Reduced Myofiber Size Research Articles

Abstract 3026: Genetic Deletion Of Skeletal Muscle Bag3 Exacerbates Ischemic Myopathy

Background: Bcl2-associated athanogene 3 (BAG3) is a multifunctional adaptor protein that regulates diverse cellular functions. Mutations in BAG3 are causally linked to myofibrillar myopathy and dilated cardiomyopathy in humans and now appear to be one of the most common to cause human disease. We previously identified a murine coding variant in Bag3 that contributes to hindlimb ischemia (HLI) pathology, however, no cell specific data on Bag3’s role in PAD pathology exists. Hypothesis: We hypothesized that muscle specific BAG3 is required for both the restoration of blood flow and muscle recovery following HLI. Methods: We generated a genetic model of lifelong muscle cell (mature fiber and progenitor) Bag3 deletion (Pax7-Cre; Bag3 -/- ) on a BL6 parental background. Male and Female mice (12-20 weeks; WT: n=45; KO: n=32) were evaluated at baseline and HLI d7 and d28 for blood flow via LDPI, isometric muscle force production, vascular density and muscle morphology. Bag3 deletion was validated by recombination, qRT-PCR and Western Blotting in whole tissue, isolated myofibers, and FACS muscle progenitor cells. Results: KO reduced muscle force production (n=18; 145.8mN Force ± 28.6 vs 56.9 ± 16.8 SEM; p=0.0208) and capillary perfusion (n=7; 0.17 CD31+/Lectin+ vessels ± 0.02 vs 0.09 ± 0.01 SEM p=0.0491) indicative of the multi-compartmental effect of BAG3 loss in skeletal muscle at HLI d7. Extended HLI (d28) revealed a KO phenotype characterized by reduced blood flow recovery (LDPI; n= 7; .68 ischemic/non limb ± 0.5 vs .38 ± 0.6 SEM p=0.0011) and sustained myopathy including reduced myofiber size ( n =7; 1585um 2 ± 161 vs 1093 ± 169 SEM; p =0.05) and increased tissue lesion size ( n =7; 2.5% muscle area ± 2.0 vs 48.3% ± 21.6; p =0.03). Conclusion: Loss of skeletal muscle Bag3 exacerbates myopathy and reduces capillary perfusion and blood flow recovery following HLI and demonstrate a requirement for BAG3 in recovery from limb ischemia.

Cyclin D3 deficiency promotes a slower, more oxidative skeletal muscle phenotype and ameliorates pathophysiology in the mdx mouse model of Duchenne muscular dystrophy.

We previously reported that cyclin D3-null mice display a shift toward the slow, oxidative phenotype in skeletal muscle, improved exercise endurance, and increased energy expenditure. Here, we explored the role of cyclin D3 in the physiologic response of skeletal muscle to external stimuli and in a model of muscle degenerative disease. We show that cyclin D3-null mice exhibit a further transition from glycolytic to oxidative muscle fiber type in response to voluntary exercise and an improved response to fasting. Since fast glycolytic fibers are known to be more susceptible to degeneration in Duchenne muscular dystrophy (DMD), we examined the effects of cyclin D3 inactivation on skeletal muscle phenotype in the mdx mouse model of DMD. Compared with control mdx mice, cyclin D3-deficient mdx mice display a higher proportion of slower and more oxidative myofibers, reduced muscle degenerative/regenerative processes, and reduced myofiber size variability, indicating an attenuation of dystrophic histopathology. Furthermore, mdx muscles lacking cyclin D3 exhibit reduced fatigability during repeated electrical stimulations. Notably, cyclin D3-null mdx mice show enhanced performance during recurrent trials of endurance treadmill exercise, and post-exercise muscle damage results decreased while the regenerative capacity is boosted. In addition, muscles from exercised cyclin D3-deficient mdx mice display increased oxidative capacity and increased mRNA expression of genes involved in the regulation of oxidative metabolism and the response to oxidative stress. Altogether, our findings indicate that depletion of cyclin D3 confers benefits to dystrophic muscle, suggesting that cyclin D3 inhibition may represent a promising therapeutic strategy against DMD.

MicroRNA-501 controls myogenin+/CD74+ myogenic progenitor cells during muscle regeneration

ObjectiveSkeletal muscle regeneration is markedly impaired during aging. How adult muscle stem cells contribute to this decrease in regenerative capacity is incompletely understood. We investigated mechanisms of age-related changes in myogenic progenitor cells using the tissue-specific microRNA 501. MethodsYoung and old C57Bl/6 mice were used (3 months or 24 months of age, respectively) with or without global or tissue-specific genetic deletion of miR-501. Muscle regeneration was induced using intramuscular cardiotoxin injection or treadmill exercise and analysed using single cell and bulk RNA sequencing, qRT-PCR and immunofluorescence. Muscle fiber damage was assessed with Evan`s blue dye (EBD). In vitro analysis was performed in primary muscle cells obtained from mice and humans. ResultsSingle cell sequencing revealed myogenic progenitor cells in miR-501 knockout mice at day 6 after muscle injury that are characterized by high levels of myogenin and CD74. In control mice these cells were less in number and already downregulated after day 3 of muscle injury. Muscle from knockout mice had reduced myofiber size and reduced myofiber resilience to injury and exercise. miR-501 elicits this effect by regulating sarcomeric gene expression through its target gene estrogen-related receptor gamma (Esrrg). Importantly, in aged skeletal muscle where miR-501 was significantly downregulated and its target Esrrg significantly upregulated, the number of myog+/CD74+ cells during regeneration was upregulated to similar levels as observed in 501 knockout mice. Moreover, myog+/CD74+-aged skeletal muscle exhibited a similar decrease in the size of newly formed myofibers and increased number of necrotic myofibers after injury as observed in mice lacking miR-501. ConclusionsmiR-501 and Esrrg are regulated in muscle with decreased regenerative capacity and loss of miR-501 is permissive to the appearance of CD74+ myogenic progenitors. Our data uncover a novel link between the metabolic transcription factor Esrrg and sarcomere formation and demonstrate that stem cell heterogeneity in skeletal muscle during aging is under miRNA control. Targeting Esrrg or myog+/CD74+ progenitor cells might improve fiber size and myofiber resilience to exercise in aged skeletal muscle.

Serine and Glycine Are Essential for Skeletal Muscle Regeneration Following Injury

Abstract Objectives Skeletal muscle (SkM) regeneration post injury is reliant on SkM-specific stem cells (muscle progenitor cells [MPCs]) and a well-orchestrated myogenic program. The regenerative process is impaired with advancing age, potentiating pathological SkM remodeling (infiltration of fat and fibrotic tissues). We have previously demonstrated that the nutritionally non-essential amino acids serine (Ser) and glycine (Gly) are required for early stages of SkM regeneration (MPC proliferation). However, Ser and Gly availability (SkM and circulating) declines with aging. The objective was to test the hypothesis that reduced endogenous Ser/Gly during regeneration promotes pathological SkM remodeling in aged animals. Methods Old mice (∼20 months of age) were given a Ser/Gly depleted diet (SGdep) or an isonitrogenous, isoenergetic diet containing Ser/Gly (SGcont) for 4 weeks followed by notexin-induced injury to the tibialis anterior (TA) SkM. At 28 days post injury the TA was harvested and histological analysis of SkM morphology (H&E and immunofluorescence [IF]) and gene expression analyses (qPCR) were completed. Results Old mice receiving the SGdep diet had a shift toward reduced myofiber size and enhanced adipocyte infiltration in the SkM. Adipocyte infiltration was confirmed with IF of perilipin-1, an adipocyte marker. Uninjured mice on the SGdep diet did not demonstrate altered SkM morphology. Gene expression analysis of differentially expressed genes underlying SkM remodeling (reduced myofiber size and increased fat infiltration) with SGdep is ongoing. Conclusions Reduced Ser and Gly availability following injury instigates SkM remodeling in old mice, which could explain in part age-related impairments in SkM regeneration. This research underscores the essentiality of Ser and Gly for the SkM regenerative process particularly with advancing age. Funding Sources Canadian Institutes of Health Research Doctoral Foreign Study Award to BG.

Impaired autophagic flux contributes to muscle atrophy in obesity by affecting muscle degradation and regeneration

Increased proteolytic activity has been widely associated with skeletal muscle atrophy. However, elevated proteolysis is also critical for the maintenance of intracellular homeostasis. In this study, we aimed to investigate the significance of autophagy in obesity-induced muscle atrophy and clarify the mechanism involved. First, high-fat diet (HFD)-fed rats were administered vehicle or chloroquine (CQ), an autophagy inhibitor, and we found that HFD inhibited autophagic flux and reduced myofiber size and function in rats. Additionally, the expression levels of MyoD were decreased whereas those of Atrogin-1 were increased in rats fed a HFD. Sustained autophagy inhibition by CQ exacerbated HFD-induced muscular damage and changes in the expression of Atrogin-1 and MyoD. Similar effects were reproduced in vitro in myotubes, which exhibited increased levels of autophagy-related proteins, but the resultant autophagic flux was reduced following exposure to palmitic acid (PA)-conditioned medium. Moreover, PA significantly decreased MyoD levels and induced Atrogin-1 expression, leading to progressive myotube atrophy; this phenomenon was aggravated by CQ but alleviated by the autophagy activator rapamycin. Taken together, these in vivo and in vitro findings suggest that autophagic flux is blocked in skeletal muscle of individuals with high lipid, and autophagy mediates high lipid-induced muscle atrophy by affecting muscle degradation and regeneration.

Mineralocorticoid Receptor Signaling Contributes to Normal Muscle Repair After Acute Injury.

Acute skeletal muscle injury is followed by a temporal response of immune cells, fibroblasts, and muscle progenitor cells within the muscle microenvironment to restore function. These same cell types are repeatedly activated in muscular dystrophy from chronic muscle injury, but eventually, the regenerative portion of the cycle is disrupted and fibrosis replaces degenerated muscle fibers. Mineralocorticoid receptor (MR) antagonist drugs have been demonstrated to increase skeletal muscle function, decrease fibrosis, and directly improve membrane integrity in muscular dystrophy mice, and therefore are being tested clinically. Conditional knockout of MR from muscle fibers in muscular dystrophy mice also improves skeletal muscle function and decreases fibrosis. The mechanism of efficacy likely results from blocking MR signaling by its endogenous agonist aldosterone, being produced at high local levels in regions of muscle damage by infiltrating myeloid cells. Since chronic and acute injuries share the same cellular processes to regenerate muscle, and MR antagonists are clinically used for a wide variety of conditions, it is crucial to define the role of MR signaling in normal muscle repair after injury. In this study, we performed acute injuries using barium chloride injections into tibialis anterior muscles both in myofiber MR conditional knockout mice on a wild-type background (MRcko) and in MR antagonist-treated wild-type mice. Steps of the muscle regeneration response were analyzed at 1, 4, 7, or 14 days after injury. Presence of the aldosterone synthase enzyme was also assessed during the injury repair process. We show for the first time aldosterone synthase localization in infiltrating immune cells of normal skeletal muscle after acute injury. MRcko mice had an increased muscle area infiltrated by aldosterone synthase positive myeloid cells compared to control injured animals. Both MRcko and MR antagonist treatment stabilized damaged myofibers and increased collagen infiltration or compaction at 4 days post-injury. MR antagonist treatment also led to reduced myofiber size at 7 and 14 days post-injury. These data support that MR signaling contributes to the normal muscle repair process following acute injury. MR antagonist treatment delays muscle fiber growth, so temporary discontinuation of these drugs after a severe muscle injury could be considered.

Prepubertal skeletal muscle growth requires Pax7-expressing satellite cell-derived myonuclear contribution.

ABSTRACTThe functional role of Pax7-expressing satellite cells (SCs) in postnatal skeletal muscle development beyond weaning remains obscure. Therefore, the relevance of SCs during prepubertal growth, a period after weaning but prior to the onset of puberty, has not been examined. Here, we have characterized mouse skeletal muscle growth during prepuberty and found significant increases in myofiber cross-sectional area that correlated with SC-derived myonuclear number. Remarkably, genome-wide RNA-sequencing analysis established that post-weaning juvenile and early adolescent skeletal muscle have markedly different gene expression signatures. These distinctions are consistent with extensive skeletal muscle maturation during this essential, albeit brief, developmental phase. Indelible labeling of SCs with Pax7CreERT2/+; Rosa26nTnG/+ mice demonstrated SC-derived myonuclear contribution during prepuberty, with a substantial reduction at puberty onset. Prepubertal depletion of SCs in Pax7CreERT2/+; Rosa26DTA/+ mice reduced myofiber size and myonuclear number, and caused force generation deficits to a similar extent in both fast and slow-contracting muscles. Collectively, these data demonstrate SC-derived myonuclear accretion as a cellular mechanism that contributes to prepubertal hypertrophic skeletal muscle growth.

Fatty Infiltration Is a Prognostic Marker of Muscle Function After Rotator Cuff Tear

Background: Massive rotator cuff tears (RCTs) begin as primary tendon injuries and cause a myriad of changes in the muscle, including atrophy, fatty infiltration (FI), and fibrosis. However, it is unclear which changes are most closely associated with muscle function. Purpose: To determine if FI of the supraspinatus muscle after acute RCT relates to short-term changes in muscle function. Study Design: Controlled laboratory study. Methods: Unilateral RCTs were induced in female rabbits via tenotomy of the supraspinatus and infraspinatus. Maximal isometric force and rate of fatigue were measured in the supraspinatus in vivo at 6 and 12 weeks after tenotomy. Computed tomography scanning was performed, followed by histologic analysis of myofiber size, FI, and fibrosis. Results: Tenotomy resulted in supraspinatus weakness, reduced myofiber size, FI, and fibrosis, but no differences were evident between 6 and 12 weeks after tenotomy except for increased collagen content at 12 weeks. FI was a predictor of supraspinatus weakness and was strongly correlated to force, even after accounting for muscle cross-sectional area. While muscle atrophy accounted for the loss in force in tenotomized muscles with minimal FI, it did not account for the greater loss in force in tenotomized muscles with the most FI. Collagen content was not strongly correlated with maximal isometric force, even when normalized to muscle size. Conclusion: After RCT, muscle atrophy results in the loss of contractile force from the supraspinatus, but exacerbated weakness is observed with increased FI. Therefore, the level of FI can help predict contractile function of torn rotator cuff muscles. Clinical Relevance: Markers to predict contractile function of RCTs will help determine the appropriate treatment to improve functional recovery after RCTs.

Ciliary Hedgehog Signaling Restricts Injury-Induced Adipogenesis

Injured skeletal muscle regenerates, but with age orin muscular dystrophies, muscle is replaced by fat. Upon injury, muscle-resident fibro/adipogenic progenitors (FAPs) proliferated and gave rise to adipocytes. These FAPs dynamically produced primary cilia, structures that transduce intercellular cues such as Hedgehog (Hh) signals. Genetically removing cilia from FAPs inhibited intramuscular adipogenesis, both after injury and in a mouse model of Duchenne muscular dystrophy. Blocking FAP ciliation also enhanced myofiber regeneration after injury and reduced myofiber size decline in the muscular dystrophy model. Hh signaling through FAP cilia regulated the expression of TIMP3, a secreted metalloproteinase inhibitor, that inhibited MMP14 to block adipogenesis. A pharmacological mimetic of TIMP3 blocked the conversion of FAPs into adipocytes, pointing to a strategy to combat fatty degeneration of skeletal muscle. We conclude that ciliary Hh signaling by FAPs orchestrates the regenerative response to skeletal muscle injury.

Nur77 deletion impairs muscle growth during developmental myogenesis and muscle regeneration in mice

Muscle atrophy is a prevalent condition in illness and aging. Identifying novel pathways that control muscle mass may lead to therapeutic advancement. We previously identified Nur77 as a transcriptional regulator of glycolysis in skeletal muscle. More recently, we showed that Nur77 expression also controls myofiber size in mice. It was unknown, however, whether Nur77’s regulation of muscle size begins during developmental myogenesis or only in adulthood. To determine the importance of Nur77 throughout muscle growth, we examined myofiber size at E18.5, 3 weeks postnatal age, and in young adult mice. Using the global Nur77-/- mice, we showed that Nur77 deficiency reduced myofiber size as early as E18.5. The reduction in myofiber size became more pronounced by 3 weeks of age. We observed comparable reduction in myofiber size in young myofiber-specific Nur77-knockout mice. These findings suggest that Nur77’s effect on muscle growth is intrinsic to its expression in differentiating myofibers, and not dependent on its expression in myogenic stem cells. To determine the importance of Nur77 expression in muscle accretion in mature mice, we generated an inducible-, muscle-specific, Nur77-deficient mouse model. We demonstrated that tamoxifen-induced deletion of Nur77 in 3-month-old mice reduced myofiber size. This change was accompanied by increased activity of Smad2 and FoxO3, two negative regulators of muscle mass. The role of Nur77 in muscle growth was further elaborated in the cardiotoxin-induced muscle regeneration model. Compared to wildtype mice, regenerated myofibers were smaller in Nur77-/- mice. However, when normalized to saline-injected muscle, the recovery of sarcoplasmic area was comparable between Nur77-/- and wildtype mice. These findings suggest that Nur77 deficiency compromises myofiber growth, but not the regenerative capacity of myogenic progenitor cells. Collectively, the findings presented here demonstrate Nur77 as an important regulator of muscle growth both during prenatal and postnatal myogenesis.

The NAD+-Dependent SIRT1 Deacetylase Translates a Metabolic Switch into Regulatory Epigenetics in Skeletal Muscle Stem Cells

Stem cells undergo a shift in metabolic substrate utilization during specification and/or differentiation, a process that has been termed metabolic reprogramming. Here, we report that during the transition from quiescence to proliferation, skeletal muscle stem cells experience a metabolic switch from fatty acid oxidation to glycolysis. This reprogramming of cellular metabolism decreases intracellular NAD(+) levels and the activity of the histone deacetylase SIRT1, leading to elevated H4K16 acetylation and activation of muscle gene transcription. Selective genetic ablation of the SIRT1 deacetylase domain in skeletal muscle results in increased H4K16 acetylation and deregulated activation of the myogenic program in SCs. Moreover, mice with muscle-specific inactivation of the SIRT1 deacetylase domain display reduced myofiber size, impaired muscle regeneration, and derepression of muscle developmental genes. Overall, these findings reveal how metabolic cues can be mechanistically translated into epigenetic modifications that regulate skeletal muscle stem cell biology.

Oxidative damage in the gastrocnemius of patients with peripheral artery disease is myofiber type selective

BackgroundPeripheral artery disease (PAD), a manifestation of systemic atherosclerosis that produces blockages in the arteries supplying the legs, affects approximately 5% of Americans. We have previously, demonstrated that a myopathy characterized by myofiber oxidative damage and degeneration is central to PAD pathophysiology. ObjectivesIn this study, we hypothesized that increased oxidative damage in the myofibers of the gastrocnemius of PAD patients is myofiber-type selective and correlates with reduced myofiber size. MethodsNeedle biopsies were taken from the gastrocnemius of 53 PAD patients (28 with early PAD and 25 with advanced PAD) and 25 controls. Carbonyl groups (marker of oxidative damage), were quantified in myofibers of slide-mounted tissue, by quantitative fluorescence microscopy. Myofiber cross-sectional area was determined from sarcolemma labeled with wheat germ agglutinin. The tissues were also labeled for myosin I and II, permitting quantification of oxidative damage to and relative frequency of the different myofiber Types (Type I, Type II and mixed Type I/II myofibers). We compared PAD patients in early (N=28) vs. advanced (N=25) disease stage for selective, myofiber oxidative damage and altered morphometrics. ResultsThe carbonyl content of gastrocnemius myofibers was higher in PAD patients compared to control subjects, for all three myofiber types (p<0.05). In PAD patients carbonyl content was higher (p<0.05) in Type II and I/II fibers compared to Type I fibers. Furthermore, the relative frequency and cross-sectional area of Type II fibers were lower, while the relative frequencies and cross-sectional area of Type I and Type I/II fibers were higher, in PAD compared to control gastrocnemius (p<0.05). Lastly, the type II-selective oxidative damage increased and myofiber size decreased as the disease progressed from the early to advanced stage. ConclusionsOur data confirm increased myofiber oxidative damage and reduced myofiber size in PAD gastrocnemius and demonstrate that the damage is selective for type II myofibers and is worse in the most advanced stage of PAD.

Cyclin D3 critically regulates the balance between self-renewal and differentiation in skeletal muscle stem cells

Satellite cells are mitotically quiescent myogenic stem cells resident beneath the basal lamina surrounding adult muscle myofibers. In response to injury, multiple extrinsic signals drive the entry of satellite cells into the cell cycle and then to proliferation, differentiation, and self-renewal of their downstream progeny. Because satellite cells must endure for a lifetime, their cell cycle activity must be carefully controlled to coordinate proliferative expansion and self-renewal with the onset of the differentiation program. In this study, we find that cyclin D3, a member of the family of mitogen-activated D-type cyclins, is critically required for proper developmental progression of myogenic progenitors. Using a cyclin D3-knockout mouse we determined that cyclin D3 deficiency leads to reduced myofiber size and impaired establishment of the satellite cell population within the adult muscle. Cyclin D3-null myogenic progenitors, studied ex vivo on isolated myofibers and in vitro, displayed impaired cell cycle progression, increased differentiation potential, and reduced self-renewal capability. Similarly, silencing of cyclin D3 in C2 myoblasts caused anticipated exit from the cell cycle and precocious onset of terminal differentiation. After induced muscle damage, cyclin D3-null myogenic progenitors exhibited proliferation deficits, a precocious ability to form newly generated myofibers and a reduced capability to repopulate the satellite cell niche at later stages of the regeneration process. These results indicate that cyclin D3 plays a cell-autonomous and nonredundant function in regulating the dynamic balance between proliferation, differentiation, and self-renewal that normally establishes an appropriate pool size of adult satellite cells.

Impaired Insulin/IGF Signaling in Experimental Alcohol-Related Myopathy

Alcohol-related myopathy (Alc-M) is highly prevalent among heavy drinkers, although its pathogenesis is not well understood. We hypothesize that Alc-M is mediated by combined effects of insulin/IGF resistance and oxidative stress, similar to the effects of ethanol on liver and brain. We tested this hypothesis using an established model in which adult rats were pair-fed for 8 weeks with isocaloric diets containing 0% (N = 8) or 35.5% (N = 13) ethanol by caloric content. Gastrocnemius muscles were examined by histology, morphometrics, qRT-PCR analysis, and ELISAs. Chronic ethanol feeding reduced myofiber size and mRNA expression of IGF-1 polypeptide, insulin, IGF-1, and IGF-2 receptors, IRS-1, and IRS-2. Multiplex ELISAs demonstrated ethanol-associated inhibition of insulin, IRS-1, Akt, and p70S6K signaling, and increased activation of GSK-3β. In addition, ethanol-exposed muscles had increased 4-hydroxy-2-nonenal immunoreactivity, reflecting lipid peroxidation, and reduced levels of mitochondrial Complex IV, Complex V, and acetylcholinesterase. These results demonstrate that experimental Alc-M is associated with inhibition of insulin/IGF/IRS and downstream signaling that mediates metabolism and cell survival, similar to findings in alcoholic liver and brain degeneration. Moreover, the increased oxidative stress, which could be mediated by mitochondrial dysfunction, may have led to inhibition of acetylcholinesterase, which itself is sufficient to cause myofiber atrophy and degeneration.

Abcg2 labels multiple cell types in skeletal muscle and participates in muscle regeneration

Skeletal muscle contains progenitor cells (satellite cells) that maintain and repair muscle. It also contains muscle side population (SP) cells, which express Abcg2 and may participate in muscle regeneration or may represent a source of satellite cell replenishment. In Abcg2-null mice, the SP fraction is lost in skeletal muscle, although the significance of this loss was previously unknown. We show that cells expressing Abcg2 increased upon injury and that muscle regeneration was impaired in Abcg2-null mice, resulting in fewer centrally nucleated myofibers, reduced myofiber size, and fewer satellite cells. Additionally, using genetic lineage tracing, we demonstrate that the progeny of Abcg2-expressing cells contributed to multiple cell types within the muscle interstitium, primarily endothelial cells. After injury, Abcg2 progeny made a minor contribution to regenerated myofibers. Furthermore, Abcg2-labeled cells increased significantly upon injury and appeared to traffic to muscle from peripheral blood. Together, these data suggest an important role for Abcg2 in positively regulating skeletal muscle regeneration.

The Transcription Factor ATF4 Promotes Skeletal Myofiber Atrophy during Fasting

Prolonged fasting alters skeletal muscle gene expression in a manner that promotes myofiber atrophy, but the underlying mechanisms are not fully understood. Here, we examined the potential role of activating transcription factor 4 (ATF4), a transcription factor with an evolutionarily ancient role in the cellular response to starvation. In mouse skeletal muscle, fasting increases the level of ATF4 mRNA. To determine whether increased ATF4 expression was required for myofiber atrophy, we reduced ATF4 expression with an inhibitory RNA targeting ATF4 and found that it reduced myofiber atrophy during fasting. Likewise, reducing the fasting level of ATF4 mRNA with a phosphorylation-resistant form of eukaryotic initiation factor 2alpha decreased myofiber atrophy. To determine whether ATF4 was sufficient to reduce myofiber size, we overexpressed ATF4 and found that it reduced myofiber size in the absence of fasting. In contrast, a transcriptionally inactive ATF4 construct did not reduce myofiber size, suggesting a requirement for ATF4-mediated transcriptional regulation. To begin to determine the mechanism of ATF4-mediated myofiber atrophy, we compared the effects of fasting and ATF4 overexpression on global skeletal muscle mRNA expression. Interestingly, expression of ATF4 increased a small subset of five fasting-responsive mRNAs, including four of the 15 mRNAs most highly induced by fasting. These five mRNAs encode proteins previously implicated in growth suppression (p21(Cip1/Waf1), GADD45alpha, and PW1/Peg3) or titin-based stress signaling [muscle LIM protein (MLP) and cardiac ankyrin repeat protein (CARP)]. Taken together, these data identify ATF4 as a novel mediator of skeletal myofiber atrophy during starvation.

Microarray Analysis Reveals Up-Regulation of Retinoic Acid and Hepatocyte Growth Factor Related Signaling Pathways by Pro-Insulin C-Peptide in Kidney Proximal Tubular Cells: Antagonism of the Pro-Fibrotic Effects of TGF-β1

Microarray Analysis Reveals Up-Regulation of Retinoic Acid and Hepatocyte Growth Factor Related Signaling Pathways by Pro-Insulin C-Peptide in Kidney Proximal Tubular Cells: Antagonism of the Pro-Fibrotic Effects of TGF-β1

The Transcription Factor ATF4 Promotes Skeletal Myofiber Atrophy during Fasting

Prolonged fasting alters skeletal muscle gene expression in a manner that promotes myofiber atrophy, but the underlying mechanisms are not fully understood. Here, we examined the potential role of activating transcription factor 4 (ATF4), a transcription factor with an evolutionarily ancient role in the cellular response to starvation. In mouse skeletal muscle, fasting increases the level of ATF4 mRNA. To determine whether increased ATF4 expression was required for myofiber atrophy, we reduced ATF4 expression with an inhibitory RNA targeting ATF4 and found that it reduced myofiber atrophy during fasting. Likewise, reducing the fasting level of ATF4 mRNA with a phosphorylation-resistant form of eukaryotic initiation factor 2α decreased myofiber atrophy. To determine whether ATF4 was sufficient to reduce myofiber size, we overexpressed ATF4 and found that it reduced myofiber size in the absence of fasting. In contrast, a transcriptionally inactive ATF4 construct did not reduce myofiber size, suggesting a re...