research into this disorder.12-month-old Sprague-Dawley rats were given a high fat content (HFD, SO group) or standard diet (DC groups) for 28 weeks (until 20 months of age). In addition, 2-month-old rats were fed a standard diet as an age control (YC group) until they reached 10 months of age. At the end of the intervention, quadriceps development in the rats was monitored using magnetic resonance examinations and MR spectroscopy. Age-related changes in muscle mass and strength, histopathology, HFD-induced adiposity, and metabolic disturbances were compared between the three groups.Comparing with DC group, rats of SO (20 months, and fed by high-fat diet) exhibited a more prominent loss of muscle mass and strength, a more pronounced decline in myofibre number, IFM, increase in myocyte apoptosis accompanied with increased visceral fat, remarkable glycolipid metabolic disorders, and insulin resistance. However, DC group rats (20 months with standard diet) only showed a decline in quadriceps cross-sectional area/body weight, forelimb grip strength, myofibre cross-sectional area and number, and intermyofibrillar mitochondria number (IFM), increased myocyte apoptosis, without significant metabolic disorder compared with YC group rats.After verifying, SO animal model was successfully set up by HFD induced obesity concomitant with aging-related sarcopenia.

The impact of senescence on muscle wasting in chronic kidney disease.

Muscle wasting is a common complication of chronic kidney disease (CKD) that is associated with higher mortality. Although the mechanisms of myofibre loss in CKD has been widely studied, the contribution of muscle precursor cell (MPC) senescence remains poorly understood. Senescent MPCs no longer proliferate and can produce proinflammatory factors or cytokines. In this study, we tested the hypothesis that the senescence associated secretory phenotype (SASP) of MPCs contributes to CKD-induced muscle atrophy and weakness. CKD was induced in mice by 5/6th nephrectomy. Kidney function, muscle size, and function were measured, and markers of atrophy, inflammation, and senescence were evaluated using immunohistochemistry, immunoblots, or qPCR. To study the impact of senescence, a senolytics cocktail of dasatinib+quercetin (D&Q) was given orally to mice for 8weeks. To investigate CKD-induced senescence at the cellular level, primary MPCs were incubated with serum from CKD or control subjects. The roles of specific proteins in MPC senescence were studied using adenoviral transduction, siRNA, and plasmid transfection. In the hindlimb muscles of CKD mice, (i) the senescence biomarker SA-β-gal was sharply increased (~30-fold); (ii) the DNA damage response marker γ-H2AX was increased 1.9-fold; and (iii) the senescence pathway markers p21 and p16INK4a were increased 1.99-fold and 2.82-fold, respectively (all values, P<0.05), whereas p53 was unchanged. γ-H2AX, p21, and p16INK4A were negatively correlated at P<0.05 with gastrocnemius weight, suggesting a causal relationship with muscle atrophy. Administration of the senolytics cocktail to CKD mice for 8weeks eliminated the disease-related elevation of p21, p16INK4a , and γ-H2AX, abolished positive SA-β-gal, and depressed the high levels of the SASP cytokines, TNF-α, IL-6, IL-1β, and IFN (all values, P<0.05). Skeletal muscle weight, myofibre cross-sectional area, and grip function were improved in CKD mice receiving D&Q. Markers of protein degradation, inflammation, and MPCs dysfunction were also attenuated by D&Q treatment compared with the vehicle treatment in 5/6th nephrectomy mice (all values, P<0.05). Uraemic serum induced senescence in cultured MPCs. Overexpression of FoxO1a in MPCs increased the number of p21+ senescent cells, and p21 siRNA prevented uraemic serum-induced senescence (P<0.05). Senescent MPCs are likely to contribute to the development of muscle wasting during CKD by producing inflammatory cytokines. Limiting senescence with senolytics ameliorated muscle wasting and improved muscle strength in vivo and restored cultured MPC functions. These results suggest potential new therapeutic targets to improve muscle health and function in CKD.

Influence of vitamin D on sarcopenia pathophysiology: A longitudinal study in humans and basic research in knockout mice.

Vitamin D is an essential nutrient in musculoskeletal function; however, its relationship to sarcopenia remains ambiguous, and the mechanisms and targets of vitamin D activity have not been elucidated. This study aimed to clarify the role of vitamin D in mature skeletal muscle and its relationship with sarcopenia. This epidemiological study included 1653 community residents who participated in both the fifth and seventh waves of the National Institute for Longevity Sciences, Longitudinal Study of Aging and had complete background data. Participants were classified into two groups: vitamin D-deficient (serum 25-hydroxyvitamin D<20ng/mL) and non-deficient (serum 25-hydroxyvitamin D≥20ng/mL); they underwent propensity-score matching for background factors (age, sex, height, weight, comorbidities, smoker, alcohol intake, energy intake, vitamin D intake, steps, activity, season and sarcopenia). Changes in muscle strength and mass over the 4-year period were compared. For basic analysis, we generated Myf6CreERT2 Vitamin D Receptor (VDR)-floxed (VdrmcKO ) mice with mature muscle fibre-specific vitamin D receptor knockout, injected tamoxifen into 8-week-old mice and analysed various phenotypes at 16weeks of age. Grip strength reduction was significantly greater in the deficient group (-1.55±2.47kg) than in the non-deficient group (-1.13±2.47kg; P=0.019). Appendicular skeletal muscle mass reduction did not differ significantly between deficient (-0.05±0.79kg) and non-deficient (-0.01±0.74kg) groups (P=0.423). The incidence of new cases of sarcopenia was significantly higher in the deficient group (15 vs. 5 cases; P=0.039). Skeletal muscle phenotyping of VdrmcKO mice showed no significant differences in muscle weight, myofibre percentage or myofibre cross-sectional area; however, both forelimb and four-limb muscle strength were significantly lower in VdrmcKO mice (males: forelimb, P=0.048; four-limb, P=0.029; females: forelimb, P<0.001; four-limb, P<0.001). Expression profiling revealed a significant decrease in expression of sarcoendoplasmic reticulum Ca2+ -ATPase (SERCA) 1 (P=0.019) and SERCA2a (P=0.049) genes in the VdrmcKO mice. In contrast, expression of non-muscle SERCA2b and myoregulin genes showed no changes. Vitamin D deficiency affects muscle strength and may contribute to the onset of sarcopenia. Vitamin D-VDR signalling has minimal influence on the regulation of muscle mass in mature myofibres but has a significant influence on muscle strength.

Changes in vastus lateralis fibre cross-sectional area, pennation angle and fascicle length do not predict changes in muscle cross-sectional area.

What is the central question of this study? Do changes in myofibre cross-sectional area, pennation angle and fascicle length predict vastus lateralis whole-muscle cross-sectional area changes following resistance training? What is the main finding and its importance? Changes in vastus lateralis mean myofibre cross-sectional area, fascicle length and pennation angle following a period of resistance training did not collectively predict changes in whole-muscle cross-sectional area. Despite the limited sample size in this study, these data reiterate that it remains difficult to generalize the morphological adaptations that predominantly drive tissue-level vastus lateralis muscle hypertrophy. Myofibre hypertrophy during resistance training (RT) poorly associates with tissue-level surrogates of hypertrophy. However, it is underappreciated that, in pennate muscle, changes in myofibre cross-sectional area (fCSA), fascicle length (Lf ) and pennation angle (PA) likely coordinate changes in whole-muscle cross-sectional area (mCSA). Therefore, we determined if changes in fCSA, PA and Lf predicted vastus lateralis (VL) mCSA changes following RT. Thirteen untrained college-aged males (23±4years old, 25.4±5.2kg/m2 ) completed 7weeks of full-body RT (twice weekly). Right leg VL ultrasound images and biopsies were obtained prior to (PRE) and 72h following (POST) the last training bout. Regression was used to assess if training-induced changes in mean fCSA, PA and Lf predicted VL mCSA changes. Correlations were also performed between PRE-to-POST changes in obtained variables. Mean fCSA (+18%), PA (+8%) and mCSA (+22%) increased following RT (P <0.05), but not Lf (0.1%, P =0.772). Changes in fCSA, Lf and PA did not collectively predict changes in mCSA (R2 =0.282, adjusted R2 = 0.013, F3,8 =1.050, P =0.422). Moderate negative correlations existed for percentage changes in PA and Lf (r =-0.548, P =0.052) and changes in fCSA and Lf (r =-0.649, P =0.022), and all other associations were weak (|r|<0.500). Although increases in mean fCSA, PA and VL mCSA were observed, inter-individual responses for each variable and limitations for each technique make it difficult to generalize the morphological adaptations that predominantly drive tissue-level VL muscle hypertrophy. However, the small subject pool is a significant limitation, and more research in this area is needed.

Neurogranin inhibits calcineurin in murine soleus muscle: Effects of heterozygous knockdown on muscle adaptations to tenotomy and fatigue resistance

Neurogranin (Ng) is a calmodulin (CaM) binding protein that negatively regulates calcineurin - a Ca2+/CaM-dependent phosphatase that can mitigate the slow-to-fast fibre type shift observed with muscle unloading. Here, we questioned whether heterozygous deletion of Ng (Ng+/−) would enhance calcineurin activity, thereby minimizing the slow-to-fast fibre type shift caused by muscle unloading. As expected, soleus muscles from young adult (3–4 months old) Ng± mice had lowered Ng content and enhanced calcineurin activity when compared to soleus muscles obtained from male age-matched wild-type (WT) mice. Two weeks after tenotomy surgery, where the soleus and gastrocnemius tendons were severed, soleus total fibre count were found to be similarly reduced across both genotypes. However, significant reductions in myofibre cross-sectional area were only found in WT mice and not Ng± mice. Furthermore, while soleus muscles from both WT and Ng± mice exhibited a slow-to-fast fibre type shift with tenotomy, soleus muscles from Ng± mice, in both sham and tenotomized conditions, had a greater proportion of oxidative fibres (type I and IIA) compared with that of WT mice. Corresponding well with this, we found that soleus muscles from Ng± mice were more fatigue resistant compared with those obtained from their WT counterparts. Collectively, these findings show that heterozygous Ng deletion increases calcineurin activation, preserves myofibre size in response to unloading, and promotes the oxidative fibre type to ultimately enhance fatigue resistance. This study demonstrates the role of Ng in regulating calcineurin in vivo and its influence on skeletal muscle form and function.

Ambra1 deficiency impairs mitophagy in skeletal muscle.

BackgroundMaintaining healthy mitochondria is mandatory for muscle viability and function. An essential surveillance mechanism targeting defective and harmful mitochondria to degradation is the selective form of autophagy called mitophagy. Ambra1 is a multifaceted protein with well‐known autophagic and mitophagic functions. However, the study of its role in adult tissues has been extremely limited due to the embryonic lethality caused by full‐body Ambra1 deficiency.MethodsTo establish the role of Ambra1 as a positive regulator of mitophagy, we exploited in vivo overexpression of a mitochondria‐targeted form of Ambra1 in skeletal muscle. To dissect the consequence of Ambra1 inactivation in skeletal muscle, we generated muscle‐specific Ambra1 knockout (Ambra1 fl/fl:Mlc1f‐Cre) mice. Mitochondria‐enriched fractions were obtained from muscles of fed and starved animals to investigate the dynamics of the mitophagic flux.ResultsOur data show that Ambra1 has a critical role in the mitophagic flux of adult murine skeletal muscle and that its genetic inactivation leads to mitochondria alterations and myofibre remodelling. Ambra1 overexpression in wild‐type muscles is sufficient to enhance mitochondria clearance through the autophagy‐lysosome system. Consistently with this, Ambra1‐deficient muscles display an abnormal accumulation of the mitochondrial marker TOMM20 by +76% (n = 6–7; P < 0.05), a higher presence of myofibres with swollen mitochondria by +173% (n = 4; P < 0.05), and an alteration in the maintenance of the mitochondrial membrane potential and a 34% reduction in the mitochondrial respiratory complex I activity (n = 4; P < 0.05). Lack of Ambra1 in skeletal muscle leads to impaired mitophagic flux, without affecting the bulk autophagic process. This is due to a significantly decreased recruitment of DRP1 (n = 6–7 mice; P < 0.01) and Parkin (n = 6–7 mice; P < 0.05) to the mitochondrial compartment, when compared with controls. Ambra1‐deficient muscles also show a marked dysregulation of the endolysosome compartment, as the incidence of myofibres with lysosomal accumulation is 20 times higher than wild‐type muscles (n = 4; P < 0.05). Histologically, Ambra1‐deficient muscles of both 3‐ and 6‐month‐old animals display a significant decrease of myofibre cross‐sectional area and a 52% reduction in oxidative fibres (n = 6–7; P < 0.05), thus highlighting a role for Ambra1 in the proper structure and activity of skeletal muscle.ConclusionsOur study indicates that Ambra1 is critical for skeletal muscle mitophagy and for the proper maintenance of functional mitochondria.

Lack of muscle stem cell proliferation and myocellular hypertrophy in sIBM patients following blood-flow restricted resistance training

Sporadic inclusion body myositis (sIBM) is characterised by skeletal muscle inflammation, progressive muscle loss and weakness, which is largely refractory to immunosuppressive treatment. Low-load blood-flow restricted (BFR) training has been shown to evoke gains in myofibre cross sectional area (mCSA) in healthy adults. This could partially be due to the activation and integration of muscle satellite cells (SC) resulting in myonuclei addition. Consequently, this study investigated the effect of 12-weeks lower limb low-load BFR resistance training in sIBM patients on SC and myonuclei content, myofibre size and capillarization. Muscle biopsies from sIBM patients randomised to 12-weeks of low-load BFR resistance training (n = 11) or non-exercising controls (CON) (n = 9) were analysed for SC and myonuclei content, myofibre size and capillarization using three-colour immunofluorescence microscopy and computerised quantification procedures. No between-group differences (time-by-group interactions) or within-groups changes were observed for resident SCs (Pax7+/Six1+), proliferating SCs (Pax7+/ Ki67+), myonuclei (Six1+), type 1 mCSA or capillary number (CD31+). However, a time-by-group interaction for type 2 mCSA was observed (p = 0.04). Satellite cell content, myonuclei number, mCSA and capillary density remained unaffected following 12-weeks low-load BFR resistance training, indicating limited myogenic capacity and satellite cell plasticity in long-term sIBM patients.

Myofibre Hypertrophy in the Absence of Changes to Satellite Cell Content Following Concurrent Exercise Training in Young Healthy Men

Concurrent exercise training has been suggested to create an ‘interference effect,’ attenuating resistance training-based skeletal muscle adaptations, including myofibre hypertrophy. Satellite cells support myofibre hypertrophy and are influenced by exercise mode. To determine whether satellite cells contribute to the ‘interference effect’ changes in satellite cell and myonuclear content were assessed following a period of training in 32 recreationally active males (age: 25 ± 5 year; body mass index: 24 ± 3 kg⋅m–2; mean ± SD) who undertook 12-week of either isolated (3 d⋅w–1) resistance (RES; n = 10), endurance (END; n = 10), or alternate day (6 d⋅w–1) concurrent (CET, n = 12) training. Skeletal muscle biopsies were obtained pre-intervention and after 2, 8, and 12 weeks of training to determine fibre type-specific cross-sectional area (CSA), satellite cell content (Pax7+DAPI+), and myonuclei (DAPI+) using immunofluorescence microscopy. After 12 weeks, myofibre CSA increased in all training conditions in type II (P = 0.0149) and mixed fibres (P = 0.0102), with no difference between conditions. Satellite cell content remained unchanged after training in both type I and type II fibres. Significant correlations were observed between increases in fibre type-specific myonuclear content and CSA of Type I (r = 0.63, P < 0.0001), Type II (r = 0.69, P < 0.0001), and mixed fibres (r = 0.72, P < 0.0001). Resistance, endurance, and concurrent training induce similar myofibre hypertrophy in the absence of satellite cell and myonuclear pool expansion. These findings suggest that myonuclear accretion via satellite cell fusion is positively correlated with hypertrophy after 12 weeks of concurrent training, and that individuals with more myonuclear content displayed greater myofibre hypertrophy.

Functional recovery from eccentric injury is maintained in sarcopenic mouse muscle.

Eccentric contractions induce muscle damage (EIMD) that compromises muscle function. Poor recovery from EIMD has been suggested to be a contributor to the decline in muscle function evident in sarcopenia, but it is unclear which aspects of muscle function are more susceptible to disruption by EIMD in old versus young muscle. The purpose of this study was to determine the extent of impairment in contractile function (force, fatigue, tetanus and twitch kinetics) during the recovery from EIMD in very old (VO) mice compared to young adult (YA). Male CB6F1 were obtained from National Institure of Aging colony. VO mice were 29-31 months of age, and YA mice were 7-9 months of age. The plantarflexor muscles were subjected to 20 eccentric contractions in vivo to induce injury (EIMD). Changes in tetanic force and kinetics were assessed before EIMD, immediately after EIMD and 3 days after EIMD (3dEIMD). Force-frequency and rates of fatigue were assessed 3d-EIMD and compared with baseline. Histological analysis was conducted in injured and non-injured contralateral gastrocnemius muscle. There was a greater loss in isometric tetanic force immediately following EIMD in VO compared with YA (-31.6% ± 10.4 vs. -21.7% ± 6.0, P < 0.05). At 3d-EIMD, the rate of contraction of tetanus began to recover in VO, but not in YA (20.8% vs. -6.8%, P < 0.05), whereas the extent of recovery of force tended to be greater in VO than YA (39.3% vs. 17.1%, P = 0.08) when compared with tetanic function immediately after injury. Compared with function pre-injury (baseline), VO and YA had similar deficits in tetanic force (-7.3% ± 5.3 vs. -9.2% ± 6.0, respectively) and kinetics at Day 3. Twitch kinetics (rate of relaxation) recovered faster in VO compared with YA. The rate of muscle fatigue was similar to baseline values, with VO continuing to be more fatigue resistant than YA 3d-EIMD. There were no detectable differences in muscle mass or myofibre cross-sectional area despite continued deficits in force following EIMD in either age group. Despite clear functional deficits and greater susceptibility to injury, aged sarcopenic muscle exhibited a similar ability to recover contractile function to younger muscle following EIMD. In addition, neither age group showed accelerated muscle fatigue in the recovery phase after EIMD; thus, sarcopenic mouse muscles do not appear to be more susceptible to long-term functional impairment than young healthy muscles.

Weight loss with exercise improves muscle architecture and progenitor cell populations compared with weight loss alone in mice with preneoplastic colorectal lesions.

Weight loss and exercise reduce colorectal cancer (CRC) risk in persons with obesity. Whether weight loss and exercise effect myofiber characteristics and muscle stem/progenitor cell populations in mice with preneoplastic colorectal lesions, a model of CRC risk, is unknown. To address this gap, male C57Bl/6J mice were fed a high-fat diet (HFD) to induce obesity or a control (CON) diet prior to azoxymethane injection to induce preneoplastic colorectal lesions. The HFD group was then randomized to weight loss conditions that included (1) switching to the CON diet only (HFD-SED) or switching to the CON diet with treadmill exercise training (HFD-EX). Average myofiber cross-sectional area was not different between groups. There were more smaller-sized fibres in HFD-EX (p < 0.05 vs. CON), and more fibrosis in HFD-SED (p < 0.05 vs. HFD-EX and CON). There was a trend for more committed (Pax7+MyoD+) myoblasts (p = 0.059) and more fibro-adipogenic progenitors in HFD-EX (p < 0.05 vs. CON). Additionally, the canonical pro-inflammatory marker p-NF-κB was markedly reduced in the interstitium of HFD-EX (p < 0.05 vs. CON and HFD-SED). Our findings suggest that in mice with preneoplastic colorectal lesions, HFD followed by weight loss with exercise reduces muscle fibrosis and results in a higher content of muscle stem/progenitor cells. Novelty: Exercise improves muscle architecture in mice with preneoplastic colorectal lesion Exercise increases fibro/adipogenic progenitors and reduces inflammatory signaling in mice with preneoplastic colorectal lesions.

Skeletal Muscle Fibre Type Changes in an Avian Model of Hepatic Fibrosis

We investigated the susceptibility of type I and type II skeletal myofibres to atrophy in hens with hepatic fibrosis induced by bile duct ligation (BDL). Seven hens, approximately 2 years old, were randomly assigned to BDL (n=4) and sham surgery (SHAM) (n=3) groups. Mean body weight and mean liver weight as a percentage of mean body weight were significantly lower in the BDL group than in the SHAM group at 4 weeks post surgery (P=0.002, P=0.005, respectively). Mean plasma aspartate aminotransferase activity was slightly higher, while total cholesterol (P<0.001), total bilirubin (P=0.022) and NH3 (P=0.048) concentrations weresignificantly higher in the BDL group than in the SHAM group. Liver lesions were induced in all hens in the BDL group. The weights of the pectoralis (PCT) (P=0.049) and flexor perforans et perforatus digiti III (FPPD III) muscles (P=0.006) as a percentage of body weight were significantly decreased in the BDL group. A significantly reduced mean myofibre cross-sectional area in the PCT of BDL hens (P=0.005) was indicative of atrophy. No significant differences were observed in the fibre type composition of the PCT, supracoracoideus or FPPD III muscles between the SHAM and BDL groups. However, there was an approximate 43% increase in the number of type I fibres in the femorotibialis lateralis of the BDL group and small angular type II fibres and large round type I fibres in this muscle were characteristic of peripheral neuropathy. The results suggest that type II fibres are more susceptible to atrophy than type I fibres in this model of hepatic fibrosis.

Dietary guanidinoacetic acid supplementation improved carcass characteristics, meat quality and muscle fibre traits in growing-finishing gilts.

This study was designed to evaluate the effects of guanidinoacetic acid (GAA) on growth performance, carcass characteristics, meat and muscle fibre traits of growing-finishing gilts. 300 female PIC pigs were randomly divided (30.10±2.94kg) into 2 treatments with 6 replicates of 25 each for a 100-day trial. Two dietary treatments were comprised of a control diet and a control diet fortified with 450mg/kg GAA. Growth performance was evaluated for each phase. Carcass characteristics and meat quality were determined at last phase. Gilts had free access to feed and water during the experiment. The result indicated that GAA did not affect growth performance (p>0.05). GAA not only increased longissimus dorsi (LM) muscle weight but also decreased its shear force, b*value and drip loss (p<0.05). Mandibular fat index was decreased by GAA (p<0.05). GAA upregulated myosin heavy chain (MyHC) I mRNA expression with lower myofibre cross-sectional area and fibre diameter in LM muscle (p<.05). In conclusion, GAA can improve carcass characteristics and meat quality by changing muscle fibre characteristics and reducing mandibular fat index in finishing gilts.

Myopalladin promotes muscle growth through modulation of the serum response factor pathway.

BackgroundMyopalladin (MYPN) is a striated muscle‐specific, immunoglobulin‐containing protein located in the Z‐line and I‐band of the sarcomere as well as the nucleus. Heterozygous MYPN gene mutations are associated with hypertrophic, dilated, and restrictive cardiomyopathy, and homozygous loss‐of‐function truncating mutations have recently been identified in patients with cap myopathy, nemaline myopathy, and congenital myopathy with hanging big toe.MethodsConstitutive MYPN knockout (MKO) mice were generated, and the role of MYPN in skeletal muscle was studied through molecular, cellular, biochemical, structural, biomechanical, and physiological studies in vivo and in vitro.ResultsMKO mice were 13% smaller compared with wild‐type controls and exhibited a 48% reduction in myofibre cross‐sectional area (CSA) and significantly increased fibre number. Similarly, reduced myotube width was observed in MKO primary myoblast cultures. Biomechanical studies showed reduced isometric force and power output in MKO mice as a result of the reduced CSA, whereas the force developed by each myosin molecular motor was unaffected. While the performance by treadmill running was similar in MKO and wild‐type mice, MKO mice showed progressively decreased exercise capability, Z‐line damage, and signs of muscle regeneration following consecutive days of downhill running. Additionally, MKO muscle exhibited progressive Z‐line widening starting from 8 months of age. RNA‐sequencing analysis revealed down‐regulation of serum response factor (SRF)‐target genes in muscles from postnatal MKO mice, important for muscle growth and differentiation. The SRF pathway is regulated by actin dynamics as binding of globular actin to the SRF‐cofactor myocardin‐related transcription factor A (MRTF‐A) prevents its translocation to the nucleus where it binds and activates SRF. MYPN was found to bind and bundle filamentous actin as well as interact with MRTF‐A. In particular, while MYPN reduced actin polymerization, it strongly inhibited actin depolymerization and consequently increased MRTF‐A‐mediated activation of SRF signalling in myogenic cells. Reduced myotube width in MKO primary myoblast cultures was rescued by transduction with constitutive active SRF, demonstrating that MYPN promotes skeletal muscle growth through activation of the SRF pathway.ConclusionsMyopalladin plays a critical role in the control of skeletal muscle growth through its effect on actin dynamics and consequently the SRF pathway. In addition, MYPN is important for the maintenance of Z‐line integrity during exercise and aging. These results suggest that muscle weakness in patients with biallelic MYPN mutations may be associated with reduced myofibre CSA and SRF signalling and that the disease phenotype may be aggravated by exercise.

Intrauterine growth restriction affects diaphragm function in adult female and male mice

In utero diaphragm development is critically important for postnatal respiratory function and any disturbance to fetal development may lead to diaphragm dysfunction and respiratory complications in the postnatal period. Intrauterine growth restriction (IUGR) has been shown to affect respiratory function in a sex-dependent manner; however, the effect of IUGR on diaphragm function is unknown. This study used a maternal hypoxia-induced mouse model of IUGR to investigate the impact of IUGR on diaphragm function and structure in male and female adult offspring. Pregnant BALB/c mice were housed under hypoxic conditions (10.5% O2 ) from gestational days 11 to 17.5 and then returned to normoxic conditions. Control mice were housed under normoxic conditions throughout pregnancy. At 8 weeks of age, offspring were euthanized and diaphragms isolated for functional assessment in organ bath experiments and for histological analysis. IUGR offspring were lighter at birth and remained lighter at 8 weeks of age compared to Controls. While diaphragm force (maximal or twitch) was not affected by treatment or sex, the IUGR group exhibited a longer half-relaxation time after twitch contractions compared to Control. Female offspring had a lower maximum rate of force development and higher fatigue resistance compared to males, independent of IUGR. There was no difference in the diaphragm myofibre cross-sectional area between groups or sexes. Sex and IUGR independently affect diaphragm contraction in adult mice without changes in structure. This study demonstrates that IUGR affects diaphragm contractile function in later life and could impair respiratory function if exacerbated under conditions of increased respiratory load.

The Late Effects of Radiation Therapy on Skeletal Muscle Morphology and Progenitor Cell Content are Influenced by Diet-Induced Obesity and Exercise Training in Male Mice

Radiation exposure during muscle development induces long-term decrements to skeletal muscle health, which contribute to reduced quality of life in childhood cancer survivors. Whether the effects of radiation on skeletal muscle are influenced by relevant physiological factors, such as obesity and exercise training remains unknown. Using skeletal muscle from our previously published work examining the effects of obesity and exercise training on radiation-exposed bone marrow, we evaluated the influence of these physiological host factors on irradiated skeletal muscle morphology and cellular dynamics. Mice were divided into control and high fat diet groups with or without exercise training. All mice were then exposed to radiation and continued in their intervention group for an additional 4 weeks. Diet-induced obesity resulted in increased muscle fibrosis, while obesity and exercise training both increased muscle adiposity. Exercise training enhanced myofibre cross-sectional area and the number of satellite cells committed to the myogenic lineage. High fat groups demonstrated an increase in p-NFĸB expression, a trend for a decline in IL-6, and increase in TGFB1. These findings suggest exercise training improves muscle morphology and satellite cell dynamics compared to diet-induced obesity in irradiated muscle, and have implications for exercise interventions in cancer survivors.