Specific Tetanic Force Research Articles

Sucla2 Knock-Out in Skeletal Muscle Yields Mouse Model of Mitochondrial Myopathy With Muscle Type-Specific Phenotypes.

Pathogenic variants in subunits of succinyl-CoA synthetase (SCS) are associated with mitochondrial encephalomyopathy in humans. SCS catalyses the conversion of succinyl-CoA to succinate coupled with substrate-level phosphorylation of either ADP or GDP in the TCA cycle. This report presents a muscle-specific conditional knock-out (KO) mouse model of Sucla2, the ADP-specific beta subunit of SCS, generating a novel invivo model of mitochondrial myopathy. The mouse model was generated using the Cre-Lox system, with the human skeletal actin (HSA) promoter driving Cre-recombination of a CRISPR-Cas9-generated Sucla2 floxed allele within skeletal muscle. Inactivation of Sucla2 was validated using RT-qPCR and western blot, and both enzyme activity and serum metabolites were quantified by mass spectrometry. To characterize the model invivo, whole-body phenotyping was conducted, with mice undergoing a panel of strength and locomotor behavioural assays. Additionally, exvivo contractility experiments were performed on the soleus (SOL) and extensor digitorum longus (EDL) muscles. SOL and EDL cryosections were also subject to imaging analyses to assess muscle fibre-specific phenotypes. Molecular validation confirmed 68% reduction of Sucla2 transcript within the mutant skeletal muscle (p < 0.001) and 95% functionally reduced SUCLA2 protein (p < 0.0001). By 3 weeks of age, Sucla2 KO mice were 44% the size of controls by body weight (p < 0.0001). Mutant mice also exhibited 34%-40% reduced grip strength (p < 0.01) and reduced spontaneous exercise, spending about 88% less cumulative time on a running wheel (p < 0.0001). Contractile function was also perturbed in a muscle-specific manner; although no genotype-specific deficiencies were seen in EDL function, SUCLA2-deficient SOL muscles generated 40% less specific tetanic force (p < 0.0001), alongside slower contraction and relaxation rates (p < 0.001). Similarly, a SOL-specific threefold increase in mitochondria (p < 0.0001) was observed, with qualitatively increased staining for both COX and SDH, and the proportion of Type 1 myosin heavy chain expressing fibres within the SOL was nearly doubled (95% increase, p < 0.0001) in the Sucla2 KO mice compared with that in controls. SUCLA2 loss within murine skeletal muscle yields a model of SCS-deficient mitochondrial myopathy with reduced body weight, muscle weakness and exercise intolerance. Physiological and morphological analyses of hindlimb muscles showed remarkable differences in exvivo function and cellular consequences between the EDL and SOL muscles, with SOL muscles significantly more impacted by Sucla2 inactivation. This novel model will provide an invaluable tool for investigations of muscle-specific and fibre type-specific pathogenic mechanisms to better understand SCS-deficient myopathy.

Isoflurane conditioning improves functional outcomes after peripheral nerve injury in a sciatic cut repair murine model.

Anesthetic conditioning has been shown to provide neuroprotection in several neurological disorders. Whether anesthetic conditioning provides protection against peripheral nerve injuries remains unknown. The aim of our current study is to investigate the impact of isoflurane conditioning on the functional outcomes after peripheral nerve injury (PNI) in a rodent sciatic nerve injury model. Adult male Lewis rats underwent sciatic nerve cut and repair and exposed to none (Group 1, sham), single isoflurane exposure (Group 2), three-time isoflurane exposure (Group 3), and six-time isoflurane exposure (Group 4). Isoflurane conditioning was established by administration of 2% isoflurane for 1 hour, beginning 1-hour post sciatic nerve cut and repair. Groups 3 and 4 were exposed to isoflurane for 1 hour, 3 and 6 consecutive days respectively. Functional outcomes assessed included compound muscle action potential (CMAP), evoked muscle force (tetanic and specific tetanic force), wet muscle mass, and axonal counting. We observed an increase in axons, myelin width and a decrease in G-ratio in the isoflurane conditioning groups (3- and 6-days). This correlated with a significant improvement in tetanic and specific tetanic forces, observed in both groups 3 and 4. Isoflurane conditioning (3- and 6-day groups) resulted in improvement in functional outcomes at 12 weeks post peripheral nerve injury and repair in a murine model. Future experiments should be focused on identifying the therapeutic window of isoflurane conditioning and exploring the underlying molecular mechanisms responsible for isoflurane conditioning induced neuroprotection in PNI.

A Phytomolecule Icariin Protects from Sarcopenia Partially by Suppressing Myosin Heavy Chain Degradation in Orchiectomized Rats.

Treatments are lacking for sarcopenia, which is an age-related disease characterized by loss of skeletal muscle mass, strength, and/or physical performance. Icariin is a phytomolecule fromherbal Epimedium, a traditional Chinese medicine widely used to treat musculoskeletal disorders for thousands of years. Here the effects of icariin against sarcopenia are investigated and the underlying mechanism is elucidated. A classic rat model of bilaterally orchiectomized (ORX) is used to induce sarcopenia. After administration for 8 weeks, compared to the control group, the forelimb grip strength, the specific tetanic forces of the soleus (SOL) and extensor digitorum longus muscle (EDL) are higher, and the fiber cross-sectional areas (CSAs) of the gastrocnemius and tibialis anterior muscle are larger in the icariin group. In addition, icariin promotes mRNA and protein expressions of myosin heavy chain (MyHC) both in SOL and EDL. Mechanistically, icariin significantly suppresses the mRNA and protein expressions of FOXO3a, atrogin-1, and MuRF-1, which are related to the degradation of myosin heavy chain. Collectively, icariin protects from sarcopenia in ORX rats characterized by enhancing grip strength and skeletal muscle contraction, as well as increasing skeletal muscle CSA by inhibiting the ubiquitination degradation of the MyHC in skeletal muscle fibers.

Numb is required for optimal contraction of skeletal muscle.

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

Magnesium supplementation alleviates corticosteroid-associated muscle atrophy in rats.

PurposeCorticosteroid (CS) therapy for infectious and rheumatological diseases showed to decrease serum magnesium (Mg++) level and induce muscle atrophy in patients. The present study investigated the effects of Mg++ supplementation on preventing CS-induced muscle atrophy in an animal model, which provided experimental data for potential clinical translation.MethodsTwelve 24-week-old male Sprague–Dawley rats were treated with lipopolysaccharide (LPS) and CS methylprednisolone (MPS) to induce muscle atrophy, with half of the rats also given daily 50 mg/kg Mg++ oral supplementation. Additional six rats without LPS + CS treatments were used as normal controls. After treatment for 6 weeks, serum was collected for Mg++ quantification, animal dual-energy X-ray absorptiometry (DXA) was performed for tissue composition, and the extensor digitorum longus (EDL) was collected for muscle functional test and histology including muscle fiber size, intramuscular fat infiltration and fiber typing. In vitro myotube atrophy model was used to study the in vitro effect associated with in vivo muscle atrophy.ResultsLPS + CS treatments induced hypomagnesemia while the serum Mg++ level was in normal range after Mg++ supplementation. DXA showed 53.0% lower fat percent and 29.7% higher lean mass in LPS + CS + Mg group when compared to LPS + CS group. Muscle functional test showed 22.2% higher specific twitch force and 40.3% higher specific tetanic force in LPS + CS + Mg group when compared to LPS + CS group. Histological analysis showed 4.1% higher proportion of muscle fibers area to total area and 63.6% lower intramuscular fat infiltration in EDL sections in LPS + CS + Mg group when compared to LPS + CS group. LPS + CS + Mg group had 33.0% higher area proportion and 29.4% higher cross-sectional area (CSA) of type IIb muscle fiber. Myoblast culture results showed that Mg++ supplementation group had larger myotube diameter. The mRNA expressions of the muscle atrophy marker genes MuRF1 and MAFbx were lower in Mg++ supplementation group both in vitro and in vivo.ConclusionThe current study demonstrated that Mg++ supplementation successfully alleviated CS-associated muscle atrophy in rats at both functional and morphology levels, indicating a translational potential for patients undergoing CS therapy. This study provided the evidence for the first time that Mg++ supplementation could prevent muscle atrophy—an adverse effect of CS therapy, currently also adopted for treating coronavirus disease 2019 (COVID-19).

Long-term human IgG treatment improves heart and muscle function in a mouse model of Duchenne muscular dystrophy.

BackgroundDuchenne muscular dystrophy (DMD) is a progressive muscle‐wasting disease caused by mutations in the dystrophin gene, which leads to structural instability of the dystrophin–glycoprotein‐complex with subsequent muscle degeneration. In addition, muscle inflammation has been implicated in disease progression and therapeutically addressed with glucocorticosteroids. These have numerous adverse effects. Treatment with human immunoglobulin G (IgG) improved clinical and para‐clinical parameters in the early disease phase in the well‐established mdx mouse model. The aim of the present study was to confirm the efficacy of IgG in a long‐term pre‐clinical study in mdx mice.MethodsIgG (2 g/kg body weight) or NaCl solution as control was administered monthly over 18 months by intraperitoneal injection in mdx mice beginning at 3 weeks of age. Several clinical outcome measures including endurance, muscle strength, and echocardiography were assessed. After 18 months, the animals were sacrificed, blood was collected for analysis, and muscle samples were obtained for ex vivo muscle contraction tests, quantitative PCR, and histology.ResultsIgG significantly improved the daily voluntary running performance (1.9 m more total daily running distance, P < 0.0001) and slowed the decrease in grip strength by 0.1 mN, (P = 0.018). IgG reduced fatigability of the diaphragm (improved ratio to maximum force by 0.09 ± 0.04, P = 0.044), but specific tetanic force remained unchanged in the ex vivo muscle contraction test. Cardiac function was significantly better after IgG, especially fractional area shortening (P = 0.012). These results were accompanied by a reduction in cardiac fibrosis and the infiltration of T cells (P = 0.0002) and macrophages (P = 0.0027). In addition, treatment with IgG resulted in a significant reduction of the infiltration of T cells (P ≤ 0.036) in the diaphragm, gastrocnemius, quadriceps, and a similar trend in tibialis anterior and macrophages (P ≤ 0.045) in gastrocnemius, quadriceps, tibialis anterior, and a similar trend in the diaphragm, as well as a decrease in myopathic changes as reflected by a reduced central nuclear index in the diaphragm, tibialis anterior, and quadriceps (P ≤ 0.002 in all).ConclusionsThe present study underscores the importance of an inflammatory contribution to the disease progression of DMD. The data demonstrate the long‐term efficacy of IgG in the mdx mouse. IgG is well tolerated by humans and could preferentially complement gene therapy in DMD. The data call for a clinical trial with IgG in DMD.

Vibration and β-hydroxy-β-methylbutyrate treatment suppresses intramuscular fat infiltration and adipogenic differentiation in sarcopenic mice.

BackgroundSarcopenia is an aging‐induced deterioration of skeletal muscle mass and function. Low‐magnitude high‐frequency vibration (LMHFV) was shown to improve muscle functions and β‐hydroxy‐β‐methylbutyrate (HMB) to increase muscle mass and strength. Muscle‐derived stem cells (MDSCs) are progenitor cells important for muscle regeneration. We hypothesized that LMHFV and HMB could retard sarcopenia by reducing fat infiltration through inhibiting adipogenesis in MDSCs.MethodsSenescence‐accelerated mouse P8 male mice were randomized into control (CTL), HMB, LMHFV (VIB), and combined (COM) groups. Interventions started at age of month 7 and assessed at 1, 2, and 3 months post‐intervention by densitometry, histology, and functional tests. In vitro, MDSCs isolated from gastrocnemius of senescence‐accelerated mouse P8 mice were characterized, randomized into CTL, VIB, HMB, and COM groups, and assessed by oil red O staining, mRNA, and protein expression.ResultsAt 2 months post‐intervention, percentage lean mass of HMB, VIB, and COM groups were significantly higher than CTL group. Twitch, tetanic, and specific tetanic forces of COM group were higher, while specific twitch force of both VIB and COM groups were higher. Grip strength of HMB, VIB, and COM groups were higher. Histologically, both VIB and COM groups presented lower oil red O area than CTL group. Type I muscle fibre in CTL group was higher than HMB, VIB, and COM groups. MDSC were detected in situ by immunofluorescence stain with stem cell antigen‐1 signals confirmed with higher β‐catenin expression in the COM group. The observations were also confirmed in vitro, MDSCs in the HMB, VIB, and COM groups presented lower adipogenesis vs. the CTL group. β‐Catenin mRNA and protein expressions were lower in the CTL group while their relationship was further validated through β‐catenin knock‐down approach.ConclusionsOur results showed that combined LMHFV and HMB interventions enhanced muscle strength and decreased percentage fat mass and intramuscular fat infiltration as compared with either treatment alone. Additive effect of LMHFV and HMB was demonstrated in β‐catenin expression than either treatment in MDSCs and altered cell fate from adipogenesis to myogenesis, leading to inhibition of intramuscular lipid accumulation. Wnt/β‐catenin signalling pathway was found to be the predominant regulatory mechanism through which LMHFV and HMB combined treatment suppressed MDSCs adipogenesis.

Skeletal Muscle Constructs Engineered from Human Embryonic Stem Cell Derived Myogenic Progenitors Exhibit Enhanced Contractile Forces When Differentiated in a Medium Containing EGM-2 Supplements.

Three-dimensional (3D) skeletal muscle constructs engineered from myogenic progenitors derived from human pluripotent stem cells (hPSCs) have a wide range of applications, but to date, such constructs generate lower specific tetanic force than adult human muscles. Methods enhancing functional muscle differentiation and force generation of these constructs are highly desirable. The finding of this study is that addition of the supplements in the endothelial cell growth medium-2 (EGM-2) to the myogenic differentiation medium can substantially enhance contractile force generation. For constructs differentiated for 4 weeks, addition of the EGM-2 supplements in the first 2 weeks leads to tenfold and sevenfold increases in twitch and tetanic forces, respectively. The specific tetanic force generated by these constructs is 33 mN mm-2 , which is significantly higher than previously reported. These constructs show wider myotubes and higher gene expression levels for all skeletal muscle-specific myosin heavy chain isoforms, suggesting that a more mature differentiation stage of the cells underlies the greater contractile force generation. The constructs exposed to these supplements for 4 weeks do not generate as high contractile forces, suggesting that prolonged treatment is not beneficial. These results suggest that temporal conditioning with the EGM-2 supplements assists functional development of hPSC-derived skeletal muscle constructs.

Utrophin haploinsufficiency does not worsen the functional performance, resistance to eccentric contractions and force production of dystrophic mice.

The lack of dystrophin in Duchenne muscular dystrophy (DMD) compromises the integrity and function of muscle fibers. Skeletal muscles, except the diaphragm, do not undergo progressive degeneration in adult mdx mice due to compensatory mechanisms, including structural protein upregulation. New mouse models, including utrophin haploinsufficient mdx (mdx/utrn+/-) mice, may better recapitulate DMD. Our goal was to determine whether mdx/utrn+/- worsens the mdx phenotype and to characterize the course of the disease on muscle function and contractility at 1, 2, and 5 months of age, which encompass all stages of development relevant to DMD therapy. The functional performances of mdx/utrn+/- mice showed that they are not more affected than mdx/utrn+/+ mice based on downhill treadmill running parameters and subsequent recovery measured by open-field voluntary activity. WT mice ran the entire distance (450 m) on the treadmill, with an additional 561 m during the 4 h of open-field while mdx/utrn+/+ and mdx/utrn+/- mice completed, respectively, 236 m and 273 m on the treadmill and 341 m and 287 m during the open-field period. In addition, isolated ex vivo contractile properties and repeated eccentric contractions showed that mdx/utrn+/- does not significantly worsen the function of dystrophic EDL muscles, which are mainly composed of fast-twitch fibers that are preferentially affected in DMD. Twitch, absolute tetanic, and specific tetanic forces were very similar in dystrophic EDL muscles from mdx/utrn+/+ and mdx utrn+/- mice at 1, 2, and 5 months of age. Five-month-old mdx/utrn+/+ and mdx/utrn+/- mice lost roughly 50% of their force due to repeated eccentric contractions. Thus, histological, morphological, biochemical functional and contractile observations showed that utrophin haploinsufficiency has a very limited impact on mdx mice.

BGP-15 improves contractile function of regenerating soleus muscle.

This study investigated the effect of the heat shock protein inducer O-[3-piperidino-2-hydroxy-1-propyl]-nicotinic amidoxime (BGP-15) on the morphology and contractile function of regenerating soleus muscles from mice. Cryolesioned soleus muscles from young mice treated daily with BGP-15 (15mg/Kg) were evaluated on post-cryolesion day 10. At this time point, there was a significant decrease in the cross-sectional area of regenerating myofibers, maximal force, specific tetanic force, and fatigue resistance of regenerating soleus muscles. BGP-15 did not reverse the decrease in myofiber cross-sectional area but effectively prevented the reduction in tetanic force and fatigue resistance of regenerating muscles. In addition, BGP-15 treatment increased the expression of embryonic myosin heavy chain (e-MyHC), MyHC-II and MyHC-I in regenerating muscles. Although BGP-15 did not alter voltage dependent anion-selective channel 2 (VDAC2) expression in cryolesioned muscles, it was able to increase inducible 70-kDaheat shock protein(HSP70) expression. Our results suggest that BGP-15 improves strength recovery in regenerating soleus muscles by accelerating the re-expression of adult MyHC-II and MyHC-I isoforms and HSP70 induction. The beneficial effects of BGP-15 on the contractile function of regenerating muscles reinforce the potential of this molecule to be used as a therapeutic agent.

Skeletal muscle function during the progression of cancer cachexia in the male ApcMin/+ mouse.

While cancer-induced skeletal muscle wasting has been widely investigated, the drivers of cancer-induced muscle functional decrements are only beginning to be understood. Decreased muscle function impacts cancer patient quality of life and health status, and several potential therapeutics have failed in clinical trials due to a lack of functional improvement. Furthermore, systemic inflammation and intrinsic inflammatory signaling's role in the cachectic disruption of muscle function requires further investigation. We examined skeletal muscle functional properties during cancer cachexia and determined their relationship to systemic and intrinsic cachexia indices. Male ApcMin/+ (MIN) mice were stratified by percent body weight loss into weight stable (WS; <5% loss) or cachectic (CX; >5% loss). Age-matched C57BL/6 littermates served as controls. Tibialis anterior (TA) twitch properties, tetanic force, and fatigability were examined in situ. TA protein and mRNA expression were examined in the nonstimulated leg. CX decreased muscle mass, tetanic force (Po), and specific tetanic force (sPo). Whole body and muscle fatigability were increased in WS and CX. CX had slower contraction rates, +dP/d t and -dP/d t, which were inversely associated with muscle signal transducer and activator of transcription 3 ( STAT3) and p65 activation. STAT3 and p65 activation were also inversely associated with Po. However, STAT3 was not related to sPo or fatigue. Muscle suppressor of cytokine signaling 3 mRNA expression was negatively associated with TA weight, Po, and sPo but not fatigue. Our study demonstrates that multiple functional deficits that occur with cancer cachexia are associated with increased muscle inflammatory signaling. Notably, muscle fatigability is increased in the MIN mouse before cachexia development. NEW & NOTEWORTHY Recent studies have identified decrements in skeletal muscle function during cachexia. We have extended these studies by directly relating decrements in muscle function to established cachexia indices. Our results demonstrate that a slow-fatigable contractile phenotype is developed during the progression of cachexia that coincides with increased muscle inflammatory signaling. Furthermore, regression analysis identified predictors of cancer-induced muscle dysfunction. Last, we report the novel finding that whole body and muscle fatigability were increased before cachexia development.

Muscle Growth by Activin Type II Receptor Blocking Ameliorates Weakness in GNE Myopathy Mice

BackgroundGNE myopathy is an autosomal recessive muscle disease clinically characterized by skeletal muscle atrophy and weakness. We have established that a key factor in the mechanism of disease is sialic acid deficiency, a target for recent and ongoing clinical trials. In GNE myopathy patients with existing severe muscle atrophy and degeneration, however, exploring additional therapeutic options may prove beneficial.ObjectiveIn this study, we demonstrate the efficacy of myostatin pathway blockade, which promotes muscle growth, on GNE myopathy model mice.MethodsCDD866, a murinized anti‐activin type II receptor antibody, were administered to GNE myopathy model mice once weekly with subcutaneous injections for 8 weeks. Physiological examination and pathological observation on the skeletal muscles of treated mice were performed.ResultsCDD866, not only promoted body weight gain and increase in muscle size, but also led to the recovery of specific tetanic force, which is a functional indicator of muscle contractile devices. In addition, we demonstrate that increased S‐nitrosylation of contractile and metabolic proteins leads to muscle weakness in GNE myopathy mice. More importantly, CDD866 treatment in mice reversed S‐nitrosylation of the muscle proteins along with the recovery of muscle function.DiscussionThis results suggest that protein synthesis via myostatin pathway blockade has a beneficial effect by providing newly‐synthesized fresh muscle proteins to chronic myopathic muscles in addition to muscle size increment. Our results also suggest that the myostatin pathway blockade may be a promising therapy on GNE myopathy in addition to sialic acid supplementation.Support or Funding InformationIntramural Research Grant (28‐6) for Neurological and Psychiatric Disorders of NCNP

Superoxide dismutase/catalase mimetic EUK-134 prevents diaphragm muscle weakness in monocrotalin-induced pulmonary hypertension.

Patients with pulmonary hypertension (PH) suffer from inspiratory insufficiency, which has been associated with intrinsic contractile dysfunction in diaphragm muscle. Here, we examined the role of redox stress in PH-induced diaphragm weakness by using the novel antioxidant, EUK-134. Male Wistar rats were randomly divided into control (CNT), CNT + EUK-134 (CNT + EUK), monocrotaline-induced PH (PH), and PH + EUK groups. PH was induced by a single intraperitoneal injection of monocrotaline (60 mg/kg body weight). EUK-134 (3 mg/kg body weight/day), a cell permeable mimetic of superoxide dismutase (SOD) and catalase, was daily intraperitoneally administered starting one day after induction of PH. After four weeks, diaphragm muscles were excised for mechanical and biochemical analyses. There was a decrease in specific tetanic force in diaphragm bundles from the PH group, which was accompanied by increases in: protein expression of NADPH oxidase 2/gp91phox, SOD2, and catalase; 3-nitrotyrosine content and aggregation of actin; glutathione oxidation. Treatment with EUK-134 prevented the force decrease and the actin modifications in PH diaphragm bundles. These data show that redox stress plays a pivotal role in PH-induced diaphragm weakness. Thus, antioxidant treatment can be a promising strategy for PH patients with inspiratory failure.

G.O.17 - The physiological consequences of different levels of dystrophin following antisense based exon-skipping in the mdx mouse

We examined the effects on muscle physiology of restoring different levels of dystrophin in mdx mice with established dystrophic pathophysiology (12 weeks and older). Dystrophin expression was induced very efficiently using cell penetrating peptides linked to an antisense sequencing targeting exon 23 which contains a premature stop mutation. We assessed muscle physiology in the tibialis anterior (TA) muscle of the mouse using a terminally anaesthetised in situ protocol. To assess muscle physiology in the diaphragm we used strips of diaphragm in an in-vitro system. In both cases we examined the force–frequency relationship and established maximum specific tetanic force. We then subjected the muscles to a 10% stretch while stimulating them to contract. This eccentric exercise was highly damaging to dystrophic muscle. We present data showing that 15% of normal levels of dystrophin were sufficient to prevent eccentric exercise induced damage following a single dose of Pip6a-PMO. Chronic intravenous (IV) administration had a cumulative effect and we show that restoration of 50% of normal levels of dystrophin produced a 40% improvement in maximum specific force. Intraperitoneal administration of a single dose of B-PMO produced an 88% increase in maximum specific force as well as protecting against eccentric exercise induced damage in the diaphragm. Similar results were obtained in the diaphragm with chronic IV delivery of Pip6a-PMO at the same dose as the studies in the TA, even when treating older mice with extensive fibrosis in the diaphragm. While caution must be applied when extrapolating these results to DMD patients, the results suggest that moderate levels of dystrophin may be sufficient to slow-down or possibly prevent disease progression whereas higher levels of dystrophin will also improve muscle force production.

Effects of exercise on soleus in severe burn and muscle disuse atrophy

BackgroundMuscle loss is a sequela of severe burn and critical illness with bed rest contributing significantly to atrophy. We hypothesize that exercise will mitigate muscle loss after burn with bed rest. Materials and methodsMale rats were assigned to sham ambulatory (S/A), burn ambulatory (B/A), sham hindlimb unloading (S/H), or burn hindlimb unloading (B/H). Rats received a 40% scald burn or sham and were ambulatory or placed in hindlimb unloading, a model of bed rest. Half from each group performed twice daily resistance climbing. Hindlimb isometric forces were measured on day 14. ResultsSoleus mass and muscle function were not affected by burn alone. Mass was significantly lower in hindlimb unloading (79 versus 139 mg, P < 0.001) and no exercise (103 versus 115 mg, P < 0.01). Exercise significantly increased soleus mass in B/H (86 versus 77 mg, P < 0.01). Hindlimb unloading significantly decreased muscle force in the twitch (12 versus 31 g, P < 0.001), tetanic (55 versus 148 g, P < 0.001), and specific tetanic measurements (12 versus 22 N/cm2, P < 0.001). Effects of exercise on force depended on other factors. In B/H, exercise significantly increased twitch (14 versus 8 g, P < 0.05) and specific tetanic force (14 versus 7 N/cm2, P < 0.01). Fatigue index was lower in ambulatory (55%) and exercise (52%) versus hindlimb (69%, P = 0.03) and no exercise (73%, P = 0.002). ConclusionsHindlimb unloading is a significant factor in muscle atrophy. Exercise increased the soleus muscle mass, twitch, and specific force in this model. However, the fatigue index decreased with exercise in all groups. This suggests exercise contributes to functional muscle change in this model of disuse and critical illness.

Deletion of connexin43 in osteoblasts/osteocytes leads to impaired muscle formation in mice.

It is well-established that muscle forces are necessary for bone development as well as proper bone modeling and remodeling. Recent work has also suggested that bone acts as an endocrine organ that can influence the development of other organs. Connexin43 (Cx43), a gap junction protein that transduces mechanical signals, is an important determinant of cortical bone modeling. Using an osteoblast/osteocyte-specific ablation of the Cx43 gene (Gja1) driven by the 2.3-kb Col1 α1 promoter (cKO) in the mouse, in this study we confirmed reduced cortical bone thickness and density with expanded bone marrow cavity in the cKO humerus. Surprisingly, Gja1 deletion in bone cells also affected skeletal muscle development, resulting in lower fast muscle weight, grip strength, and maximum absolute and specific tetanic forces (60% to 80%, 85%, and 50%, respectively, of WT mice). The normally fast twitch extensor digitorum longus (EDL) muscle exhibited increased slow twitch fibers in cKO mice. These muscle defects were accompanied by a 40% to 60% reduction in mRNA abundance for genes encoding osteocalcin in the humerus, relative to WT mice. Accordingly, both carboxylated and undercarboxylated isoforms of osteocalcin were reduced by over 30% in the circulation of cKO mice. Moreover, the active, undercarboxylated isoform of osteocalcin (glu-OC) promoted myotube formation in C2C12 myoblast cultures, and glu-OC injections to cKO mice rescued EDL muscle cross-sectional area and grip strength in vivo. These findings demonstrate that Cx43 in osteoblasts/osteocytes indirectly modulates skeletal muscle growth and function, potentially via an endocrine effect of glu-OC.

G.P.85: Truncated dystrophin with exon 45–55 deletion induced muscle atrophy and fiber type change through the hyper-nitrosylation of the ryanodine receptor type-1 and constant release of Ca2+to the cytosol

Duchenne muscular dystrophy (DMD), the most common form of muscular dystrophy, is caused by lack of dystrophin. Exon skipping has been developed as a possible treatment of DMD and exon 45–55 skipping strategy could be one of goals of this therapy, partly because patients with exon 45–55 in-frame deletion have shown very mild skeletal muscle symptoms. Moreover, this strategy could theoretically rescue up to 63% of DMD deletion patients, however function of exon 45–55 deleted dystrophin is not well understood. To clarify the functional role of truncated dystrophin, we generated the transgenic (Tg) mice which expressed the exon 45–55 lacking human dystrophin and two Tg lines were established. We, then, mated Tg male mice with <i>mdx</i> female mice to get Tg/<i>mdx</i> male mice and extensively analyzed the phenotypes. These Tg/<i>mdx</i> mice expressed only truncated dystrophin, accompanied by recovery of the dystrophin–glycoprotein complex at the sarcolemma. The Tg/<i>mdx</i> also showed improvement of the function, including serum creatine kinase level, muscle pathology and specific tetanic force, to a level of the wild type. These results suggest that truncated dystrophin with exon 45–55 deletion has a equivalent function to the full length dystrophin. On the other hand, atrophy of type2B fibers and increasing of type2A fibers were observed at 20weeks of Tg/<i>mdx</i> mice. Moreover, the localization of neuronal NOS (nNOS) in Tg/<i>mdx</i> was changed from sarcolemma to cytosol and nNOS itself was activated. This activation induced hyper-nitrosylation of the ryanodine receptor type-1 and Ca²⁺ was constantly released from sarcoplasmic reticulum to cytosol. This Ca²⁺ release might be related to the findings, up-reguletion of Atrogin-1 and MuRF1, which could induce muscle atrophy and PGC1<i>α</i>, which could alter the muscle fiber types. This Tg/<i>mdx</i> mouse could be also a very useful model for analyzing pathogenesis of BMD phenotypes.

Mechanical ventilation impairs sarcomeric protein function in rat diaphragm single fibers

Mechanical ventilation (MV) is a widely used clinical intervention for patients unable to maintain alveolar ventilation. Although a life‐saving measure for patients suffering from respiratory failure, MV promotes diaphragm atrophy and contractile dysfunction. The cellular basis of contractile dysfunction remains unclear. The mechanisms of contractile dysfunction are important to understand because MV‐induced diaphragm weakness can delay the weaning process. We studied triton‐permeabilized diaphragm single fibers from rats after 12 hrs of spontaneous breathing (SB) and MV (n = 4/group). Key parameters of isometric contractile properties were maximal specific tetanic force (sPo), rate constant of tension redevelopment (ktr), and calcium sensitivity (pCa50). In type I diaphragm fibers, sPo did not differ between groups. MV slowed ktr by ~6% and decreased the pCa50 such that calcium concentration eliciting 50% sPo increased by ~20%. In type II diaphragm fibers, MV diminished sPo by ~32% and slowed ktr by ~22%. However, pCa50 did not differ between groups. Together, our data suggest that MV impairs diaphragm sarcomeric protein function as evidenced by diminished specific force and calcium sensitivity, and slowing of cross bridge cycling kinetics in membrane‐permeabilized single fibers.Funding support: NIH R00‐HL098453 (LFF) and HL087839 (SKP)

Neuromuscular Electrical Stimulation as a Method to Maximize the Beneficial Effects of Muscle Stem Cells Transplanted into Dystrophic Skeletal Muscle

Cellular therapy is a potential approach to improve the regenerative capacity of damaged or diseased skeletal muscle. However, its clinical use has often been limited by impaired donor cell survival, proliferation and differentiation following transplantation. Additionally, functional improvements after transplantation are all-too-often negligible. Because the host microenvironment plays an important role in the fate of transplanted cells, methods to modulate the microenvironment and guide donor cell behavior are warranted. The purpose of this study was to investigate whether the use of neuromuscular electrical stimulation (NMES) for 1 or 4 weeks following muscle-derived stem cell (MDSC) transplantation into dystrophic skeletal muscle can modulate the fate of donor cells and enhance their contribution to muscle regeneration and functional improvements. Animals submitted to 4 weeks of NMES after transplantation demonstrated a 2-fold increase in the number of dystrophin+ myofibers as compared to control transplanted muscles. These findings were concomitant with an increased vascularity in the MDSC+NMES group when compared to non-stimulated counterparts. Additionally, animals subjected to NMES (with or without MDSC transplantation) presented an increased maximal specific tetanic force when compared to controls. Although cell transplantation and/or the use of NMES resulted in no changes in fatigue resistance, the combination of both MDSC transplantation and NMES resulted in a faster recovery from fatigue, when compared to non-injected and non-stimulated counterparts. We conclude that NMES is a viable method to improve MDSC engraftment, enhance dystrophic muscle strength, and, in combination with MDSC transplantation, improve recovery from fatigue. These findings suggest that NMES may be a clinically-relevant adjunct approach for cell transplantation into skeletal muscle.

Gender differences in contractile and passive properties of mdx extensor digitorum longus muscle

Duchenne muscular dystrophy (DMD) is a severe, muscle-wasting disease caused by mutations in the dystrophin gene. The mdx mouse is the first and perhaps the most commonly used animal model for study of DMD. Both male and female mdx mice are used. However, it is not completely clear whether gender influences contraction and the passive mechanical properties of mdx skeletal muscle. We compared isometric tetanic forces and passive forces of the extensor digitorum longus muscle between male and female mdx mice. At age 6 months, female mdx mice showed better-preserved specific tetanic force. Interestingly, at 20 months of age, female mdx muscle appeared stiffer. Our results suggest that gender may profoundly influence physiological measurement outcomes in mdx mice.