Skeletal Muscle-specific Knockout Mice Research Articles

Sex-Specific Effect of CARM1 in Skeletal Muscle Adaptations to Exercise.

The purpose of this study was to determine how the intersection of coactivator-associated arginine methyltransferase 1 (CARM1) and biological sex affects skeletal muscle adaptations to chronic physical activity. Twelve-week-old female (F) and male (M) wild-type (WT) and CARM1 skeletal muscle-specific knockout (mKO) mice were randomly assigned to sedentary (SED) or voluntary wheel running (VWR) experimental groups. For 8 wk, the animals in the VWR cohort had volitional access to running wheels. Subsequently, we performed whole-body functional tests, and 48 h later muscles were harvested for molecular analysis. Western blotting, enzyme activity assays, as well as confocal and transmission electron microscopy were used to examine skeletal muscle biology. Our data reveal a sex-dependent reduction in VWR volume caused by muscle-specific ablation of CARM1, as F CARM1 mKO mice performed less chronic, volitional exercise than their WT counterparts. Regardless of VWR output, exercise-induced adaptations in physiological function were similar between experimental groups. A broad panel of protein arginine methyltransferase (PRMT) biology measurements, including markers of arginine methyltransferase expression and activity, were unaffected by VWR, except for CARM1 and PRMT7 protein levels, which decreased and increased with VWR, respectively. Changes in myofiber morphology and mitochondrial protein content showed similar trends among animals. However, a closer examination of transmission electron microscopy images revealed contrasting responses to VWR in CARM1 mKO mice compared with WT littermates, particularly in mitochondrial size and fractional area. The present findings demonstrate that CARM1 mKO reduces daily running volume in F mice, as well as exercise-evoked skeletal muscle mitochondrial plasticity, which indicates that this enzyme plays an essential role in sex-dependent differences in exercise performance and mitochondrial health.

1940-P: Disruption of the Exocyst Trafficking Complex Results in Impaired Glucose Uptake in Skeletal Muscle

Objectives: Skeletal muscle cells are responsible for 80-90% of the insulin-induced glucose uptake in the body. Insulin signaling in skeletal muscle results in the targeted trafficking of glucose transporter type 4 (GLUT4) onto the cell membrane, enabling glucose uptake. Insulin resistant cells show defects in insulin-induced GLUT4 exocytosis. The eight-protein exocyst complex has an essential role in the insulin-induced exocytosis of GLUT4 vesicles in cultured adipocytes but it is not known if the exocyst-mediated molecular mechanism is conserved in other, insulin-responsive tissues, such as the skeletal muscle. We hypothesized that the exocyst complex is essential for the insulin-induced exocytosis of GLUT4-containing vesicles in skeletal muscle as well and that the exocyst is a master regulator of glucose homeostasis in insulin-responsive tissues. Methods/Results: We have generated a tamoxifen-inducible skeletal muscle-specific knockout mouse strain of exocyst central subunit Exoc5 (Exoc5-SMKO) to assess the exocyst’s role in glucose homeostasis in vivo. Exoc5 knockout does not affect grip strength, motor coordination or locomotor activity levels in these animals. Both male and female Exoc5-SMKO mice present with elevated fasting glucose levels, as compared to control littermates. Glucose tolerance testing revealed an impaired glucose clearance in Exoc5-SMKO mice, while insulin tolerance, fasting insulin levels, and A1C levels were similar between knockouts and controls. Conclusion: Our findings suggest that Exoc5 and the exocyst are necessary for insulin-stimulated glucose uptake in skeletal muscle. Ongoing work will further investigate the molecular mechanism of exocyst-mediated GLUT4 trafficking in skeletal muscle. Disclosure B. Fujimoto: None. L.T. Carter: None. A.M. Wong: None. M.W. Pitts: None. R.K. Villiger: None. M. Young: None. B. Fogelgren: None. N. Polgar: None. Funding National Institute of General Medical Sciences (5P20GM113134)

Mitophagy Receptor FUNDC1 Links Mitochondrial Quality Control to Muscle–Adipose Dialogue in Alleviating Dietary Obesity

The quality of mitochondria in skeletal muscle is essential for maintaining metabolic homeostasis during adaptive stress responses. However, the precise control mechanism of muscle mitochondrial quality and its physiological impacts remain unclear. Here, using two independent skeletal muscle-specific knockout mouse lines, we demonstrate that FUNDC1, a mediator of mitophagy, plays an essential role in controlling muscle mitochondrial quality as well as metabolic homeostasis. Mice with FUNDC1 deficiency in skeletal muscle (FUNDC1 mKO) showed decreased muscle fat utilization and endurance capacity (∼40%) compared to WT littermates during exercise. Analysis of biochemical markers of mitophagy revealed a marked decrease in mitochondrial localization of LC3-II and P62 protein levels, and this caused impaired mitochondrial respiration capacity. FUNDC1 loss-of-function studies in primary skeletal myocyte recapitulated defective mitophagy and impaired mitochondrial energetic as observed in FUNDC1 mKO muscle. Surprisingly, mice lacking muscle FUNDC1 were resistant to high fat diet (HFD) induced obesity despite reduced muscle mitochondrial energetics. Moreover, glucose and insulin tolerance tests demonstrated that HFD-fed FUNDC1 mKO mice were more tolerant to glucose challenge and more sensitive to insulin stimulation compared to HFD-fed WT controls. This seemingly paradoxical phenotype was due to enhanced fat burning in in adipose tissue. HFD-fed FUNDC1 mKO mice had markedly elevated expression of key thermogenic genes such as Ucp1 (17-fold), Cidea (13-fold), and Dio2 (9-fold) in white adipose. Mechanistically, FUNDC1 deficiency elicited a retrograde response in muscle that upregulated FGF-21 expression, thereby promoting the thermogenic remodeling of adipose tissue. Thus, these findings reveal a pivotal role of FUNDC1-dependent mitochondrial quality-control in mediating the muscle-adipose dialogue to regulate systemic metabolism. Disclosure T. Fu: None. Z. Xu: None. L. Liu: None. X. Liang: None. D. Zhou: None. Q. Guo: None. L. Xiao: None. Z. Gan: None.

Muscle specific MnSOD deficiency leads to complex II‐specific inactivity of ETC and contractile dysfunction, but increases muscle mass

Multiple pathological conditions exhibit excess mitochondrial ROS and muscle weakness in parallel, including aging, denervation and cancer cachexia. To directly test the role of mitochondrial ROS in skeletal muscle weakness and atrophy we generated a skeletal muscle specific knockout mouse model in which MnSOD (Sod2), the primary superoxide anion scavenger in the mitochondrial matrix, was deleted using a Cre‐Lox approach driven by a constitutively active human skeletal actin promoter (HSA‐Cre). Female wild type and MnSOD deficient (mSod2 KO) mice were studied at 6–8 months of age. In mSod2 KO mice, the observed rate of superoxide generation from isolated skeletal muscle mitochondria was significantly elevated, and hydrogen peroxide generation using permeabilized fibers increased by ~5 fold. ATP generation rate and electron transport chain (ETC) activity were severely reduced in response to Complex II linked substrate, suggesting superoxide‐induced complex II inactivity. Reduced ATP generation rate and ETC activity were consistent with severe exercise intolerance in mSod2 KO mice. To determine the effects of mitochondrial ROS on skeletal muscle weakness, we first tested in situ contractile function using gastrocnemius. Maximum isometric specific force was decreased by ~30 % in mSod2 KO mice with direct stimulation on muscle, but the reduction in force was further diminished (~50%) after stimulation of the sciatic nerve suggesting neuromuscular disruption. Morphological assessment exhibited increases in NMJ size and fragmentation in mSod2 KO mice supporting that mitochondrial dysfunction can feed back to initiate NMJ disruption. Surprisingly, gastrocnemius muscle mass was increased in mSod2 KO mice, which was associated with increased numbers of central nuclei and hyperplasia via fiber branching. In summary, our data suggest that excess mitochondrial superoxide caused a selective loss of complex II activity and mitochondrial defects, leading to severe exercise intolerance. Increased cytoplasmic hydrogen peroxide induced necrosis‐mediated regeneration, which increased muscle mass by fiber branching.Support or Funding InformationNIH/NIA to Dr. Van Remmen (R01 AG050676) and a VA Merit grant to HVRThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Deletion of estrogen receptor α in skeletal muscle results in impaired contractility in female mice.

Estradiol deficiency in females can result in skeletal muscle strength loss, and treatment with estradiol mitigates the loss. There are three primary estrogen receptors (ERs), and estradiol elicits effects through these receptors in various tissues. Ubiquitous ERα-knockout mice exhibit numerous biological disorders, but little is known regarding the specific role of ERα in skeletal muscle contractile function. The purpose of this study was to determine the impact of skeletal muscle-specific ERα deletion on contractile function, hypothesizing that ERα is a main receptor through which estradiol affects muscle strength in females. Deletion of ERα specifically in skeletal muscle (skmERαKO) did not affect body mass compared with wild-type littermates (skmERαWT) until 26 wk of age, at which time body mass of skmERαKO mice began to increase disproportionally. Overall, skmERαKO mice had low strength demonstrated in multiple muscles and by several contractile parameters. Isolated extensor digitorum longus muscles from skmERαKO mice produced 16% less eccentric and 16-26% less submaximal and maximal isometric force, and isolated soleus muscles were more fatigable, with impaired force recovery relative to skmERαWT mice. In vivo maximal torque productions by plantarflexors and dorsiflexors were 16% and 12% lower in skmERαKO than skmERαWT mice, and skmERαKO muscles had low phosphorylation of myosin regulatory light chain. Plantarflexors also generated 21-32% less power, submaximal isometric and peak concentric torques. Data support the hypothesis that ablation of ERα in skeletal muscle results in muscle weakness, suggesting that the beneficial effects of estradiol on muscle strength are receptor mediated through ERα. NEW & NOTEWORTHY We comprehensively measured in vitro and in vivo skeletal muscle contractility in female estrogen receptor α (ERα) skeletal muscle-specific knockout mice and report that force generation is impaired across multiple parameters. These results support the hypothesis that a primary mechanism through which estradiol elicits its effects on strength is mediated by ERα. Evidence is presented that estradiol signaling through ERα appears to modulate force at the molecular level via posttranslational modifications of myosin regulatory light chain.

Identification of mechanically regulated phosphorylation sites on tuberin (TSC2) that control mechanistic target of rapamycin (mTOR) signaling

Mechanistic target of rapamycin (mTOR) signaling is necessary to generate a mechanically induced increase in skeletal muscle mass, but the mechanism(s) through which mechanical stimuli regulate mTOR signaling remain poorly defined. Recent studies have suggested that Ras homologue enriched in brain (Rheb), a direct activator of mTOR, and its inhibitor, the GTPase-activating protein tuberin (TSC2), may play a role in this pathway. To address this possibility, we generated inducible and skeletal muscle-specific knock-out mice for Rheb (iRhebKO) and TSC2 (iTSC2KO) and mechanically stimulated muscles from these mice with eccentric contractions (EC). As expected, the knock-out of TSC2 led to an elevation in the basal level of mTOR signaling. Moreover, we found that the magnitude of the EC-induced activation of mTOR signaling was significantly blunted in muscles from both inducible and skeletal muscle-specific knock-out mice for Rheb and iTSC2KO mice. Using mass spectrometry, we identified six sites on TSC2 whose phosphorylation was significantly altered by the EC treatment. Employing a transient transfection-based approach to rescue TSC2 function in muscles of the iTSC2KO mice, we demonstrated that these phosphorylation sites are required for the role that TSC2 plays in the EC-induced activation of mTOR signaling. Importantly, however, these phosphorylation sites were not required for an insulin-induced activation of mTOR signaling. As such, our results not only establish a critical role for Rheb and TSC2 in the mechanical activation of mTOR signaling, but they also expose the existence of a previously unknown branch of signaling events that can regulate the TSC2/mTOR pathway.

PDLIM7 is a novel target of the ubiquitin ligase Nedd4-1in skeletal muscle.

Skeletal muscle atrophy remains a complication occurring both as a natural response to muscle disuse and as a pathophysiological response to illness such as diabetes mellitus and nerve injury, such as traumatic muscle denervation. The ubiquitin-proteasome system (UPS) is the predominant proteolytic machinery responsible for atrophy of skeletal muscle, and Nedd4-1 (neural precursor cell-expressed developmentally down-regulated 4-1) is one of a series of E3 ubiquitin ligases identified to mediate inactivity-induced muscle wasting. Targets of Nedd4-1 mediated ubiquitination in skeletal muscle remain poorly understood. In the present study, we identified PDLIM7 (PDZ and LIM domain 7, Enigma), a member of the PDZ-LIM family of proteins, as a novel target of Nedd4-1in skeletal muscle. The PDZ-LIM family of proteins is known to regulate muscle development and function. We show that Nedd4-1 expression in muscle atrophied by denervation is co-incident with a decrease in PDLIM7 and that PDLIM7 protein levels are stabilized in denervated muscle of Nedd4-1 skeletal muscle-specific knockout mice (SMS-KO). Exogenous PDLIM7 and Nedd4-1 transfected into human embryonic kidney (HEK)293 cells co-immunoprecipitate through binding between the PY motif of PDLIM7 and the second and third WW domains of Nedd4-1 and endogenous PDLIM7 and Nedd4-1 interact in the cytoplasm of differentiated C2C12 myotubes, leading to PDLIM7 ubiquitination. These results identify PDLIM7 as a bona fide skeletal muscle substrate of Nedd4-1 and suggest that this interaction may underlie the progression of skeletal muscle atrophy. This offers a novel therapeutic target that could be potentially used to attenuate muscle atrophy.

Egr3‐dependent muscle stretch receptor morphogenesis and innervation homeostasis

Muscle spindles are skeletal muscle receptors that mediate stretch reflexes and proprioception in vertebrates. Proprioception abnormalities are observed in many human neuropathies with unknown etiology and factors involved in sensory nerve‐stretch receptor homeostasis are poorly understood. During skeletal muscle development, sensory (Ia‐afferent) innervation is restricted to only a few myotubes which induces their transformation into intrafusal muscle fibers that form the stretch receptor core. The transcriptional regulator Egr3 is induced by sensory innervation and it has an essential role in stretch receptor morphogenesis, but whether its function is restricted to developing receptors and whether it has a role in innervation homeostasis is not known. Ablation of Egr3 using skeletal muscle specific knockout mice demonstrated its autonomous function in developing stretch receptors. Lineage tracing experiments using Egr3‐Cre knock‐in mice showed that stretch receptor differentiation is blocked in the absence of Egr3 and innervation fails to be maintained. Thus, Egr3 has an essential function in intrafusal muscle fiber differentiation and gene regulation required to maintain innervation homeostasis. (Support: NIH R01‐NS063078 and K26‐OD010945).

Myoblast expansion defects leads to muscle growth delay and subsequent compensatory adaptation in adult Atrx cKO skeletal muscle

Background The growth of muscle tissue and its regeneration from injury is crucially dependent on a self-renewing population of muscle progenitor cells called satellite cells. Activated satellite cells give rise to a rapidly expanding population of myoblasts that increase muscle mass by differentiating into new or adding into pre-existing muscle fibers. Patients with mutations in the chromatin remodeling gene ATRX are clinically characterised by severe cognitive disabilities and muscular hypotonia, thus dramatically compromising their independent locomotory ability. Materials and methods We generated a skeletal muscle specific knockout mouse model by interbreeding Myf5-Cre mice with mice harbouring the Atrx floxed conditional knockout allele (herein referred to as Atrx cKO). Body mass of Atrx cKO and control littermates were measured along with general morphometric analysis of select hindlimb muscles at 3weeks, 5-weeks, and greater than 8-weeks of age. Methods of analysis included single muscle fiber preparations from the extensor digitorum longus (EDL) and soleus muscles as well as RNA expression analysis from the EDL and soleus muscles. Primary myoblasts were also cultured from dissociated muscle tissue from the hindlimbs and analyzed via immuno-fluorescent microscopy. Neuromuscular acuity and endurance was assessed in Atrx cKO and control littermates by the roto-rod apparatus. Muscle strength was assessed by utilizing a digital grip strength measuring apparatus. Results Atrx cKO mice presented with telltale characteristics of weaker musculature, exemplified by spinal kyphosis and reduced body mass at 3-weeks of age. Satellite cell derived myoblasts from Atrx cKO mice were incapable of rapid expansion in culture but were fully capable of terminally differentiating. Atrx cKO myoblasts displayed delayed cell cycle progression through mid-late S-phase and rampant signs of genomic instability characterised by fragmented nuclei, g-H2AX foci, and telomeric aberrations. Despite inefficient myoblast proliferative capacity, Atrx cKO animals were able to re-establish normal body mass by adulthood. Muscular fitness and function in Atrx cKO mice was also age dependent, as younger 3-week old Atrx cKO mice had a reduced capacity to stay on the roto-rod and poorer gripping strength. However, differences in grip strength of adult Atrx cKO mice were almost indistinguishable from their control littermates. Data regarding pathways mediating the hypertrophic compensatory adaptation in Atrx cKO mice will also be presented. Conclusions Inefficient expansion of activated satellite cells in Atrx cKO mice results in delayed muscle development up to 3-weeks of age, when myonuclear accretion reaches its plateau. Reduced muscle mass at 3-weeks of age also correlated with poorer performance in physical tasks that require muscular force, endurance, and coordination. Compensatory mechanisms are triggered after 3-weeks of age in Atrx cKO mice that allow for the eventual recovery of body mass and muscle functionality in adults.

The Ubiquitin Ligase Nedd4-1 Participates in Denervation-Induced Skeletal Muscle Atrophy in Mice

Skeletal muscle atrophy is a consequence of muscle inactivity resulting from denervation, unloading and immobility. It accompanies many chronic disease states and also occurs as a pathophysiologic consequence of normal aging. In all these conditions, ubiquitin-dependent proteolysis is a key regulator of the loss of muscle mass, and ubiquitin ligases confer specificity to this process by interacting with, and linking ubiquitin moieties to target substrates through protein∶protein interaction domains. Our previous work suggested that the ubiquitin-protein ligase Nedd4-1 is a potential mediator of skeletal muscle atrophy associated with inactivity (denervation, unloading and immobility). Here we generated a novel tool, the Nedd4-1 skeletal muscle-specific knockout mouse (myoCre;Nedd4-1flox/flox) and subjected it to a well validated model of denervation induced skeletal muscle atrophy. The absence of Nedd4-1 resulted in increased weights and cross-sectional area of type II fast twitch fibres of denervated gastrocnemius muscle compared with wild type littermates controls, at seven and fourteen days following tibial nerve transection. These effects are not mediated by the Nedd4-1 substrates MTMR4, FGFR1 and Notch-1. These results demonstrate that Nedd4-1 plays an important role in mediating denervation-induced skeletal muscle atrophy in vivo.