Skeletal Muscle-specific Knockout Research Articles

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)

The RNA binding protein HuR influences skeletal muscle metabolic flexibility in rodents and humans

BackgroundMetabolic flexibility can be assessed by changes in respiratory exchange ratio (RER) following feeding. Though metabolic flexibility (difference in RER between fasted and fed state) is often impaired in individuals with obesity or type 2 diabetes, the cellular processes contributing to this impairment are unclear. Materials and methodsFrom several clinical studies we identified the 16 most and 14 least metabolically flexible male and female subjects out of >100 participants based on differences between 24-hour and sleep RER measured in a whole-room indirect calorimeter. Global skeletal muscle gene expression profiles revealed that, in metabolically flexible subjects, transcripts regulated by the RNA binding protein, HuR, are enriched. We generated and characterized mice with a skeletal muscle-specific knockout of the HuR encoding gene, Elavl1 (HuRm−/−). ResultsMale, but not female, HuRm−/− mice exhibit metabolic inflexibility, with mild obesity, impaired glucose tolerance, impaired fat oxidation and decreased in vitro palmitate oxidation compared to HuRfl/fl littermates. Expression levels of genes involved in mitochondrial fatty acid oxidation and oxidative phosphorylation are decreased in both mouse and human muscle when HuR is inhibited. ConclusionsHuR inhibition results in impaired metabolic flexibility and decreased lipid oxidation, suggesting a role for HuR as an important regulator of skeletal muscle metabolism.

Life-long reduction in myomiR expression does not adversely affect skeletal muscle morphology

We generated an inducible, skeletal muscle-specific Dicer knockout mouse to deplete microRNAs in adult skeletal muscle. Following tamoxifen treatment, Dicer mRNA expression was significantly decreased by 87%. Wild-type (WT) and Dicer knockout (KO) mice were subjected to either synergist ablation or hind limb suspension for two weeks. There was no difference in muscle weight with hypertrophy or atrophy between WT and KO groups; however, even with the significant loss of Dicer expression, myomiR (miR-1, -133a and -206) expression was only reduced by 38% on average. We next aged WT and KO mice for ~22 months following Dicer inactivation to determine if myomiR expression would be further reduced over a prolonged timeframe and assess the effects of myomiR depletion on skeletal muscle phenotype. Skeletal muscle Dicer mRNA expression remained significantly decreased by 80% in old KO mice and sequencing of cloned Dicer mRNA revealed the complete absence of the floxed exons in KO skeletal muscle. Despite a further reduction of myomiR expression to ~50% of WT, no change was observed in muscle morphology between WT and KO groups. These results indicate the life-long reduction in myomiR levels did not adversely affect skeletal muscle phenotype and suggest the possibility that microRNA expression is uniquely regulated in skeletal muscle.

Skeletal muscle–specific knockout of DEP domain containing 5 protein increases mTORC1 signaling, muscle cell hypertrophy, and mitochondrial respiration

mTORC1 regulates protein synthesis and in turn is regulated by growth factors, energy status, and amino acid availability. In kidney cell (HEK293-T) culture, the GAP activity toward RAG (GATOR1) protein complex suppresses activation of the RAG A/B-RAG C/D heterodimer when amino acids are insufficient. During amino acid sufficiency, the RAG heterodimer recruits mTORC1 to the lysosomal membrane where its interaction with Ras homolog enriched in brain (Rheb) stimulates mTORC1's kinase activity. The DEP domain containing 5 (DEPDC5) protein, a GATOR1 subunit, causes familial focal epilepsy when mutated, and global knockout of the Depdc5 gene is embryonically lethal. To study the function of DEPDC5 in skeletal muscle, we generated a muscle-specific inducible Depdc5 knockout mouse, hypothesizing that knocking out Depdc5 in muscle would make mTORC1 constitutively active, causing hypertrophy and improving muscle function. Examining mTORC1 signaling, morphology, mitochondrial respiratory capacity, contractile function, and applied physical function (e.g. rotarod, treadmill, grip test, and wheel running), we observed that mTORC1 activity was significantly higher in knockout (KO) mice, indicated by the increased phosphorylation of mTOR and its downstream effectors (by 118% for p-mTOR/mTOR, 114% for p-S6K1/S6K1, and 35% for p-4E-BP1/4E-BP1). The KO animals also exhibited soleus muscle cell hypertrophy and a 2.5-fold increase in mitochondrial respiratory capacity. However, contrary to our hypothesis, neither physical nor contractile function improved. In conclusion, DEPDC5 depletion in adult skeletal muscle removes GATOR1 inhibition of mTORC1, resulting in muscle hypertrophy and increased mitochondrial respiration, but does not improve overall muscle quality and function.

Skeletal muscle-specific Prmt1 deletion causes muscle atrophy via deregulation of the PRMT6-FOXO3 axis

ABSTRACTProtein arginine methyltransferases (PRMTs) have emerged as important regulators of skeletal muscle metabolism and regeneration. However, the direct roles of the various PRMTs during skeletal muscle remodeling remain unclear. Using skeletal muscle-specific prmt1 knockout mice, we examined the function and downstream targets of PRMT1 in muscle homeostasis. We found that muscle-specific PRMT1 deficiency led to muscle atrophy. PRMT1-deficient muscles exhibited enhanced expression of a macroautophagic/autophagic marker LC3-II, FOXO3 and muscle-specific ubiquitin ligases, TRIM63/MURF-1 and FBXO32, likely contributing to muscle atrophy. The mechanistic study reveals that PRMT1 regulates FOXO3 through PRMT6 modulation. In the absence of PRMT1, increased PRMT6 specifically methylates FOXO3 at arginine 188 and 249, leading to its activation. Finally, we demonstrate that PRMT1 deficiency triggers FOXO3 hyperactivation, which is abrogated by PRMT6 depletion. Taken together, PRMT1 is a key regulator for the PRMT6-FOXO3 axis in the control of autophagy and protein degradation underlying muscle maintenance.Abbreviations: Ad-RNAi: adenovirus-delivered small interfering RNA; AKT: thymoma viral proto-oncogene; AMPK: AMP-activated protein kinase; Baf A1: bafilomycin A1; CSA: cross-sectional area; EDL: extensor digitorum longus; FBXO32: F-box protein 32; FOXO: forkhead box O; GAS: gatrocnemieus; HDAC: histone deacetylase; IGF: insulin-like growth factor; LAMP: lysosomal-associated membrane protein; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; mKO: Mice with skeletal muscle-specific deletion of Prmt1; MTOR: mechanistic target of rapamycin kinase; MYH: myosin heavy chain; MYL1/MLC1f: myosin, light polypeptide 1; PRMT: protein arginine N-methyltransferase; sgRNA: single guide RNA; SQSTM1: sequestosome 1; SOL: soleus; TA: tibialis anterior; TRIM63/MURF-1: tripartite motif-containing 63; YY1: YY1 transcription factor

Induced in vivo knockdown of the Brca1 gene in skeletal muscle results in skeletal muscle weakness.

Breast cancer 1 early onset gene codes for the DNA repair enzyme, breast cancer type 1 susceptibility protein (BRCA1). The gene is prone to mutations that cause a loss of protein function. BRCA1/Brca1 has recently been found to regulate several cellular pathways beyond DNA repair and is expressed in skeletal muscle. Skeletal muscle specific knockout of Brca1 in mice caused a loss of muscle quality, identifiable by reductions in muscle force production and mitochondrial respiratory capacity. Loss of muscle quality was associated with a shift in muscle phenotype and an accumulation of mitochondrial DNA mutations. These results demonstrate that BRCA1 is necessary for skeletal muscle function and that increased mitochondrial DNA mutations may represent a potential underlying mechanism. Recent evidence suggests that the breast cancer 1 early onset gene (BRCA1) influences numerous peripheral tissues, including skeletal muscle. The present study aimed to determine whether induced-loss of the breast cancer type 1 susceptibility protein (Brca1) alters skeletal muscle function. We induced genetic ablation of exon 11 in the Brca1 gene specifically in the skeletal muscle of adult mice to generate skeletal muscle-specific Brca1 homozygote knockout (Brca1KOsmi ) mice. Brca1KOsmi exhibited kyphosis and decreased maximal isometric force in limb muscles compared to age-matched wild-type mice. Brca1KOsmi skeletal muscle shifted toward an oxidative muscle fibre type and, in parallel, increased myofibre size and reduced capillary numbers. Unexpectedly, myofibre bundle mitochondrial respiration was reduced, whereas contraction-induced lactate production was elevated in Brca1KOsmi muscle. Brca1KOsmi mice accumulated mitochondrial DNA mutations and exhibited an altered mitochondrial morphology characterized by distorted and enlarged mitochondria, and these were more susceptible to swelling. In summary, skeletal muscle-specific loss of Brca1 leads to a myopathy and mitochondriopathy characterized by reductions in skeletal muscle quality and a consequent kyphosis. Given the substantial impact of BRCA1 mutations on cancer development risk in humans, a parallel loss of BRCA1 function in patient skeletal muscle cells would potentially result in implications for human health.

Adult skeletal muscle deletion of Mitofusin 1 and 2 impedes exercise performance and training capacity.

Endurance exercise has been shown to be a positive regulator of skeletal muscle metabolic function. Changes in mitochondrial dynamics (fusion and fission) have been shown to influence mitochondrial oxidative capacity. We therefore tested whether genetic disruption of mitofusins (Mfns) affected exercise performance in adult skeletal muscle. We generated adult-inducible skeletal muscle-specific Mfn1 (iMS-Mfn1KO), Mfn2 (iMS-Mfn2KO), and Mfn1/2 (iMS-MfnDKO) knockout mice. We assessed exercise capacity by performing a treadmill time to exhaustion stress test before deletion and up to 8 wk after deletion. Analysis of either the iMS-Mfn1KO or the iMS-Mfn2KO did not reveal an effect on exercise capacity. However, analysis of iMS-MfnDKO animals revealed a progressive reduction in exercise performance. We measured individual electron transport chain (ETC) complex activity and observed a reduction in ETC activity in both the subsarcolemmal and intermyofibrillar mitochondrial fractions specifically for NADH dehydrogenase (complex I) and cytochrome- c oxidase (complex IV), which was associated with a decrease in ETC subunit expression for these complexes. We also tested whether voluntary exercise training would prevent the decrease in exercise capacity observed in iMS-MfnDKO animals ( n = 10/group). However, after 8 wk of training we did not observe any improvement in exercise capacity or ETC subunit parameters in iMS-MfnDKO animals. These data suggest that the decrease in exercise capacity observed in the iMS-MfnDKO animals is in part the result of impaired ETC subunit expression and ETC complex activity. Taken together, these results provide strong evidence that mitochondrial fusion in adult skeletal muscle is important for exercise performance. NEW & NOTEWORTHY This study is the first to utilize an adult-inducible skeletal muscle-specific knockout model for Mitofusin (Mfn)1 and Mfn2 to assess exercise capacity. Our findings reveal a progressive decrease in exercise performance with Mfn1 and Mfn2 deletion. The decrease in exercise capacity was accompanied by impaired oxidative phosphorylation specifically for complex I and complex IV. Furthermore, voluntary exercise training was unable to rescue the impairment, suggesting that normal fusion is essential for exercise-induced mitochondrial adaptations.

Perturbed Redox Signaling Exacerbates a Mitochondrial Myopathy

SummaryAlternative oxidases (AOXs) bypass respiratory complexes III and IV by transferring electrons from coenzyme Q directly to O2. They have therefore been proposed as a potential therapeutic tool for mitochondrial diseases. We crossed the severely myopathic skeletal muscle-specific COX15 knockout (KO) mouse with an AOX-transgenic mouse. Surprisingly, the double KO-AOX mutants had decreased lifespan and a substantial worsening of the myopathy compared with KO alone. Decreased ROS production in KO-AOX versus KO mice led to impaired AMPK/PGC-1α signaling and PAX7/MYOD-dependent muscle regeneration, blunting compensatory responses. Importantly, the antioxidant N-acetylcysteine had a similar effect, decreasing the lifespan of KO mice. Our findings have major implications for understanding pathogenic mechanisms in mitochondrial diseases and for the design of therapies, highlighting the benefits of ROS signaling and the potential hazards of antioxidant treatment.

Impact of skeletal muscle IL-6 on regulation of liver and adipose tissue metabolism during fasting.

The liver and adipose tissue are important tissues in whole-body metabolic regulation during fasting. Interleukin 6 (IL-6) is a cytokine shown to be secreted from contracting muscle in humans and suggested to signal to the liver and adipose tissue. Furthermore, skeletal muscle IL-6 has been proposed to play a role during fasting. Therefore the aim of the present study was to investigate the role of skeletal muscle IL-6 in the regulation of substrate production in the liver and adipose tissue during fasting. Male skeletal muscle-specific IL-6 knockout (IL-6 MKO) mice and littermate floxed (control) mice fasted for 6 or 18h (6h fasting or 18h fasting) with corresponding fed control groups (6h fed or 18h fed) and liver and adipose tissue were quickly obtained. Plasma β-hydroxybutyrate increased and hepatic glucose, lactate and glycogen decreased with fasting. In addition, fasting increased phosphoenolpyruvate carboxykinase protein and phosphorylation of pyruvate dehydrogenase (PDH) in the liver as well as hormone-sensitive lipase (HSL)Ser660 and HSLSer563 phosphorylation, PDH phosphorylation, adipose triglyceride lipase phosphorylation and perilipin phosphorylation and protein content in adipose tissue without any effect of lack of skeletal muscle IL-6. In conclusion, fasting induced regulation of enzymes in adipose tissue lipolysis and glyceroneogenesis as well as regulation of hepatic gluconeogenic capacity and hepatic substrate utilization in mice. However, skeletal muscle IL-6 was not required for these fasting-induced effects, but had minor effects on markers of lipolysis and glyceroneogenesis in adipose tissue as well as markers of hepatic gluconeogenesis in the fed state.

ATAD3 controls mitochondrial cristae structure in mouse muscle, influencing mtDNA replication and cholesterol levels.

Mutations in the mitochondrial inner membrane ATPase ATAD3A result in neurological syndromes in humans. In mice, the ubiquitous disruption of Atad3 (also known as Atad3a) was embryonic lethal, but a skeletal muscle-specific conditional knockout (KO) was viable. At birth, ATAD3 muscle KO mice had normal weight, but from 2 months onwards they showed progressive motor-impaired coordination and weakness. Loss of ATAD3 caused early and severe mitochondrial structural abnormalities, mitochondrial proliferation and muscle atrophy. There was dramatic reduction in mitochondrial cristae junctions and overall cristae morphology. The lack of mitochondrial cristae was accompanied by a reduction in high molecular weight mitochondrial contact site and cristae organizing system (MICOS) complexes, and to a lesser extent in OPA1. Moreover, muscles lacking ATAD3 showed altered cholesterol metabolism, accumulation of mitochondrial DNA (mtDNA) replication intermediates, progressive mtDNA depletion and deletions. Unexpectedly, decreases in the levels of some OXPHOS components occurred after cristae destabilization, indicating that ATAD3 is not crucial for mitochondrial translation, as previously suggested. Our results show a critical early role of ATAD3 in regulating mitochondrial inner membrane structure, leading to secondary defects in mtDNA replication and complex V and cholesterol levels in postmitotic tissue.This article has an associated First Person interview with the first author of the paper.

HuR Influences Metabolic Flexibility in Skeletal Muscle

Metabolic Flexibility allows for the oxidation of fuels based on their availability. Respiratory Quotient (RQ) values from subjects with type 2 diabetes mellitus in both basal and insulin stimulated states remain consistent, indicating a decreased ability to switch between available substrates. The molecular mechanisms governing defects in this switching ability are unclear at present. We therefore compared global gene expression in skeletal muscle between obese subjects classified by their sleep and 24 hour RQ as either metabolically flexible or inflexible. From this data we identified a number of transcripts stabilized by the RNA binding protein, HuR, which were enriched in metabolically flexible subjects. This finding prompted us to generate mice with a skeletal muscle specific knockout of Elavl1, the gene encoding HuR (HuRm-/-). HuRm-/- mice have a metabolically inflexible phenotype with mild obesity and impaired glucose tolerance compared to control littermates. HuRm-/- mice have an increase in RER; and decreased expression of genes involved in oxidative phosphorylation, fatty acid transport, and mitochondrial fatty acid metabolism, suggesting a decreased ability to use fats as an energy source. HuRm-/- mouse skeletal muscle also shows decreases in protein levels of important modulators of the mTOR signaling pathway, and compensatory increases in transcript levels of HuR target mRNAs. These data highlight the importance of HuR function in skeletal muscle metabolism; and suggest HuR as a mediator of metabolite selection. Disclosure J.D. Warfel: None.

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.

Deubiquitinase UCHL1 Regulates Myogenesis in Skeletal Muscles

Myogenesis is crucial for skeletal muscle regeneration in injury and disease. This multi‐step process is closely regulated by several myogenic factors. Timely activation and inactivation of these factors require ubiquitin‐proteasome system (UPS)‐mediated protein degradation. In this aspect, deubiquitinases (DUBs) could also play a role in myogenesis. Ubiquitin C‐terminal hydrolase L1 (UCHL1) is a DUB that is highly expressed in the brain. Though undetectable in adult skeletal muscles, UCHL1 protein level is significantly increased in injured muscles. However, the functional significance of UCHL1 in skeletal muscles is unknown. Using in vitro and in vivo approaches, this study tested a hypothesis that UCHL1 can play a role in skeletal muscle myogenesis and regeneration.Our data show that UCHL1 protein level was gradually downregulated during myoblast C2C12 differentiation. Knockdown of UCHL1 expression significantly reduced C2C12 cell proliferation. In contrast, UCHL1 knockdown markedly accelerated cell differentiation, evidenced by more myotubes in UCHL1 deficient cells, which was accompanied by significant upregulation of myogenic factors.In mouse muscles, UCHL1 was expressed by type I and IIa muscle fibers. Denervation increased UCHL1 protein levels in muscle fibers, which was completely abolished in muscles from the mice with UCHL1 skeletal muscle specific knockout (smKO). smKO mice showed reduced satellite cells in soleus and declined type I fiber number. Moreover, smKO muscles exhibited right shift of fiber distribution and a remarkable loss of type I muscle fiber upon denervation as compared with wildtype ones. These observations further suggest the potential involvement of UCHL1 in myogenesis for the regeneration of type I fibers.Support or Funding InformationNIH grants No. 1R15HL118696 and 1R03AG051926 and USD SSOM BBS bridge grant, CBBRe pilot grant, PQCD pilot grant.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of gp130 in basal and exercise-trained skeletal muscle mitochondrial quality control.

The IL-6 cytokine family activates intracellular signaling pathways through glycoprotein-130 (gp130), and this signaling has established regulatory roles in muscle glucose metabolism and proteostasis. Although the IL-6 family has been implicated as myokines regulating the muscles' metabolic response to exercise, gp130's role in mitochondrial quality control involving fission, fusion, mitophagy, and biogenesis is not well understood. Therefore, we examined gp130's role in basal and exercise-trained muscle mitochondrial quality control. Muscles from C57BL/6, skeletal muscle-specific gp130 knockout (KO) mice, and C2C12 myotubes, were examined. KO did not alter treadmill run-to-fatigue or indices of mitochondrial content [cytochrome- c oxidase (COX) activity] or biogenesis (AMPK, peroxisome proliferator-activated receptor-γ coactivator-1α, mitochondrial transcription factor A, and COX IV). KO increased mitochondrial fission 1 protein (FIS-1) while suppressing mitofusin-1 (MFN-1), which was recapitulated in myotubes after gp130 knockdown. KO induced ubiquitin-binding protein p62, Parkin, and ubiquitin in isolated mitochondria from gastrocnemius muscles. Knockdown of gp130 in myotubes suppressed STAT3 and induced accumulation of microtubule-associated protein-1 light chain 3B (LC3)-II relative to LC3-I. Suppression of myotube STAT3 did not alter FIS-1 or MFN-1. Exercise training increased muscle gp130 and suppressed STAT3. KO did not alter the exercise-training induction of COX activity, biogenesis, FIS-1, or Beclin-1. KO increased MFN-1 and suppressed 4-hydroxynonenal after exercise training. These findings suggest a role for gp130 in the modulation of mitochondrial dynamics and autophagic processes. NEW & NOTEWORTHY Although the IL-6 family of cytokines has been implicated in the regulation of skeletal muscle protein turnover and metabolism, less is understood about its role in mitochondrial quality control. We examined the glycoprotein-130 receptor in the regulation of skeletal muscle mitochondria quality control in the basal and exercise-trained states. We report that the muscle glycoprotein-130 receptor modulates basal mitochondrial dynamics and autophagic processes and is not necessary for exercise-training mitochondrial adaptations to quality control.

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.

The role of AMP-activated protein kinase in the expression of the dystrophin-associated protein complex in skeletal muscle.

Stimulation of AMPK induces the expression of dystrophin-associated protein complex (DAPC) components in skeletal muscle, whereas reductions in AMPK are associated with DAPC dysfunction. We sought to determine whether AMPK was necessary for the maintenance of DAPC expression in skeletal muscle. Fast, glycolytic extensor digitorum longus (EDL) and slow, oxidative soleus (Sol) muscles from wild-type mice and from littermates with skeletal muscle-specific knockout of the AMPK β1 and β2 subunits (AMPK β1 β2M-KO; MKO) were analyzed. DAPC mRNA and protein expression were similar between genotypes, with the exception of elevated neuronal nitric oxide synthase expression at the sarcolemma in MKO muscles. The content of transcriptional and post-transcriptional regulators of the DAPC was also not affected by the loss of AMPK. However, MyoD and myogenin expression was diminished in MKO muscles, consistent with previous reports of myopathy in these animals. Furthermore, we observed decrements in extrasynaptic utrophin expression selectively in MKO Sol muscles, likely due to the adaptive accumulation of peroxisome proliferator-activated receptor γ coactivator-1α at the sarcolemma of MKO EDL muscles. Collectively, the evidence indicates that AMPK is sufficient but not essential for the maintenance of DAPC expression in skeletal muscle, yet it is required for preserving extrasynaptic utrophin levels in slow oxidative muscles.-Dial, A. G., Rooprai, P., Lally, J. S., Bujak, A. L., Steinberg, G. R., Ljubicic, V. The role of AMP-activated protein kinase in the expression of the dystrophin-associated protein complex in skeletal muscle.

Loss of Mitochondrial RNA Binding Protein PTCD1 Leads to Cardiomyopathy

In eukaryotic cells mitochondria produce energy at the electron transport chain (ETC). ETC assembly requires regulated mitochondrial gene expression, which is predominantly controlled post-transcriptionally by RNA-binding proteins, including the pentatricopeptide repeat (PPR) domain proteins. The PPR domain 1 (PTCD1) protein is involved in RNA metabolism and stability, and Ptcd1 haploinsufficiency in mice causes an increase in transcription, accumulation of precursor transcripts, and decreased protein synthesis. Furthermore, whole-exome analysis has identified a PTCD1 mutation in a patient with cardiomyopathy. To further characterise the molecular role of PTCD1, we investigated a homozygous heart and skeletal muscle-specific knockout in mice. Echocardiography revealed that PTCD1 loss led to dilated cardiomyopathy. Specifically, Ptcd–/– mice (n = 5) showed a significant decrease in fractional shortening (15.7 ± 1.53%) compared with wild-type Ptcd1+/+ mice (n = 5) (24.9 ± 0.91%), and a significant increase in ventricle diameter. Immunoblotting revealed a decrease in ETC subunit abundance, as well as altered expression of various nuclear-encoded ribosomal proteins and proteases, in Ptcd1–/– mice (n = 3) compared with Ptcd1+/+ (n = 3). Northern blotting revealed a compensatory increase in transcription, evident as an increase in mature mRNA and tRNA expression in Ptcd1–/– (n = 3) compared with Ptcd1+/+ mice (n = 3). Interestingly, abundance of the 16S rRNA was decreased, indicating that PTCD1 is required for its stability, and therefore mitoribosome assembly, which could be contributing to the decrease in mitochondrial protein translation observed. These results suggest that PTCD1 is essential for assembly of the mitoribosome and translation, which is necessary for ETC assembly and, ultimately, heart function.

Lack of O-GlcNAcylation enhances exercise-dependent glucose utilization potentially through AMP-activated protein kinase activation in skeletal muscle

O-GlcNAcylation is a post-translational modification that is characterized by the addition of N-acetylglucosamine (GlcNAc) to proteins by O-GlcNAc transferase (Ogt). The degree of O-GlcNAcylation is thought to be associated with glucotoxicity and diabetic complications, because GlcNAc is produced by a branch of the glycolytic pathway. However, its role in skeletal muscle has not been fully elucidated. In this study, we created skeletal muscle-specific Ogt knockout (Ogt-MKO) mice and analyzed their glucose metabolism. During an intraperitoneal glucose tolerance test, blood glucose was slightly lower in Ogt-MKO mice than in control Ogt-flox mice. High fat diet-induced obesity and insulin resistance were reversed in Ogt-MKO mice. In addition, 12-month-old Ogt-MKO mice had lower adipose and body mass. A single bout of exercise significantly reduced blood glucose in Ogt-MKO mice, probably because of higher AMP-activated protein kinase α (AMPKα) protein expression. Furthermore, intraperitoneal injection of 5-aminoimidazole-4-carboxamide ribonucleotide, an AMPK activator, resulted in a more marked decrease in blood glucose levels in Ogt-MKO mice than in controls. Finally, Ogt knockdown by siRNA in C2C12 myotubes significantly increased protein expression of AMPKα, glucose uptake and oxidation. In conclusion, loss of O-GlcNAcylation facilitates glucose utilization in skeletal muscle, potentially through AMPK activation. The inhibition of O-GlcNAcylation in skeletal muscle may have an anti-diabetic effect, through an enhancement of glucose utilization during exercise.

Overload-mediated skeletal muscle hypertrophy is not impaired by loss of myofiber STAT3.

Although the signal pathways mediating muscle protein synthesis and degradation are well characterized, the transcriptional processes modulating skeletal muscle mass and adaptive growth are poorly understood. Recently, studies in mouse models of muscle wasting or acutely exercised human muscle have suggested a potential role for the transcription factor signal transducer and activator of transcription 3 (STAT3), in adaptive growth. Hence, in the present study we sought to define the contribution of STAT3 to skeletal muscle adaptive growth. In contrast to previous work, two different resistance exercise protocols did not change STAT3 phosphorylation in human skeletal muscle. To directly address the role of STAT3 in load-induced (i.e., adaptive) growth, we studied the anabolic effects of 14 days of synergist ablation (SA) in skeletal muscle-specific STAT3 knockout (mKO) mice and their floxed, wild-type (WT) littermates. Plantaris muscle weight and fiber area in the nonoperated leg (control; CON) was comparable between genotypes. As expected, SA significantly increased plantaris weight, muscle fiber cross-sectional area, and anabolic signaling in WT mice, although interestingly, this induction was not impaired in STAT3 mKO mice. Collectively, these data demonstrate that STAT3 is not required for overload-mediated hypertrophy in mouse skeletal muscle.