IL-15R? deficiency in skeletal muscle alters respiratory function and the proteome of mitochondrial subpopulations independent of changes to the mitochondrial genome.
ABSTRACT: Interleukin-15 receptor alpha knockout (IL15R?KO) mice exhibit a greater skeletal muscle mitochondrial density with an altered mitochondrial morphology. However, the mechanism and functional impact of these changes have not been determined. In this study, we characterized the functional, proteomic, and genomic alterations in mitochondrial subpopulations isolated from the skeletal muscles of IL15R?KO mice and B6129 background control mice. State 3 respiration was greater in interfibrillar mitochondria and whole muscle ATP levels were greater in IL15R?KO mice supporting the increases in respiration rate. However, the state 3/state 4 ratio was lower, suggesting some degree of respiratory uncoupling. Proteomic analyses identified several markers independently in mitochondrial subpopulations that are associated with these functional alterations. Next Generation Sequencing of mtDNA revealed a high degree of similarity between the mitochondrial genomes of IL15R?KO mice and controls in terms of copy number, consensus coding and the presence of minor alleles, suggesting that the functional and proteomic alterations we observed occurred independent of alterations to the mitochondrial genome. These data provide additional evidence to implicate IL-15R? as a regulator of skeletal muscle phenotypes through effects on the mitochondrion, and suggest these effects are driven by alterations to the mitochondrial proteome.
Project description:Analysis of the proteome of myostatin (MSTN) knockout (KO) mouse C2C12 cells has proven valuable to studies investigating the molecular mechanisms by which MSTN regulates skeletal muscle development. To identify new protein/pathway alterations and candidate biomarkers for skeletal muscle development, we compared proteomic profiles of MSTN KO C2C12 cells (KO) with corresponding wild-type cells (NC) using a label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique. A total of 2637 proteins were identified and quantified in KO cells. Among these proteins, 77 proteins were significantly differentially expressed, 38 upregulated, and 39 downregulated, in MSTN KO C2C12 cells. These significantly altered proteins are involved in metabolic processes, developmental processes, immune system processes, and the regulation of other biological processes. Enrichment analysis was utilized to link these alterations to biological pathways, which are predominantly related to oxidative phosphorylation, protein digestion and absorption, mitochondrion localisation, antigen processing and presentation, the MAPK signaling pathway, the PPAR signaling pathway, the PI3K-Akt signaling pathway, and the JAK-STAT signaling pathway. Upregulation of several proteins, including epoxide hydrolase, tropomyosin 1, Cyb5a, HTRA1, Cox6a1, CD109, Synap29, and Ugt1a6, likely enhanced skeletal muscle development, the immune system, and energy metabolism. Collectively, our results present a comprehensive proteomics analysis of MSTN KO C2C12 myoblast cells; we hypothesize that MSTN KO could activate p38MAPK signaling pathway by CDC42, and we further deciphered the function of MSTN in the regulation of skeletal muscle development, immune processes, and mitochondrial energy metabolism.
Project description:Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2) catalyzes the oxidative decarboxylation of isocitrate into ?-ketoglutarate with concurrent reduction of NADP+ to NADPH. However, it is not fully understood how IDH2 is intertwined with muscle development and fatty acid metabolism. Here, we examined the effects of IDH2 knockout (KO) on skeletal muscle energy homeostasis. Calf skeletal muscle samples from 10-week-old male IDH2 KO and wild-type (WT; C57BL/6N) mice were harvested, and the ratio of skeletal muscle weight to body and the ratio of mitochondrial to nucleic DNA were measured. In addition, genes involved in myogenesis, mitochondria biogenesis, adipogenesis, and thermogenesis were compared. Results showed that the ratio of skeletal muscle weight to body weight was lower in IDH2 KO mice than those in WT mice. Of note, a noticeable shift in fiber size distribution was found in IDH2 KO mice. Additionally, there was a trend of a decrease in mitochondrial content in IDH2 KO mice than in WT mice (p = 0.09). Further, mRNA expressions for myogenesis and mitochondrial biogenesis were either decreased or showed a trend of decrease in IDH2 KO mice. Moreover, genes for adipogenesis pathway (Pparg, Znf423, and Fat1) were downregulated in IDH2 KO mice. Interestingly, mRNA and protein expression of uncoupling protein 1 (UCP1), a hallmark of thermogenesis, were remarkably increased in IDH2 KO mice. In line with the UCP1 expression, IDH2 KO mice showed higher rectal temperature than WT mice under cold stress. Taken together, IDH2 deficiency may affect myogenesis, possibly due to impairments of muscle generation and abnormal fatty acid oxidation as well as thermogenesis in muscle via upregulation of UCP1.
Project description:The role of Nrf2 in disease prevention and treatment is well documented; however the specific role of Nrf2 in skeletal muscle is not well described. The current study investigated whether Nrf2 plays a protective role in an STZ-induced model of skeletal muscle atrophy. Modulation of Nrf2 through siRNA resulted in a more robust differentiation of C2C12s, whereas increasing Nrf2 with sulforaphane treatment inhibited differentiation. Diabetic muscle atrophy was not dramatically influenced by Nrf2 genotype, since no differences were observed in total atrophy (all fiber types combined) between WT+STZ and KO+STZ animals. Nrf2-KO animals however illustrated alterations in muscle size of Fast, Type II myosin expressing fibers. KO+STZ animals show significant alterations in myosin isoform expression in the GAST. Similarly, KO controls mimic both WT+STZ and KO+STZ muscle alterations in mitochondrial subunit expression. PGC-1?, a well-established player in mitochondrial biogenesis and myosin isoform expression, was decreased in KO control, WT+STZ and KO+STZ SOL muscle. Similarly, PGC-1? protein levels are correlated with Nrf2 levels in C2C12s after modulation by Nrf2 siRNA or sulforaphane treatment. We provide experimental evidence indicating Nrf2 plays a role in myocyte differentiation and governs molecular alterations in contractile and metabolic properties in an STZ-induced model of muscle atrophy.
Project description:A single-nucleotide polymorphism in the human arylamine N-acetyltransferase 2 (Nat2) gene has recently been identified as associated with insulin resistance in humans. To understand the cellular and molecular mechanisms by which alterations in Nat2 activity might cause insulin resistance, we examined murine ortholog Nat1 knockout (KO) mice. Nat1 KO mice manifested whole-body insulin resistance, which could be attributed to reduced muscle, liver, and adipose tissue insulin sensitivity. Hepatic and muscle insulin resistance were associated with marked increases in both liver and muscle triglyceride (TAG) and diacylglycerol (DAG) content, which was associated with increased PKC? activation in liver and increased PKC? activation in skeletal muscle. Nat1 KO mice also displayed reduced whole-body energy expenditure and reduced mitochondrial oxygen consumption in white adipose tissue, brown adipose tissue, and hepatocytes. Taken together, these studies demonstrate that Nat1 deletion promotes reduced mitochondrial activity and is associated with ectopic lipid-induced insulin resistance. These results provide a potential genetic link among mitochondrial dysfunction with increased ectopic lipid deposition, insulin resistance, and type 2 diabetes.
Project description:AMP-activated protein kinase (AMPK) ?1 or ?2 subunits are required for assembling of AMPK heterotrimers and are important for regulating enzyme activity and cellular localization. In skeletal muscle, ?2?2?3-containing heterotrimers predominate. However, compensatory up-regulation and redundancy of AMPK subunits in whole-body AMPK ?2, ?2, and ?3 null mice has made it difficult to determine the physiological importance of AMPK in regulating muscle metabolism, because these models have normal mitochondrial content, contraction-stimulated glucose uptake, and insulin sensitivity. In the current study, we generated mice lacking both AMPK ?1 and ?2 isoforms in skeletal muscle (?1?2M-KO). ?1?2M-KO mice are physically inactive and have a drastically impaired capacity for treadmill running that is associated with reductions in skeletal muscle mitochondrial content but not a fiber-type switch. Interestingly, young ?1?2M-KO mice fed a control chow diet are not obese or insulin resistant but do have impaired contraction-stimulated glucose uptake. These data demonstrate an obligatory role for skeletal muscle AMPK in maintaining mitochondrial capacity and contraction-stimulated glucose uptake, findings that were not apparent in mice with single mutations or deletions in muscle ?, ?, or ? subunits.
Project description:<h4>Objective</h4>Estrogen receptor-? (ER?) is a nuclear receptor family member thought to substantially contribute to the metabolic regulation of skeletal muscle. However, previous mouse models utilized to assess the necessity of ER? signaling in skeletal muscle were confounded by altered developmental programming and/or influenced by secondary effects, making it difficult to assign a causal role for ER?. The objective of this study was to determine the role of skeletal muscle ER? in regulating metabolism in the absence of confounding factors of development.<h4>Methods</h4>A novel mouse model was developed allowing for induced deletion of ER? in adult female skeletal muscle (ER?KO<sup>ism</sup>). ER?shRNA was also used to knockdown ER? (ER?KD) in human myotubes cultured from primary human skeletal muscle cells isolated from muscle biopsies from healthy and obese insulin-resistant women.<h4>Results</h4>Twelve weeks of HFD exposure had no differential effects on body composition, VO<sub>2</sub>, VCO<sub>2</sub>, RER, energy expenditure, and activity counts across genotypes. Although ER?KO<sup>ism</sup> mice exhibited greater glucose intolerance than wild-type (WT) mice after chronic HFD, ex vivo skeletal muscle glucose uptake was not impaired in the ER?KO<sup>ism</sup> mice. Expression of pro-inflammatory genes was altered in the skeletal muscle of the ER?KO<sup>ism</sup>, but the concentrations of these inflammatory markers in the systemic circulation were either lower or remained similar to the WT mice. Finally, skeletal muscle mitochondrial respiratory capacity, oxidative phosphorylation efficiency, and H<sub>2</sub>O<sub>2</sub> emission potential was not affected in the ER?KO<sup>ism</sup> mice. ER?KD in human skeletal muscle cells neither altered differentiation capacity nor caused severe deficits in mitochondrial respiratory capacity.<h4>Conclusions</h4>Collectively, these results suggest that ER? function is superfluous in protecting against HFD-induced skeletal muscle metabolic derangements after postnatal development is complete.
Project description:Exercise confers numerous health benefits, many of which are thought to stem from exercise-induced mitochondrial biogenesis (EIMB) in skeletal muscle. The transcriptional coactivator PGC-1?, a potent regulator of metabolism in numerous tissues, is widely believed to be required for EIMB. We show here that this is not the case. Mice engineered to lack PGC-1? specifically in skeletal muscle (Myo-PGC-1?KO mice) retained intact EIMB. The exercise capacity of these mice was comparable to littermate controls. Induction of metabolic genes after 2 weeks of in-cage voluntary wheel running was intact. Electron microscopy revealed no gross abnormalities in mitochondria, and the mitochondrial biogenic response to endurance exercise was as robust in Myo-PGC-1?KO mice as in wildtype mice. The induction of enzymatic activity of the electron transport chain by exercise was likewise unperturbed in Myo-PGC-1?KO mice. These data demonstrate that PGC-1? is dispensable for exercise-induced mitochondrial biogenesis in skeletal muscle, in sharp contrast to the prevalent assumption in the field.
Project description:Mice deficient in pregnancy-associated plasma protein-A (PAPP-A) have extended lifespan associated with decreased incidence and severity of degenerative diseases of age, such as cardiomyopathy and nephropathy. In this study, the effect of PAPP-A deficiency on aging skeletal muscle was investigated. Whole-genome expression profiling was performed on soleus muscles from 18-month-old wild-type (WT) and PAPP-A knock-out (KO) mice of the same sex and from the same litter ('womb-mates') to identify potential mechanisms of skeletal muscle aging and its retardation in PAPP-A deficiency. Top genes regulated in PAPP-A KO compared to WT muscle were associated with increased muscle function, increased metabolism, in particular lipid metabolism, and decreased stress. Fiber cross-sectional area was significantly increased in solei from PAPP-A KO mice. In vitro contractility experiments indicated increased specific force and decreased fatigue in solei from PAPP-A KO mice. Intrinsic mitochondrial oxidative capacity was significantly increased in skeletal muscle of aged PAPP-A KO compared to WT mice. Moreover, 18-month-old PAPP-A KO mice exhibited significantly enhanced endurance running on a treadmill. Thus, PAPP-A deficiency in mice is associated with indices of healthy skeletal muscle function with age.
Project description:Muscle weakness and exercise intolerance are hallmark symptoms in mitochondrial disorders. Little is known about the mechanisms leading to impaired skeletal muscle function and ultimately muscle weakness in these patients. In a mouse model of lethal mitochondrial myopathy, the muscle-specific Tfam knock-out (KO) mouse, we previously demonstrated an excessive mitochondrial Ca(2+) uptake in isolated muscle fibers that could be inhibited by the cyclophilin D (CypD) inhibitor, cyclosporine A (CsA). Here we show that the Tfam KO mice have increased CypD levels, and we demonstrate that this increase is a common feature in patients with mitochondrial myopathy. We tested the effect of CsA treatment on Tfam KO mice during the transition from a mild to terminal myopathy. CsA treatment counteracted the development of muscle weakness and improved muscle fiber Ca(2+) handling. Importantly, CsA treatment prolonged the lifespan of these muscle-specific Tfam KO mice. These results demonstrate that CsA treatment is an efficient therapeutic strategy to slow the development of severe mitochondrial myopathy.
Project description:While ?-actin isoforms predominate in adult striated muscle, skeletal muscle-specific knockouts (KOs) of nonmuscle cytoplasmic ?cyto - or ?cyto -actin each cause a mild, but progressive myopathy effected by an unknown mechanism. Using transmission electron microscopy, we identified morphological abnormalities in both the mitochondria and the sarcoplasmic reticulum (SR) in aged muscle-specific ?cyto - and ?cyto -actin KO mice. We found ?cyto - and ?cyto -actin proteins to be enriched in isolated mitochondrial-associated membrane preparations, which represent the interface between mitochondria and sarco-endoplasmic reticulum important in signaling and mitochondrial dynamics. We also measured significantly elongated and interconnected mitochondrial morphologies associated with a significant decrease in mitochondrial fission events in primary mouse embryonic fibroblasts lacking ?cyto - and/or ?cyto -actin. Interestingly, mitochondrial respiration in muscle was not measurably affected as oxygen consumption was similar in skeletal muscle fibers from 12 month-old muscle-specific ?cyto - and ?cyto -actin KO mice. Instead, we found that the maximal rate of relaxation after isometric contraction was significantly slowed in muscles of 12-month-old ?cyto - and ?cyto -actin muscle-specific KO mice. Our data suggest that impaired Ca2+ re-uptake may presage development of the observed SR morphological changes in aged mice while providing a potential pathological mechanism for the observed myopathy.