Muscle Function In Mice Research Articles

The gut microbiota from maintenance hemodialysis patients with sarcopenia influences muscle function in mice.

Sarcopenia is a common complication in patients undergoing maintenance hemodialysis (MHD). Growing evidence suggests a close relationship between the gut microbiota and skeletal muscle. However, research on gut microbiota in patients with sarcopenia undergoing MHD (MS) remains scarce. To bridge this knowledge gap, we aimed to evaluate the pathogenic influence of gut microbiota in the skeletal muscle of patients with MS, to clarify the causal association between gut microbiota and skeletal muscle symptoms in patients with MS and identify the potential mechanisms underlying this causal association. Fecal samples were collected from 10 patients with MS and 10 patients without MS (MNS). Bacteria were extracted from these samples for transplantation. Mice (n=42) were randomly divided into three groups and, after antibiotic treatment, fecal microbiota transplantation (FMT) was performed once a day for 3 weeks. Skeletal muscle and fecal samples from the mice were collected for 16S rRNA gene sequencing and for histological, real-time PCR, and metabolomic analyses. Mice colonized with gut microbiota from MS patients exhibited notable decreases in muscle function and muscle mass, compared with FMT from patients with MNS. Moreover, 16S rRNA sequencing revealed that the colonization of MS gut microbiota reduced the abundance of Akkermansia in the mouse intestines. Metabolome analysis revealed that seven metabolic pathways were notably disrupted in mice transplanted with MS microbiota. This study established a connection between skeletal muscle and the gut microbiota of patients with MS, implying that disruption of the gut microbiota may be a driving factor in the development of skeletal muscle disorders in patients undergoing MHD. This finding lays the foundation for understanding the pathogenesis and potential treatment methods for sarcopenia in patients undergoing MHD.

Genetic deletion of Kvβ2 (AKR6) causes loss of muscle function and increased inflammation in mice.

The voltage-gated potassium channels (Kv) are complex ion channels with distinct roles in neurotransmission, electrical conductivity of the heart, and smooth and striated muscle functions. Previously, we demonstrated that deletion of Kvβ2 in mice results in decreased Pax7 protein levels, hindlimb muscles and body weights, and fiber type switching. In the present study, we tested the hypothesis that Kvβ2 regulates skeletal muscle function in mice. The young and old Kvβ2 knockout (KO) and wildtype (WT) mice were utilized to test the aging phenotype and skeletal muscle function. Consistent with our previous finding, we found a significant reduction in hindlimb skeletal muscles mass and body weight in young Kvβ2 KO mice, which was also significantly reduced in old Kvβ2 KO mice compared with age-matched WT mice. Forelimb grip strength, and the hindleg extensor digitorum longus (EDL) muscles force-frequency relations were significantly decreased in young and old Kvβ2 KO mice compared to age-matched WT mice. Analysis of transmission electron microscopy images of EDL muscles in young mice revealed a significant reduction in the sarcomere length for Kvβ2 KO vs. WT. Hematoxylin and eosin-stained tibialis anterior muscles cryosections displayed a significant decrease in the number of medium (2,000-4,000µm2) and largest (>4,000µm2) myofibers area in young Kvβ2 KO vs. WT mice. We also found a significant increase in fibrotic tissue area in young Kvβ2 KO mice compared with age-matched WT mice. Analysis of RNA Seq data of the gastrocnemius muscles (GAS) identified significant increase in genes involved in skeletal muscle development, proliferation and cell fate determination, atrophy, energy metabolism, muscle plasticity, inflammation, and a decrease in circadian core clock genes in young Kvβ2 KO vs. WT mice. Several genes were significantly upregulated (384 genes) and downregulated (40 genes) in young Kvβ2 KO mice compared to age-matched WT mice. Further, RT-qPCR analysis of the GAS muscles displayed a significant increase in pro-inflammatory marker Il6 expression in young Kvβ2 KO mice compared to age-matched WT mice. Overall, the present study shows that deletion of Kvβ2 leads to decreased muscles strength and increased inflammation.

AAV-vectored base editor trans-splicing delivers dystrophin repair

AAV-vectored base editor trans-splicing delivers dystrophin repair

Pharmacological TRPC6 inhibition improves survival and muscle function in mice with Duchenne muscular dystrophy.

Gene mutations causing loss of dystrophin result in the severe muscle disease known as Duchenne muscular dystrophy (DMD). Despite efforts at genetic repair, DMD therapy remains largely palliative. Loss of dystrophin destabilizes the sarcolemmal membrane, inducing mechanosensitive cation channels to increase calcium entry and promote cell damage and, eventually, muscle dysfunction. One putative channel is transient receptor potential canonical 6 (TRPC6); we have shown that TRPC6 contributed to abnormal force and calcium stress-responses in cardiomyocytes from mice lacking dystrophin that were haplodeficient for utrophin (mdx/utrn+/- [HET] mice). Here, we show in both the HET mouse and the far more severe homozygous mdx/utrn-/- mouse that TRPC6 gene deletion or its selective pharmacologic inhibition (by BI 749327) prolonged survival 2- to 3-fold, improving skeletal and cardiac muscle and bone defects. Gene pathways reduced by BI 749327 treatment most prominently regulated fat metabolism and TGF-β1 signaling. These results support the testing of TRPC6 inhibitors in human trials for other diseases as a novel DMD therapy.

AAV-mediated expression of PFKFB3 in myofibers, but not endothelial cells, improves ischemic muscle function in mice with critical limb ischemia.

6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) is a powerful driver of angiogenesis through its modulation of glycolytic metabolism within endothelial cells. Recent work has demonstrated that PFKFB3 modulates the response to muscle ischemia, however the cell specificity of these effects is not fully understood. In this study, we tested the impact of viral mediated expression of PFKFB3, driven by gene promoters specific for myofibers or endothelial cells, on ischemic hindlimb revascularization and muscle function. We hypothesized that both endothelium- and muscle-specific expression of PFKFB3 would attenuate limb pathology following femoral artery ligation. Male and female BALB/cJ mice were injected with adeno-associated virus encoding the either a green fluorescent protein (GFP) or PFKFB3 driven by either the human skeletal actin (ACTA1) or cadherin-5 (Cdh5) promoters. Four weeks after AAV treatment, mice were subjected to unilateral femoral artery ligation and limb perfusion and muscle function were assessed. Both endothelium- and muscle-specific PFKFB3 expression resulted in significantly more perfused capillaries within the ischemic limb muscle, but neither changed myofiber size/area. Muscle-specific, but not endothelium-specific, PFKFB3 expression significantly improved maximal force production in ischemic muscle (P = 0.0005). Notably, there was a significant effect of sex on maximal force levels in both cohorts of mice (P = 0.0075 and P = 0.0481), indicating that female mice had higher ischemic muscle strength compared with male mice, regardless of treatment group. Taken together, these data demonstrate that although both muscle- and endothelium-specific expression of PFKFB3 enhanced ischemic revascularization, only muscle-specific PFKFB3 expression improved muscle function.NEW & NOTEWORTHY Critical limb ischemia (CLI) carries a significant risk for limb amputation, and treatment options remain limited. We tested the impact of expression of PFKFB3 in myofibers or endothelial cells on limb pathology in mice with CLI. Although both muscle and endothelium-specific PFKFB3 expression increased perfused capillary density, only muscle-specific PFKFB3 expression improve contractile function. Regardless of treatment, female mice demonstrated better recovery from limb ischemic compared with male mice.

Effects of Hydroxyurea on Skeletal Muscle Energetics and Function in a Mildly Anemic Mouse Model.

Hydroxyurea (HU) is a ribonucleotide reductase inhibitor most commonly used as a therapeutic agent in sickle cell disease (SCD) with the aim of reducing the risk of vaso-occlusion and improving oxygen transport to tissues. Previous studies suggest that HU may be even beneficial in mild anemia. However, the corresponding effects on skeletal muscle energetics and function have never been reported in such a mild anemia model. Seventeen mildly anemic HbAA Townes mice were subjected to a standardized rest-stimulation (transcutaneous stimulation)-protocol while muscle energetics using 31Phosphorus magnetic resonance spectroscopy and muscle force production were assessed and recorded. Eight mice were supplemented with hydroxyurea (HU) for 6 weeks while 9 were not (CON). HU mice displayed a higher specific total force production compared to the CON, with 501.35 ± 54.12 N/mm3 and 437.43 ± 57.10 N/mm3 respectively (+14.6%, p < 0.05). Neither the total rate of energy consumption nor the oxidative metabolic rate were significantly different between groups. The present results illustrated a positive effect of a HU chronic supplementation on skeletal muscle function in mice with mild anemia.

Exercise attenuates juvenile irradiation-induced skeletal muscle decline by improving calcium handling and decreasing mitochondrial stress

Proper skeletal muscle development, maintenance, and function is necessary for movement. Decline in muscle function with age and disease is directly associated with a diminished quality of life. Radiation therapy is commonly used to treat certain forms of childhood cancer based on the cytotoxic effects of radiation on cancerous tissue. However, the adverse effects elicited by radiation are not always constrained to the diseased tissue and can accelerate muscle wasting and decline, which is particularly detrimental to juvenile cancer survivors. Exercise is effective at limiting muscle decline and improving muscle function in various diseases. Thus, we hypothesized 1 mo of voluntary endurance exercise following juvenile radiation treatment will reduce muscle damage and restore functional deficits that occur following radiation. Here, we show that following juvenile radiation, 1 mo of voluntary wheel running significantly improved muscle function in mice by promoting adaptations in intracellular calcium handling, improving mitochondrial turnover and reducing oxidative stress resulting from radiation-induced mitochondrial damage. These findings help guide caregivers in their approach to childhood cancer survivor recovery and have implications for other diseases where similar mechanisms of calcium handling and mitochondrial function are disrupted.

Cardiosphere-derived cells, with and without a biological scaffold, stimulate myogenesis and recovery of muscle function in mice with volumetric muscle loss

Extremity trauma to military personnel and civilians commonly results in volumetric muscle loss (VML), leaving patients suffering chronic physical disability. Biomaterial-based technologies such as extracellular matrices (ECMs) are currently in clinical testing for soft tissue repair, but, in preclinical models of VML, the efficacy of ECMs is equivocal. In a murine model of VML, we investigated the effects of ECM and/or cardiosphere-derived cell (CDC) therapy; the latter improves skeletal myogenesis and muscle function in mdx mice, so we reasoned that CDCs may exert disease-modifying bioactivity in VML. While ECM alone improves functional recovery, CDCs have no additive or synergistic benefits with ECM transplantation following VML injury. However, CDCs alone are sufficient to promote muscle recovery, leading to sustained increases in muscle function throughout the study period. Notably, CDCs stimulate satellite cell accumulation in the muscle defect area and hasten myogenic progression (as evidenced by qPCR gene expression profiling), leading to global increases in myofiber numbers and anterior muscle compartment volume. Together, these data implicate CDCs as a viable therapeutic candidate to regenerate skeletal muscle injured by VML.

In vivo Measurement of Knee Extensor Muscle Function in Mice.

Skeletal muscle plasticity in response to countless conditions and stimuli mediates concurrent functional adaptation, both negative and positive. In the clinic and the research laboratory, maximal muscular strength is widely measured longitudinally in humans, with knee extensor musculature the most reported functional outcome. Pathology of the knee extensor muscle complex is well documented in aging, orthopedic injury, disease, and disuse; knee extensor strength is closely related to functional capacity and injury risk, underscoring the importance of reliable measurement of knee extensor strength. Repeatable, in vivo assessment of knee extensor strength in pre-clinical rodent studies offers valuable functional endpoints for studies exploring osteoarthritis or knee injury. We report an in vivo and non-invasive protocol to repeatedly measure isometric peak tetanic torque of the knee extensors in mice across time. We demonstrate consistency using this novel method to measure knee extensor strength with repeated assessment in multiple mice producing similar results.

In vivo Measurement of Knee Extensor Muscle Function in Mice

In vivo Measurement of Knee Extensor Muscle Function in Mice

Therapeutic Targeting of PTEN in Duchenne Muscular Dystrophy

Therapeutic Targeting of PTEN in Duchenne Muscular Dystrophy

Stem Cell-Derived Exosomes Ameliorate Doxorubicin-Induced Muscle Toxicity through Counteracting Pyroptosis.

Doxorubicin (Dox)-induced muscle toxicity (DIMT) is a common occurrence in cancer patients; however, the cause of its development and progression is not established. We tested whether inflammation-triggered cell death, “pyroptosis” plays a role in DIMT. We also examined the potential role of exosomes derived from embryonic stem cells (ES-Exos) in attenuating DIMT. C57BL/6J mice (10 ± 2 wks age) underwent the following treatments: Control (saline), Dox, Dox+ES-Exos, and Dox+MEF-Exos (mouse-embryonic fibroblast-derived exosomes, negative control). Our results demonstrated that Dox significantly reduced muscle function in mice, which was associated with a significant increase in NLRP3 inflammasome and initiation marker TLR4 as compared with controls. Pyroptosis activator, ASC, was significantly increased compared to controls with an upregulation of specific markers (caspase-1, IL-1β, and IL-18). Treatment with ES-Exos but not MEF-Exos showed a significant reduction in inflammasome and pyroptosis along with improved muscle function. Additionally, we detected a significant increase in pro-inflammatory cytokines (TNF-α and IL-6) and inflammatory M1 macrophages in Dox-treated animals. Treatment with ES-Exos decreased M1 macrophages and upregulated anti-inflammatory M2 macrophages. Furthermore, ES-Exos showed a significant reduction in muscular atrophy and fibrosis. In conclusion, these results suggest that DIMT is mediated through inflammation and pyroptosis, which is attenuated following treatment with ES-Exos.

PTEN Inhibition Ameliorates Muscle Degeneration and Improves Muscle Function in a Mouse Model of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is caused by a mutation of the muscle membrane protein dystrophin and characterized by severe degeneration of myofibers, progressive muscle wasting, loss of mobility, and, ultimately, cardiorespiratory failure and premature death. Currently there is no cure for DMD. Herein, we report that skeletal muscle-specific knockout (KO) of the phosphatase and tensin homolog (Pten) gene in an animal model of DMD (mdx mice) alleviates myofiber degeneration and restores muscle function without increasing tumor incidence. Specifically, Pten KO normalizes myofiber size and prevents muscular atrophy, and it improves grip strength and exercise performance in mdx mice. Pten KO also reduces fibrosis and inflammation, and it ameliorates muscle pathology in mdx mice. Unbiased RNA sequencing reveals that Pten KO upregulates extracellular matrix and basement membrane components positively correlated with wound healing and suppresses negative regulators of wound healing and lipid biosynthesis, thus improving the integrity of muscle basement membrane at the ultrastructural level. Importantly, pharmacological inhibition of PTEN similarly ameliorates muscle pathology and improves muscle integrity and function in mdx mice. Our findings provide evidence that PTEN inhibition may represent a potential therapeutic strategy to restore muscle function in DMD.

Sialic Acid Alterations in Mouse Models of Atrophy and Hypertrophy

Sialic acids (SAs) are negatively charged nine‐carbon sugars that are typically found at the ends of glycan chains on glycolipids and glycoproteins. As such, they play important structural and signaling roles for the cell. Skeletal muscle seems to be particularly sensitive to SA perturbations; in GNE myopathy, hypomorphic mutations in a key enzyme of SA biosynthesis leads to muscle weakness and wasting, and SA abundance has been shown to correlate with muscle function in mice. Additionally, enzymatic activity of sialidases, which removes SAs, is altered in both atrophy and hypertrophy. However, linkage‐specific SA changes have not yet been described in atrophy and hypertrophy. Here, we have used a panel of lectins to probe alterations in SA abundance in mouse models of hind‐limb suspension atrophy and hind‐limb overload‐induced hypertrophy.

Exercise May Ameliorate the Detrimental Side Effects of High Vitamin D Supplementation on Muscle Function in Mice.

ABSTRACTVitamin D is commonly prescribed to normalize deficiencies and to treat osteoporosis. However, the effect vitamin D supplements have on skeletal muscle health is equivocal. Although vitamin D is known to play a role in the various processes that maintain muscle integrity and function, recent studies utilizing high bolus dose vitamin D supplementation has demonstrated an increased risk of falls. Thus, the aim of this study was to investigate the effects of high vitamin D supplementation on skeletal muscle function with and without exercise enrichment. Four‐week old C57BL/10 mice (n = 48) were separated into either normal vitamin D (1500 IU/kg diet; unsupplemented) or high vitamin D (20,000 IU/kg diet; supplemented) treatment groups. Each dietary group was further separated into interventional subgroups where mice either remained sedentary or received exercise‐enrichment for 8 weeks in the form of voluntary running. Following the intervention period, whole body in vivo and ex vivo contractile analysis were performed. High vitamin D supplementation decreased force production in the slow‐twitch soleus muscles of sedentary mice (p < .01); however, exercise normalized this effect. Eight weeks of exercise did not improve fatigue resistance of the extensor digitorum longus (EDL) or soleus muscles in unsupplemented mice, likely due to low levels of activation in these muscles. In contrast, fatigability was improved in the EDL (p < .01) and even more so in the soleus (p < .001) in the supplemented exercise‐enriched group. Our data highlights that increasing vitamin D levels above normal reduces postural muscle force as seen in the soleus. Thus, unnecessary vitamin D supplementation may contribute to the increased risk of falls observed in some studies. Interestingly, when vitamin D supplementation was combined with exercise, force production was effectively restored, and fatigue resistance improved, even in muscles lowly activated. Regular exercise may modulate the effects of vitamin D on skeletal muscle, and be recommended for individuals receiving vitamin D supplements. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

Zoledronic Acid Improves Muscle Function in Healthy Mice Treated with Chemotherapy.

Carboplatin is a chemotherapy drug used to treat solid tumors but also causes bone loss and muscle atrophy and weakness. Bone loss contributes to muscle weakness through bone-muscle crosstalk, which is prevented with the bisphosphonate zoledronic acid (ZA). We treated mice with carboplatin in the presence or absence of ZA to assess the impact of bone resorption on muscle. Carboplatin caused loss of body weight, muscle mass, and bone mass, and also led to muscle weakness as early as 7 days after treatment. Mice treated with carboplatin and ZA lost body weight and muscle mass but did not lose bone mass. In addition, muscle function in mice treated with ZA was similar to control animals. We also used the anti-TGFβ antibody (1D11) to prevent carboplatin-induced bone loss and showed similar results to ZA-treated mice. We found that atrogin-1 mRNA expression was increased in muscle from mice treated with carboplatin, which explained muscle atrophy. In mice treated with carboplatin for 1 or 3 days, we did not observe any bone or muscle loss, or muscle weakness. In addition, reduced caloric intake in the carboplatin treated mice did not cause loss of bone or muscle mass, or muscle weakness. Our results show that blocking carboplatin-induced bone resorption is sufficient to prevent skeletal muscle weakness and suggests another benefit to bone therapy beyond bone in patients receiving chemotherapy. © 2019 American Society for Bone and Mineral Research.

P.328In vivo comparison of different AAV-microutrophin constructs

Over-expression of the dystrophin-related gene utrophin is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). The strategy is based on the ability of utrophin to functionally replace defective dystrophin. AAV-mediated delivery of utrophin to the mdx/utrn double-knockout mice showed improvements in dystrophic phenotype (Odom et al). The aim of this study was to create the microutrophin-coding AAV vectors for effective gene delivery to muscles, evaluate microutrophin gene expression from prepared constructions and assess the functional response after intravenous (iv) administration of microutrophin under constitutive (CMV) and muscle-specific (SPc5-12) promoters in comparison with microdystrophin gene delivery to adult mdx mice. Using bioinformatic approaches we developed constructs, encoding human microutrophin, and performed codon-optimization for effective expression in muscle tissues. Obtained codon-optimized human microutrophin sequence was cloned in AAV vector under muscle-specific SPc5-12 promoter. Proteins expression was confirmed using IHC and WB analyses. To assess muscle function in mice we used hanging wire test and force deficit measurement after lengthening contraction of hindlimb muscle. Results: Five weeks after iv administration in adult mice three types of microutrophin were detected in skeletal and heart muscles. The maximum hanging time was 3-fold increased after iv administration of human codon-optimized microutrophin and 2,5-fold increased after administration of murine microutrophin. Increase in hanging time of mice injected with microutrophin under SPc512 promoter was not significant. Differences in force deficit after lengthening contraction and creatine kinase level were not detected. Iv administration of microdystrophin under CMV promoter led to more significant increase in hanging time and reduction in CK level in with group injected with microutrophin, but because of lower immune toxicity of microutrophin, delivery of this gene is still promising.

The Effect of Yearly-Dose Vitamin D Supplementation on Muscle Function in Mice.

Supplementation with vitamin D helps to alleviate weakness and fatigue seen with deficiency. However, large bolus doses appear to worsen the risk of falls. Whether this occurs as a direct result of muscle weakness is currently unknown. Thus, the aims of this study were to examine the muscle function following administration of high doses of vitamin D. Given the safety issues associated with bolus doses, experiments were conducted on C57BL6 mice. Mice at eight weeks of age with otherwise normal levels of vitamin D were supplemented for four weeks with a high dose (HIGH; n = 12) of vitamin D (20000 IU/kg food) designed to provide a year’s worth of vitamin D. These mice were compared to another group who received that same yearly dose in a single bolus i.p. injection (YEAR; n = 12). Mice provided with standard mouse chow, which contained 1000 IU/kg food, and injected with the vitamin D vehicle were used as controls (CON; n = 16). Force and fatigue properties of hind limb fast- and slow-twitch muscles were measured. CON animals ingested vitamin D consistent with typical human supplementation. HIGH animals consumed significantly more food than the CON animals, such that they ingested more than a year’s worth of vitamin D in four weeks. Despite this, there were few differences in the muscle function compared with CON. YEAR animals demonstrated lower absolute and relative forces in both muscles compared to the HIGH animals, as well as lower force during fatigue and early recovery. Large bolus doses of vitamin D appear to have detrimental effects on the skeletal muscle function, likely being a contributor to increased risk of falls observed with similar doses in humans. Mice ingesting the same amount over four weeks did not demonstrate the same deleterious effects, suggesting this may be a safe way to provide high vitamin D if required.

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice.

Assessment of skeletal muscle contractile function is an important measurement for both clinical and research purposes. Numerous conditions can negatively affect skeletal muscle. This can result in a loss of muscle mass (atrophy) and/or loss of muscle quality (reduced force per unit of muscle mass), both of which are prevalent in chronic disease, muscle-specific disease, immobilization, and aging (sarcopenia). Skeletal muscle function in animals can be evaluated by a range of different tests. All tests have limitations related to the physiological testing environment, and the selection of a specific test often depends on the nature of the experiments. Here, we describe an in vivo, non-invasive technique involving a helpful and easy assessment of force frequency-curve (FFC) in mice that can be performed on the same animal over time. This permits monitoring of disease progression and/or efficacyof a potential therapeutic treatment.

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice

Non-invasive Assessment of Dorsiflexor Muscle Function in Mice