American Journal of Physiology-Cell Physiology

The effect of chronic contractile activity (CCA) on the biophysical properties and functional activity of skeletal muscle extracellular vesicles (Skm-EVs) is poorly understood due to challenges in distinguishing Skm-EVs originating from exercising muscle in vivo. To address this, myoblasts were differentiated into myotubes, and electrically paced (3 h/day, 4 days @ 14 V). CCA evoked an increase in mitochondrial biogenesis in stimulated vs. non-stimulated (CON) myotubes as expected. EVs were isolated from conditioned media from control and stimulated myotubes using differential ultracentrifugation and characterized biophysically using tunable resistive pulse sensing (TRPS, Exoid), TEM and western blotting. TEM images confirmed isolated round-shaped vesicles of about 30 - 150 nm with an intact lipid bilayer. The mean size of EVs ranged from 98 -138 nm, and was not altered by CCA. Zeta potential and total EV protein yield remained unchanged between groups, and total EV secretion increased after 4 days of CCA. Concomitant analysis of EVs after each day of CCA also demonstrated a progressive increase in CCA-EV concentration, while size and zeta potential remained unaltered, and EV protein yield increased in both CON-EVs and CCA groups. CCA-EVs were enriched with small-EVs vs. CON-EVs, concomitant with higher expression of small-EV markers CD81, Tsg101 and HSP70. In whole cell lysates, CD63 and ApoA1 were reduced with CCA in myotubes, whereas CD81, Tsg101, Flotillin-1 and HSP70 levels remained unchanged. To evaluate the functional effect of EVs secreted post-CCA, we treated C2C12 myoblasts with all EVs isolated from CON or CCA myotubes after each day of stimulation, and measured cell count, cell viability, protein yield and mitochondrial biogenesis in recipient cells. There was no effect on cell count, viability and protein yield. Myoblasts treated with CCA-EVs exhibited increased mitochondrial biogenesis as indicated by enhanced MitoTracker Red staining, cytochrome c oxidase activity, and protein expression of electron transport chain subunit, CIV-MTCO1. Further, CCA-EV treatment enhanced maximal oxygen consumption rates (OCR), and ATP production in treated myoblasts. This increase in maximal OCR was abrogated when CCA-EVs pre-treated with proteinase K were co-cultured with myoblasts, indicating the pro-metabolic effect was likely mediated by transmembrane or peripheral membrane proteins in CCA-EVs. Our data highlight the novel effect of Skm-EVs isolated post-CCA in mediating pro-metabolic effects in recipient cells and thereby transmitting the effects associated with traditional exercise. Further investigation to interrogate the underlying mechanisms involved in downstream cellular metabolic adaptations is warranted.

ObjectiveWe evaluated associations between vitamin D status and skeletal muscle, strength, and bone mineral density (BMD) outcomes after ACL reconstruction (ACLR) in an observational study. MethodsSerum measures included 25-hydroxyvitamin D (25(OH)D; free and total), vitamin D binding protein (DBP), and 1,25-dihydroxy vitamin D (1,25(OH)2D) at baseline, 1 week, 4 months, and 6 months post-ACLR. Vastus lateralis biopsies were collected from the healthy and ACL-injured limb of 21 young, healthy participants (62% female; 17.8 [3.2] yr, BMI: 26.0 [3.5] kg/m2) during ACLR and the injured limb only at 1 week and 4 month follow ups. RNA and protein were isolated from biopsies and assessed for vitamin D receptor [VDR], and vitamin D-activating enzymes. Quadriceps fiber cross-sectional area (CSA) was determined with immunohistochemistry. BMD of femur and tibia were determined at baseline and 6 months post-ACLR; strength was assessed with an isokinetic dynamometer. Results1,25(OH)2D decreased from baseline to one week after ACLR (21.6 [7.9] vs. 13.8 [5.5] pg/mL; p<0.0001). VDR and 25-hydroxylase transcript abundance and VDR and DBP proteins were elevated one week after ACLR compared with baseline (FDR<0.05; p<0.05). Participants with an average total 25(OH)D <30 ng/mL showed significant decreases in CSA 1 week and 4 months after ACLR (p<0.01; p=0.041 for time x D status interaction), whereas those with total 25(OH)D [&ge;]30ng/mL showed no significant differences (p>0.05 for all comparisons). BMD and strength measures were lower at follow up but did not associate with vitamin D status. ConclusionACLR promotes vitamin D pathways in the quadriceps and low status is associated with loss of skeletal muscle both 1 week and 4 months after ACLR. Summary BoxO_LIWhat is already known on this topic - Quadriceps muscle atrophy, strength loss, and reduced bone mineral density persist for many years after ACL tear and reconstruction (ACLR) leading to poorer function and long term knee health outcomes. Circulating 25-hydroxyvitamin D concentrations [&ge;]30ng/mL (75nmol/L) have been associated with reduced risk of stress fracture and injury and greater strength, but it is not known how vitamin D status, which is easily modified with supplementation, may affect ACLR outcomes. C_LIO_LIWhat this study adds - Our work shows that ACLR surgery reduces biologically active vitamin D in circulation and promotes vitamin D receptor and activating enzyme expression in skeletal muscle one week after surgery. Circulating concentrations of 25(OH)D <30 ng/mL associate with greater loss of quadriceps fiber CSA both one week and 4 months after ACLR. C_LIO_LIHow this study might affect research, practice or policy - Results suggest that correcting vitamin D status prior to ACLR may support retention of skeletal muscle size in recovery, which should be tested in a randomized clinical trial to begin to establish vitamin D cut points optimizing recovery from ACL tear and reconstruction. C_LI

Extracellular vesicles (EVs) released from all cells, are essential to cellular communication, and contain biomolecular cargo that can affect recipient cell function. Studies on the effects of contractile activity (exercise) on EVs usually rely on plasma/serum-based assessments, which contain EVs from many different cells. To specifically characterize skeletal muscle-derived vesicles and the effect of acute contractile activity, we used an in vitro model where C2C12 mouse myoblasts were differentiated to form myotubes. EVs were isolated from conditioned media from muscle cells, pre-differentiation (myoblasts) and post-differentiation (myotubes), as well as from acutely stimulated myotubes (1hr @ 14V, C-Pace EM, IonOptix) using total exosome isolation reagent (TEI, ThermoFisher, referred to as extracellular particles [EPs]) and differential ultracentrifugation (dUC; EVs). Myotube-EPs (~98 nm) were 41% smaller than myoblast-EPs (~167 nm, p<0.001, N=8-10). Two-way ANOVA showed a significant main effect for size distribution of myotube vs. myoblast-EPs (p<0.01, N=10-13). Myoblast-EPs displayed a bimodal size distribution profile with peaks at <200 nm and 400-600 nm, compared to myotube-EPs that were largely 50-300 nm in size. Total protein yield from myotube-EPs was nearly 15-fold higher than myoblast-EPs, (p<0.001 N=6-9). Similar biophysical characteristics were observed when EVs were isolated using dUC: myotube-EVs (~195 nm) remained 41% smaller in average size than myoblast-EVs (~330 nm, p=0.07, N=4-6) and had comparable size distribution profiles as EPs isolated via TEI. Myotube-EVs also had 4.7-fold higher protein yield vs. myoblast EVs (p<0.05, N=4-6). Myotube-EPs had significantly decreased expression of exosomal marker proteins TSG101, CD63, ALIX and CD81 compared to myoblast-EPs (p<0.05, N=7-12). Conversely, microvesicle marker ARF6, and lipoprotein marker APO-A1was only found in the myotube-EPs (p<0.05, N=4-12). There was no effect of acute stimulation on myotube-EP biophysical characteristics (N=7), nor on expression of TSG101, ARF6 or CD81 (N=5-6). Myoblasts treated with control or acute stimulation-derived EPs (13 g/well) for 48hrs and 72hrs showed no changes in mitochondrial mass (MitoTracker Red), cell viability or cell count (N=3-4). Myoblasts treated with EP-depleted media (72hrs) had ~90% lower cell counts (p<0.01, N=3). Our data show that EVs differ in size, distribution, protein yield and expression of subtype markers pre- vs. post-skeletal muscle differentiation. There was no effect of acute stimulation on biophysical profile or protein markers in EPs. Acute stimulation-derived EPs did not alter mitochondrial mass nor cell count/viability. Further investigation into the effects of chronic contractile activity on the biophysical characteristics and cargo of skeletal muscle-specific EVs are warranted.

Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder, for which no cure exists. This study investigates the effects of 12-week strength training on mitochondrial oxidative phosphorylation in skeletal muscle in a cohort of DM1 patients (n=11, males) in comparison to untrained sex-matched healthy subjects. Immunofluorescence was used to assess protein levels of key respiratory chain subunits of complex I (CI) and complex IV (CIV), and markers of mitochondrial mass and cell membrane in individual myofibers sampled from biopsies. We classified each patient myofiber as having normal, low or high levels of CI and CIV and compared the proportions of affected fibers before and after exercise training. The significance of changes observed between pre- and post-exercise training within patients was estimated using a permutation test. At baseline, DM1 patients present with significantly decreased mitochondrial mass, and isolated or combined CI and CIV deficiency. After strength training, in most patients a significant increase in mitochondrial mass was observed, and all patients showed a significant increase in CI and/or CIV protein levels. Remarkably, 12-week strength training is sufficient to partially rescue mitochondrial dysfunction in DM1 patients, suggesting exercise as an inexpensive and accessible therapy option.

Cells rapidly lose their physiological phenotype upon disruption of their extracellular matrix (ECM)-intracellular cytoskeleton interactions. Here, we investigated acute effects of ECM disruption on cellular and mitochondrial morphology, transcriptomic signatures, and Ca2+ handling in adult mouse skeletal muscle fibers. Adult skeletal muscle fibers were isolated from mouse toe muscle either by collagenase-induced dissociation of the ECM or by mechanical dissection that leaves the contiguous ECM intact. Experiments were generally performed four hours after cell isolation. At this time, there were striking differences in the gene expression patterns between fibers isolated with the two methods; 24h after cell isolation, enzymatically dissociated fibers had transcriptomic signatures resembling dystrophic phenotypes. Mitochondrial appearance was grossly similar in the two groups, but 3D electron microscopy revealed shorter and less branched mitochondria in enzymatically dissociated than in mechanically dissected fibers. Similar increases in free cytosolic [Ca2+] during repeated tetanic stimulation were accompanied by marked mitochondrial Ca2+ uptake only in enzymatically dissociated muscle fibers. The aberrant mitochondrial Ca2+ uptake was partially prevented by the mitochondrial Ca2+ uniporter inhibitor Ru360 and by cyclosporine A and NV556, which inhibit the mitochondrial protein Ppif (also called cyclophilin D). Importantly, inhibition of Ppif with NV556 significantly improved survival of mice with mitochondrial myopathy in which muscle mitochondria take up excessive amounts of Ca2+ even with an intact ECM. In conclusion, skeletal muscle fibers isolated by collagenase-induced dissociation of the ECM display aberrant mitochondrial Ca2+ uptake, which involves a Ppif-dependent mitochondrial Ca2+ influx resembling that observed in mitochondrial myopathies.

BackgroundSkeletal muscle in wasting conditions often exhibits a common set of phenotypes that include atrophy, mitochondrial respiratory dysfunction, and fragmentation of the acetylcholine receptor (AChR) cluster at the endplate. Mitochondria are frequently implicated in driving muscle pathology in these conditions, although which aspects of mitochondrial function are most relevant is poorly understood. MethodsTo address this gap, we focused on mitochondrial permeability transition (mPT), a well-established pathological mechanism in ischemia-reperfusion injury and neurodegeneration but poorly studied in skeletal muscle. We performed a broad assessment of the consequences of mPT in skeletal muscle, focusing on features that are common in wasting conditions. We then tested whether tumor-host factors could promote mPT and compared differentially expressed genes (DEGs) with mPT and a mouse model of pancreatic cancer cachexia. ResultsInducing mPT in mouse skeletal muscle bundles in a Ca2+ retention capacity assay progressively altered mitochondrial morphology, beginning with cristae swirling and condensation, progressing to mitochondrial cristae displacement, and culminating in breach of the outer mitochondrial membrane; features that are common in wasting conditions. Inducing mPT with Bz423 in single mouse muscle fibers increased mROS and Caspase 3 (Casp3) activity and was prevented by inhibitors of mPT, mROS or Casp3. Incubating single muscle fibers with Bz423 for 24 h reduced fiber diameter by [~]20% which was prevented by inhibiting mPT, mROS, or Casp3. Inducing mPT caused a complex I-specific mitochondrial respiratory impairment and increased co-localization of lysosomes with mitochondria. Inducing mPT also fragmented the AChR cluster at the muscle endplate and was prevented by inhibiting mPT or Casp3. The Ca2+ threshold for mPT and mitochondrial calcein colocalization were reduced by pancreatic tumor-conditioned media in skeletal muscle or C2C12 myoblasts, respectively, and these effects were counteracted by mPT inhibition or cyclophilin D knockout. Finally, there was significant overlap between the transcriptome of mPT and that seen in diaphragm muscle in a mouse model of pancreatic cancer cachexia, particularly during the muscle wasting phase. ConclusionsWe conclude that inducing mPT in skeletal muscle recapitulates muscle phenotypes common with muscle wasting conditions like cachexia. Furthermore, mPT is engaged by tumor-host factors and had significant overlap with DEGs seen during the muscle wasting phase in a mouse model of pancreatic cancer cachexia, warranting further investigation of mPT as a therapeutic target.

The ERG1A K+ channel modulates the protein degradation that contributes to skeletal muscle atrophy by increasing intracellular calcium concentration ([Ca2+]i) and enhancing calpain activity, but the mechanism by which the channel regulates the [Ca2+]i is not known. Here, we have investigated the effect of human ERG1A (HERG) on [Ca2+]i in C2C12 myotubes, using Fura-2 calcium assays, immunoblot, RT-qPCR, and electrophysiology. We hypothesized that HERG would modulate L-type calcium channel activity, specifically the Cav1.1 channel known to carry signal from the sarcoplasmic membrane of skeletal muscle to the sarcomeres of the myofibrils. However, we find that HERG has no effect on the amplitude of L-type channel current nor does it affect the mRNA levels nor protein abundance of the Cav1.1 channel. Instead we find that, although the rise in [Ca2+]i (induced by depolarization) is greater in myotubes over-expressing HERG relative to controls, the difference between the KCl-stimulated Ca2+ increase in control and HERG over-expressing cells cannot be accounted for by L-type channel mediated Ca2+ influx, which suggests that HERG could modulate excitation coupled calcium entry (ECCE). Indeed, the HERG-enhanced increase in [Ca2+]i induced by depolarization is blocked by 2-APB, an inhibitor of ECCE (and SOCE). Further, we show data suggesting that HERG also modulates the activity of ryanodine receptors, a component of ECCE, as well as store operated calcium entry (SOCE). Therefore, we investigated the effect of HERG on calsequestrin1, a calcium buffering/binding protein known to modulate ryanodine receptor 1 and store operated Ca2+ entry activities. Indeed, we find that calsequestrin1 mRNA levels are decreased 0.83-fold (p<0.05) and the total protein abundance is lowered 77% (p<0.05) in myotubes over-expressing HERG relative to controls. In summary, the data show that ERG1A overexpression modulates [Ca2+]i in skeletal muscle cells by lowering the abundance of the calcium buffering/binding protein calsequestrin1.

Non-insertional Achilles tendinopathy (NIAT) induces morpho-mechanical changes to the Achilles tendon (AT). However, evidence on how triceps surae motor unit firing properties are influenced by altered tendon mechanics in NIAT is limited. This study investigated motor unit firing properties (mean discharge rate (DR), recruitment and de-recruitment thresholds, and discharge rate variability (COVisi)), motor unit firing-torque relationships (cross-correlation coefficient between cumulative spike train (CST) and torque, and neuromechanical delay), and neural drive distribution (connectivity strength and functional networks) of the medial gastrocnemius (MG), lateral gastrocnemius (LG), and soleus (SO) muscles during isometric plantarflexion contractions at 10%, 40%, and 70% maximal voluntary contraction (MVC) using high-density surface electromyography on 26 individuals with NIAT and 25 healthy controls. Furthermore, ATs morpho-mechanical properties (thickness, cross-sectional area, length and stiffness) were assessed via ultrasound imaging. NIAT individuals showed reduced tendon stiffness and increased thickness (p<0.01). Motor unit properties changed in a load and muscle-dependent manner. LG DR increased (p=0.002) and de-recruitment threshold decreased (p=0.039) at 70%MVC in the NIAT group compared to controls. The CST-torque cross-correlation coefficient of the LG decreased at 10%MVC (p<0.0001) and increased at 70%MVC (p=0.013) in the NIAT group. Connectivity strength for the 0-5 Hz and 5-15 Hz frequency bands decreased (p<0.01) in the NIAT group at 10%MVC. This study shows that individuals with NIAT exhibit load-dependent changes in motor unit firing properties, motor unit-torque relationships, and neural drive distribution to the triceps surae. These alterations may be due to muscle-specific compensations for the modified mechanical properties of the AT. KEY POINTS- Individuals with non-insertional Achilles tendinopathy (NIAT) have changes of the neural drive to the lateral gastrocnemius (LG) muscle and altered contribution of the LG to the net plantarflexion torque. - Individuals with NIAT show a more uneven distribution of neural drive to the triceps surae muscle at low force levels, characterized by reduced intermuscular coherence between the medial and lateral gastrocnemius in the 0-5 Hz and 5-15 Hz bands compared to the control group. - Our findings support the idea that the LG may have a central role in the pathophysiology of this condition, possibly affecting the load transmission to the Achilles tendon (AT).

ObjectivesDeficits in muscle performance could be a consequence of a reduced ability of a motor neuron to increase the rate in which it discharges. This study aimed to investigate motor unit (MU) discharge properties of each Triceps surae muscle (TS), and TS torque steadiness during submaximal intensities in runners with Achilles tendinopathy (AT). MethodsWe recruited runners with (n=12) and without (n=13) mid-portion AT. MU discharge rate was analysed for each of the TS muscles, using High-Density surface electromyography during 10 and 20% isometric plantar flexor contractions. ResultsMU mean discharge rate was lower in the Gastrocnemius lateralis (GL) in AT compared to controls. In AT, GL MU mean discharge rate did not increase as torque increased from 10% peak torque, 8.24pps (95%CI: 7.08 to 9.41) to 20%, 8.52pps (7.41 to 9.63, p=0.540); however, in controls, MU discharge rate increased as torque increased from 10%, 8.39pps (7.25 to 9.53) to 20%, 10.07pps (8.89 to 11.25, p<0.001). There were no between-group difference in Gastrocnemius medialis (GM) or Soleus (SOL) MU discharge rates. We found no between-groups differences in coefficient of variation of MU discharge rate in any of the TS muscles nor in TS torque steadiness. ConclusionOur data demonstrates that runners with AT may have a reduced neural drive to GL, failing to increase MU discharge rate to adjust for the increase in torque demand. Further research is needed to understand how interventions focusing on increasing neural drive to GL would affect muscle function in runners with AT.

BackgroundEccentric exercise (ECC) is widely recognized as an effective treatment for non-insertional Achilles tendinopathy (NIAT); however, the mechanisms underlying its apparent superiority over concentric exercise (CON) remain poorly understood. This randomized controlled trial aimed to investigate changes in triceps surae motor unit firing properties, pain, function, and AT morpho-mechanical properties following a 6-week intervention involving torque feedback training with isolated ECC and CON contractions in individuals with NIAT. MethodsTwenty-six individuals with NIAT were randomized to ECC or CON training. Motor unit firing properties (mean discharge rate [MDR], recruitment and de-recruitment thresholds, and torque-firing cross-correlation and neuromechanical delay) were assessed in the medial gastrocnemius (MG), lateral gastrocnemius (LG), and soleus (SO) muscles using high-density surface electromyography (HD-sEMG) during isometric plantarflexion at 10%, 40%, and 70% of maximal voluntary contraction (MVC). Pain, function (VISA-A), and tendon properties (ultrasound, elastography) were measured at baseline, week 3, and week 6. ResultsBoth groups showed similar improvements in pain (P < 0.0001) and VISA-A scores (P < 0.001). Tendon stiffness increased in both groups by week 3 but was higher in ECC by week 6 (P = 0.02). Motor unit adaptations differed: CON demonstrated an increase in MG MDR at 40% MVC, while ECC showed a decrease (interaction: P = 0.0008). Only ECC led to increased de-recruitment thresholds in the LG at 70% MVC (P < 0.0001). However, both groups exhibited reduced MDR in the LG at high-force levels (P < 0.05). Additionally, both interventions reduced GL torque-firing relationships (P = 0.025) and decreased SO neuromechanical delay (P = 0.031). ConclusionA 6-week visuo-motor torque feedback training program involving isolated CON or ECC contractions leads to comparable improvements in clinical outcomes. However, contraction-specific and muscle-specific changes in motor unit function and tendon stiffness suggest distinct neuromechanical adaptations. These differences may underlie the observed effects and warrant further investigation in longer-term studies to determine their impact on long-term clinical outcomes. HIGHLIGHTS- Changes in clinical outcomes, tendon morphomechanical properties, and triceps surae motor unit behavior were assessed for the first time following a 6-week rehabilitation protocol for non-insertional Achilles tendinopathy, involving isolated concentric (CON) or eccentric (ECC) contractions. - Both CON and ECC training led to comparable improvements in pain, self-reported outcomes, and perceived function. - Despite these similar clinical improvements, CON and ECC protocols produced distinct adaptations in estimated tendon stiffness and motor unit firing characteristics. These neuromechanical differences may indicate potential long-term divergences between training modalities, which should be explored in studies with extended intervention periods.

Insulin resistance in skeletal muscle is a hallmark of type 2 diabetes mellitus (T2D). While two-dimensional myotube cultures offer a controlled environment for studying T2D-related metabolic dysfunction, insulin-dependent glucose transporter type 4 (GLUT4) levels are limited and insulin-independent glucose transporter type 1 (GLUT1) expression dominates; reducing physiological relevance. Three-dimensional skeletal muscle microtissue cultures offer a promising alternative, and unlike 2D myotubes, are amenable to repeated contractile stimulation. However, microtissue GLUT1 and GLUT4 glucose transporter profiles remain under-characterized, particularly under physiological glucose and insulin conditions, which is evaluated herein. We report that GLUT1 levels trended [~]3.0-fold lower in microtissues compared with myotubes in 2D culture, although not statistically significant (p = 0.072), while GLUT4 levels were [~]12-fold higher (p < 0.0001), leading to a [~]60-fold increase in the GLUT4:GLUT1 ratio (p = 0.023). Notably, the microtissue GLUT4:GLUT1 profile approached, but did not match that of native human muscle. Microtissues required supraphysiological insulin conditions for the development of maximal contractility, while physiological glucose levels were sufficient. Insulin withdrawal restored insulin responsiveness but impaired microtissue contractile strength (p < 0.0001) and fatigue resistance (p = 0.015). Our findings indicate that the glucose transporter profile of microtissues offers improved physiological relevance. However, their reliance on insulin to maintain contractile function limits their suitability for modeling T2D. The implementation of a robust, insulin-free differentiation protocol would facilitate the development of a microtissue-based T2D model which can be applied to study contraction-mediated increases in insulin sensitivity as a therapeutic approach.

Following anabolic stimuli (e.g. mechanical loading and/or amino acid provision) the mechanistic target of rapamycin complex 1 (mTORC1), a master regulator of protein synthesis, translocates toward the cell periphery. However, it is unknown if mTORC1 activity occurs prior to or following this translocation. We therefore aimed to determine the cellular location of mTORC1 activity in human skeletal muscle following anabolic stimuli. Fourteen young, healthy males either ingested a protein-carbohydrate beverage (0.25g/kg protein, 0.75g/kg carbohydrate) alone (n=7, 23{+/-}5yrs, 76.8{+/-}3.6kg, 13.6{+/-}3.8%BF, FED) or following a whole-body resistance exercise bout (n=7, 22{+/-}2yrs, 78.1{+/-}3.6kg, 12.2{+/-}4.9%BF, EXFED). Vastus lateralis muscle biopsies were obtained at rest (PRE) and 120 and 300min following anabolic stimuli. The spatial regulation of mTORC1 activity was assessed through immunofluorescent staining of p-RPS6Ser240/244, an mTORC1-specific phosphorylation event. p-RPS6Ser240/244 measured by immunofluorescent staining or immunoblot was positively correlated (r=0.76, p<0.001). Peripheral staining intensity of p-RPS6Ser240/244 increased above PRE in both FED and EXFED at 120min (~54% and ~138% respectively, p<0.05) but was greater in EXFED at both post-stimuli time points (p<0.05). The peripheral-central ratio of p-RPS6240/244 staining was displayed a similar pattern, suggesting mTORC1 activity occurs predominantly in the periphery of fibers. Moreover, p-RPS6Ser240/244 intensity within paxillin-positive regions, a marker of focal adhesion complexes, was elevated at 120min irrespective of stimulus (p=0.006) before returning to PRE at 300min. These data confirm that mTORC1 activity occurs in the region of human muscle fibers to which mTORC1 translocates following anabolic stimuli and identifies focal adhesion complexes as a potential site of mTORC1 activation in vivo.

BackgroundCritical illness myopathy (CIM) is a debilitating condition characterized by the preferential loss of the motor protein myosin. CIM is a byproduct of critical care, attributed to impaired recovery, long-term complications, and mortality. CIM pathophysiology is complex, heterogeneous and remains incompletely understood, however loss of mechanical stimuli contributes to critical illness associated muscle atrophy and weakness. Passive mechanical loading (ML) and electrical stimulation (ES) therapies augment muscle mass and function. While having beneficial outcomes, the mechanistic underpinning of these therapies is less known. Therefore, here we aimed to assess the mechanism by which chronic supramaximal ES ameliorates CIM in a unique experimental rat model of critical care. MethodsRats were subjected to 8 days critical care conditions entailing deep sedation, controlled mechanical ventilation, and immobilization with and without direct soleus ES. Muscle size and function were assessed at the single cell level. RNAseq and Western blotting were employed to understand the mechanisms driving ES muscle outcomes in CIM. ResultsFollowing 8 days of controlled mechanical ventilation and immobilization, soleus muscle mass, Myosin:Actin ratio and single muscle fiber maximum force normalized to cross-sectional area (specific force) were reduced by 40-50% (p< 0.0001). ES significantly reduced the loss of soleus muscle fiber cross-sectional area (CSA) and Myosin:Actin ratio by approximately 30% (p< 0.05) yet failed to effect specific force. RNAseq pathway analysis revealed downregulation of insulin signaling in the soleus muscle following critical care and GLUT4 trafficking was reduced by 55% leading to an 85% reduction of muscle glycogen content (p< 0.01). ES promoted phosphofructokinase and insulin signaling pathways to control levels (p< 0.05), consistent with the maintenance of GLUT4 translocation and glycogen levels. AMPK, but not AKT, signaling pathway was stimulated following ES, where the downstream target TBC1D4 increased 3 logFC (p= 0.029) and AMPK-specific P-TBC1D4 levels were increased approximately 2-fold (p= 0.06). Reduction of muscle protein degradation rather than protein synthesis promoted soleus CSA, as ES reduced E3 ubiquitin proteins, Atrogin-1 (p= 0.006) and MuRF1 (p= 0.08) by approximately 50%, downstream of AMPK-FoxO3. ConclusionsES maintained GLUT4 translocation through increased AMPK-TBC1D4 signaling leading to improved muscle glucose homeostasis. Soleus CSA and myosin content was promoted through reduced protein degradation via AMPK-FoxO3 E3 ligases, Atrogin-1 and MuRF1. These results demonstrate chronic supramaximal ES reduces critical care associated muscle wasting, preserved glucose signaling and reduced muscle protein degradation in CIM.

BackgroundStatins are the drugs most commonly used for lowering plasma low-density lipoprotein (LDL) cholesterol levels and reducing cardiovascular disease risk. Although generally well tolerated, statins can induce myopathy, a major cause of non-adherence to treatment. Impaired mitochondrial function has been implicated as a cause of statin-induced myopathy, but the underlying mechanism remains unclear. We have shown that simvastatin downregulates transcription of TOMM40 and TOMM22, genes that encode major subunits of the translocase of outer mitochondrial membrane (TOM) complex which is responsible for importing nuclear-encoded proteins and maintaining mitochondrial function. We therefore investigated the role of TOMM40 and TOMM22 in mediating statin effects on mitochondrial function, dynamics, and mitophagy. MethodsCellular and biochemical assays and transmission electron microscopy were used to investigate effects of simvastatin and TOMM40 and TOMM22 expression on measures of mitochondrial function and dynamics in C2C12 and primary human skeletal cell myotubes. ResultsKnockdown of TOMM40 and TOMM22 in skeletal cell myotubes impaired mitochondrial oxidative function, increased production of mitochondrial superoxide, reduced mitochondrial cholesterol and CoQ levels, disrupted mitochondrial dynamics and morphology, and increased mitophagy, with similar effects resulting from simvastatin treatment. Overexpression of TOMM40 and TOMM22 in simvastatin-treated muscle cells rescued statin effects on mitochondrial dynamics, but not on mitochondrial function or cholesterol and CoQ levels. Moreover, overexpression of these genes resulted in an increase in number and density of cellular mitochondria. ConclusionThese results confirm that TOMM40 and TOMM22 are central in regulating mitochondrial homeostasis and demonstrate that downregulation of these genes by statin treatment mediates disruption of mitochondrial dynamics, morphology, and mitophagy, effects that may contribute to statin-induced myopathy. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/546411v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1845842org.highwire.dtl.DTLVardef@1be8a26org.highwire.dtl.DTLVardef@5fd618org.highwire.dtl.DTLVardef@1e0f523_HPS_FORMAT_FIGEXP M_FIG C_FIG

In Duchenne muscular dystrophy (DMD), a lack of functional dystrophin leads to myofiber instability and progressive muscle damage that results in fibrosis. While fibrosis is primarily characterized by an accumulation of extracellular matrix (ECM) components, there are changes in ECM architecture during fibrosis that relate more closely to functional muscle stiffness. One of these architectural changes in dystrophic muscle is collagen cross-linking, which has been shown to increase the passive muscle stiffness in models of fibrosis including the mdx mouse, a model of DMD. We tested whether the intraperitoneal injections of beta-aminopropionitrile (BAPN), an inhibitor of the cross-linking enzyme lysyl oxidase, would reduce collagen cross- linking and passive stiffness in young and adult mdx mice compared to saline-injected controls. We found no significant differences between BAPN treated and saline treated mice in collagen cross-linking and stiffness parameters. However, we observed that while collagen cross-linking and passive stiffness scaled positively in dystrophic muscles, collagen fiber alignment scaled with passive stiffness distinctly between muscles. We also observed that the dystrophic diaphragm showed the most dramatic fibrosis in terms of collagen content, cross-linking, and stiffness. Overall, we show that while BAPN was not effective at reducing collagen cross- linking, the positive association between collagen cross-linking and stiffness in dystrophic muscles still show cross-linking as a viable target for reducing passive muscle stiffness in DMD or other fibrotic muscle conditions. Key PointsO_LIBAPN did not reduce collagen cross-linking or passive stiffness in any muscle C_LIO_LICollagen cross-links scaled with passive stiffness in dystrophic muscles C_LIO_LIThe mdx diaphragm showed the most dramatic fibrosis related to collagen content, cross- linking, and passive stiffness C_LIO_LICollagen fiber alignment scales with passive stiffness differently between muscles C_LI

Extracellular vesicles (EVs) are small lipid bilayer-delimited particles that are secreted by all cells, playing a central role in cellular communication. EVs are released from skeletal muscle during exercise, but the effects of contractile activity on skeletal muscle-derived EVs (Skm-EVs) are poorly understood due to the challenges in distinguishing Skm-EVs derived from exercising muscle in vivo. Using tunable resistive pulse sensing (TRPS), we previously demonstrated that chronic contractile activity (CCA) increased the secretion of Skm-EVs from C2C12 myotubes, while their size and zeta potential remained unchanged. Here, we aimed to determine whether similar results could be obtained using an alternative method of EV characterization, dynamic light scattering (DLS). C2C12 myoblasts were differentiated into myotubes, and electrically paced (3h/day x 4days @14V, C-PACE EM, IonOptix) to mimic chronic exercise in vitro. EVs were isolated from conditioned media of control and stimulated myotubes using differential ultracentrifugation, and characterized based on size and zeta potential. The mean size of EVs from chronically stimulated myotubes (CCA-EVs, 132 nm) was 26% smaller than control (CON-EVs, 178 nm). Size distribution analysis revealed that CCA-EVs were enriched in small EVs (100-150 nm), while CON-EVs were largely composed of 200-250 nm sized vesicles. Additionally, zeta potential was 27% lower in CCA-EVs compared to CON-EVs. Our data indicate that the effect of CCA on facilitating the release of smaller, more stable EVs, is a robust finding, reproducible by multiple methods of EV characterization. Future studies investigating the mechanisms by which CCA influences Skm-EV biogenesis and secretion are warranted.

We have previously shown that skeletal muscle-derived extracellular vesicles (EVs) released post-chronic contractile activity (CCA) increased mitochondrial biogenesis in murine myoblasts, and decreased cell viability and induced apoptosis and senescence in non-small cell lung cancer cells. While the underlying mechanisms are unknown, the effects perpetuated were dependent on membrane-bound proteins. Here, we performed an extensive LC-MS/MS proteomic analysis on EVs from control and CCA myotubes. A total of 2900 proteins were identified in CON-EVs and CCA-EVs, including EV-associated proteins such as TSG101, tetraspanins (CD9, CD81, and CD63), flotillin-1, and annexins. Of these, 856 proteins are novel and not listed in EV databases (ExoCarta and Vesiclepedia), indicating that myotube-EVs harbor proteins not yet identified in EVs of different origin. Additionally, we identified 2062 unique proteins that have not yet been previously reported in myotube-EVs to date. Remarkably, of the 2900 total proteins identified, we observed 46 upregulated, and 25 downregulated differentially expressed proteins (DEPs) in CCA-EVs vs. control-EVs. Most of upregulated DEPs include EV-associated proteins. Comparing the 71 DEPs with proteins expressed in skeletal muscle indicated 61 of these as potential myokines. We identified actin cytoskeleton signaling, integrin signaling and muscle contraction as the most enriched pathways among the DEPs using different databases/software including FunRich, KEGG, STRING and Ingenuity Pathway Analysis. Using a relevance score that prioritized membrane-bound proteins with known function in mitochondrial biogenesis and inhibition of cancer growth, we identified top-scoring highly enriched DEPs of interest: IGF1R, ATP7A, PFN1, GJA1, PRKCA and ITGA6. We confirmed upregulation of these targets in EVs using immunoblotting. Among these top-scoring DEPs, PFN1, and ITGA6 are associated with EVs, with expression upregulated following acute exercise. In summary, we report the first comprehensive analysis of skeletal muscle-EV proteome following CCA, with identification of putative protein targets and signaling pathways that may execute the pro-metabolic and anti-tumorigenic effects of CCA-EVs.

BackgroundSkeletal muscle dysfunction is a major peripheral determinant of exercise intolerance and physical disability in heart failure with preserved ejection fraction (HFpEF). Metabolic and mitochondrial dysfunction are considered to be key components of skeletal muscle dysfunction, but comprehensive profiling of metabolic pathways has not been conducted. Elucidation of dysregulated metabolic pathways is essential to determine viable targets for the treatment of exercise intolerance in HFpEF. MethodsMale ZSF1 Obese rats (HFpEF) and Wistar Kyoto (WKY) lean normotensive controls were studied at 26 weeks of age. Gastrocnemius was subjected to bulk RNA-seq, proteomics, metabolomics, and lipidomics analysis. The R package limma was used to determine differential expression in all omics layers (absolute fold-change>1.5, FDR0.05, unless otherwise indicated). Additional targeted plasma and skeletal muscle (soleus and EDL) metabolomics and lipidomics were performed on HFpEF and control rats. ResultsPathway level analysis for RNA seq and proteomics revealed significant downregulation of oxidative phosphorylation (NES -2.1, p<0.005), electron transport chain (NES -2.0, p<0.005), and TCA cycle (-1.8, p<0.05). The most upregulated pathways were PPAR signaling (NES 2.2, p<0.0001), tryptophan metabolism (NES 1.8, P<0.005), and amino acid oxidation (NES 1.8, p<0.005) pathways. Metabolomics revealed an accumulation of TCA cycle intermediate, isocitrate, and phosphate reduction. Branched-chain amino acids were significantly increased, whereas amino acids related to tryptophan metabolism were reduced and shifted towards increased serotonin accumulation. Phospholipid species were differentially regulated with increased palmitoylated phosphatidylcholines but reduced arachidonoyl-PC species. Phosphatidylethanolamines (PE) species (16:0/16:1-18:0/18:2) were increased. ConclusionOur multiomics analysis of skeletal muscle in HFpEF revealed severe mitochondrial dysfunction that was characterized by reduced complex I and II activity. Mitochondrial and peroxisomal lipid overload results in a shift in membrane phospholipid accumulation and composition. Reduced BCAA oxidation and dysregulation of tryptophan metabolism are key features of amino acid metabolism that reduce anaplerosis and promote the accumulation of toxic metabolites. Comparative analysis of other skeletal muscle disorders suggests that an acquired metabolic myopathy exists in cardiometabolic HFpEF.

The provisional matrix protein versican is upregulated in Duchenne muscular dystrophy. Versican heightens inflammation in fibrotic diseases and is involved in myogenesis. In fibrotic diaphragm muscles from dystrophic mdx mice, versican reduction attenuated macrophage infiltration and improved contractile function. We investigated the association between versican and mdx hindlimb muscle pathology, where inflammation and regeneration are increased but fibrosis is minimal. Immunohistochemistry and qRT-PCR were used to assess how fiber type and glucocorticoids (-methylprednisolone) modulate versican expression. Female mdx and male versican haploinsufficient (hdf) mice were bred resulting in male mdx-hdf and mdx (control) pups. Versican expression, contractile function, and pathology were evaluated in fast extensor digitorum longus (EDL) and slow soleus muscles, excised under medetomidine-midazolam- fentanyl anesthesia. Versican immunoreactivity was highest in soleus muscles. Versican mRNA transcripts were reduced by -methylprednisolone in soleus, but not EDL, muscles. Versican expression was decreased in soleus muscles from 6-week-old mdx-hdf mice leading to increased force output and a modest reduction in fatiguability. These functional benefits were not accompanied by decreased inflammation; muscle architecture, regeneration markers, and fiber type also did not differ between genotypes. Improvements in soleus function were lost in adult (20-week-old) mdx-hdf mice with no significant effect of versican haploinsufficiency on macrophage infiltration and regeneration markers. Soleus muscles from juvenile mdx mice were most responsive to pharmacological or genetic approaches targeting versican; however, the benefits of versican reduction were limited due to low fibrosis. Pre-clinical matrix research in dystrophy should account for muscle phenotype and the interdependence between the fibrosis and inflammation. NEW & NOTEWORTHYThe proteoglycan versican is upregulated in muscular dystrophy. In fibrotic diaphragm muscles from mdx mice, versican reduction attenuated macrophage infiltration and improved performance. Here, in hindlimb muscles from 6- and 20-week-old mdx mice, where pathology is mild, versican reduction did not decrease inflammation and contractile function improvements were limited to juvenile mice. In dystrophic mdx muscles, the association between versican and inflammation is mediated by fibrosis, demonstrating interdependence between the immune system and extracellular matrix.

Functional loss of the motor protein, Myosin Vb (MYO5B), induces various defects in intestinal epithelial function and causes a congenital diarrheal disorder, microvillus inclusion disease (MVID). Utilizing the MVID model mice, Vil1-CreERT2;Myo5bflox/flox (MYO5B{Delta}IEC) and Vil1-CreERT2;Myo5bflox/G519R(MYO5B(G519R)), we previously reported that functional MYO5B loss disrupts progenitor cell differentiation and enterocyte maturation that result in villus blunting and deadly malabsorption symptoms. In this study, we determined that both absence and a point mutation of MYO5B impair lipid metabolism and alter mitochondrial structure, which may underlie the progenitor cell malfunction observed in MVID intestine. Along with a decrease in fatty acid oxidation, the lipogenesis pathway was enhanced in the MYO5B{Delta}IEC small intestine. Consistent with these observations in vivo, RNA-sequencing of enteroids generated from two MVID mouse strains showed similar downregulation of energy metabolic enzymes, including mitochondrial oxidative phosphorylation genes. In our previous studies, lysophosphatidic acid (LPA) signaling ameliorates epithelial cell defects in MYO5B{Delta}IEC tissues and enteroids. The present study demonstrates that the highly soluble LPAR5-preferred agonist, Compound-1, improved sodium transporter localization and absorptive function, and tuft cell differentiation in patient-modeled MVID animals that carry independent mutations in MYO5B. Body weight loss in male MYO5B(G519R) mice was ameliorated by Compound-1. These observations suggest that Compound-1 treatment has a trophic effect on intestine with MYO5B functional loss through epithelial cell-autonomous pathways that may improve the differentiation of progenitor cells and the maturation of enterocytes. Targeting LPAR5 may represent an effective therapeutic approach for treatment of MVID symptoms induced by different point mutations in MYO5B. NEW & NOTEWOTHYThis study demonstrates the importance of MYO5B for cellular lipid metabolism and mitochondria in intestinal epithelial cells, a previously unexplored function of MYO5B. Alterations in cellular metabolism may underlie the progenitor cell malfunction observed in microvillus inclusion disease (MVID). To examine the therapeutic potential of progenitor-targeted treatments, the effects of LPAR5-preferred agonist, Compound-1, was investigated utilizing several MVID model mice and enteroids. Our observations suggests that Compound-1 may provide a therapeutic approach for treating MVID. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/610579v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@54b5borg.highwire.dtl.DTLVardef@1966a5aorg.highwire.dtl.DTLVardef@2073f1org.highwire.dtl.DTLVardef@9c12e1_HPS_FORMAT_FIGEXP M_FIG C_FIG