Leslie M. Baehr

The glucocorticoid receptor and FOXO1 synergistically activate the skeletal muscle atrophy-associated MuRF1 gene

The muscle specific ubiquitin E3 ligase MuRF1 has been implicated as a key regulator of muscle atrophy under a variety of conditions, such as during synthetic glucocorticoid treatment. FOXO class transcription factors have been proposed as important regulators of MuRF1 expression, but its regulation by glucocorticoids is not well understood. The MuRF1 promoter contains a near-perfect palindromic glucocorticoid response element (GRE) 200 base pairs upstream of the transcription start site. The GRE is highly conserved in the mouse, rat, and human genes along with a directly adjacent FOXO binding element (FBE). Transient transfection assays in HepG2 cells and C(2)C(12) myotubes demonstrate that the MuRF1 promoter is responsive to both the dexamethasone (DEX)-activated glucocorticoid receptor (GR) and FOXO1, whereas coexpression of GR and FOXO1 leads to a dramatic synergistic increase in reporter gene activity. Mutation of either the GRE or the FBE significantly impairs activation of the MuRF1 promoter. Consistent with these findings, DEX-induced upregulation of MuRF1 is significantly attenuated in mice expressing a homodimerization-deficient GR despite no effect on the degree of muscle loss in these mice vs. their wild-type counterparts. Finally, chromatin immunoprecipitation analysis reveals that both GR and FOXO1 bind to the endogenous MuRF1 promoter in C(2)C(12) myotubes, and IGF-I inhibition of DEX-induced MuRF1 expression correlates with the loss of FOXO1 binding. These findings present new insights into the role of the GR and FOXO family of transcription factors in the transcriptional regulation of the MuRF1 gene, a direct target of the GR in skeletal muscle.

Skeletal muscle atrophy and the E3 ubiquitin ligases MuRF1 and MAFbx/atrogin-1

Muscle RING finger 1 (MuRF1) and muscle atrophy F-box (MAFbx)/atrogin-1 were identified more than 10 years ago as two muscle-specific E3 ubiquitin ligases that are increased transcriptionally in skeletal muscle under atrophy-inducing conditions, making them excellent markers of muscle atrophy. In the past 10 years much has been published about MuRF1 and MAFbx with respect to their mRNA expression patterns under atrophy-inducing conditions, their transcriptional regulation, and their putative substrates. However, much remains to be learned about the physiological role of both genes in the regulation of mass and other cellular functions in striated muscle. Although both MuRF1 and MAFbx are enriched in skeletal, cardiac, and smooth muscle, this review will focus on the current understanding of MuRF1 and MAFbx in skeletal muscle, highlighting the critical questions that remain to be answered.

Muscle sparing in muscle RING finger 1 null mice: response to synthetic glucocorticoids

Non‐Technical Summary  Skeletal muscle has the capacity to modify its size in response to external cues such as mechanical load, neural activity, hormones, stress and nutritional status. Pathological muscle loss or ‘atrophy’ occurs as the result of a number of disparate conditions including ageing, immobilization, diabetes, cancer, sepsis and as a serious side effect of corticosteroid hormone treatment. Synthetic glucocorticoids are often used to treat inflammation; however, high doses and chronic use of these hormones can lead to the loss of skeletal muscle mass and weakness. We show that in mice with a deletion of the MuRF1 protein, but not the MAFbx protein, the loss of muscle mass is attenuated relative to normal mice following 14 days of glucocorticoid treatment. Knowledge of how the MuRF1 protein functions in skeletal muscle to regulate skeletal muscle mass could lead to the development of therapeutics to prevent muscle atrophy under various conditions including glucocorticoid treatment.

A cell-autonomous role for the glucocorticoid receptor in skeletal muscle atrophy induced by systemic glucocorticoid exposure

Glucocorticoids (GCs) are important regulators of skeletal muscle mass, and prolonged exposure will induce significant muscle atrophy. To better understand the mechanism of skeletal muscle atrophy induced by elevated GC levels, we examined three different models: exogenous synthetic GC treatment [dexamethasone (DEX)], nutritional deprivation, and denervation. Specifically, we tested the direct contribution of the glucocorticoid receptor (GR) in skeletal muscle atrophy by creating a muscle-specific GR-knockout mouse line (MGR(e3)KO) using Cre-lox technology. In MGR(e3)KO mice, we found that the GR is essential for muscle atrophy in response to high-dose DEX treatment. In addition, DEX regulation of multiple genes, including two important atrophy markers, MuRF1 and MAFbx, is eliminated completely in the MGR(e3)KO mice. In a condition where endogenous GCs are elevated, such as nutritional deprivation, induction of MuRF1 and MAFbx was inhibited, but not completely blocked, in MGR(e3)KO mice. In response to sciatic nerve lesion and hindlimb muscle denervation, muscle atrophy and upregulation of MuRF1 and MAFbx occurred to the same extent in both wild-type and MGR(e3)KO mice, indicating that a functional GR is not required to induce atrophy under these conditions. Therefore, we demonstrate conclusively that the GR is an important mediator of skeletal muscle atrophy and associated gene expression in response to exogenous synthetic GCs in vivo and that the MGR(e3)KO mouse is a useful model for studying the role of the GR and its target genes in multiple skeletal muscle atrophy models.

Maintenance of muscle mass and load‐induced growth in Muscle RING Finger 1 null mice with age

Age‐related loss of muscle mass occurs to varying degrees in all individuals and has a detrimental effect on morbidity and mortality. Muscle RING Finger 1 (MuRF1), a muscle‐specific E3 ubiquitin ligase, is believed to mediate muscle atrophy through the ubiquitin proteasome system (UPS). Deletion of MuRF1 (KO) in mice attenuates the loss of muscle mass following denervation, disuse, and glucocorticoid treatment; however, its role in age‐related muscle loss is unknown. In this study, skeletal muscle from male wild‐type (WT) and MuRF1 KO mice was studied up to the age of 24 months. Muscle mass and fiber cross‐sectional area decreased significantly with age in WT, but not in KO mice. In aged WT muscle, significant decreases in proteasome activities, especially 20S and 26S β5 (20–40% decrease), were measured and were associated with significant increases in the maladaptive endoplasmic reticulum (ER) stress marker, CHOP. Conversely, in aged MuRF1 KO mice, 20S or 26S β5 proteasome activity was maintained or decreased to a lesser extent than in WT mice, and no increase in CHOP expression was measured. Examination of the growth response of older (18 months) mice to functional overload revealed that old WT mice had significantly less growth relative to young mice (1.37‐ vs. 1.83‐fold), whereas old MuRF1 KO mice had a normal growth response (1.74‐ vs. 1.90‐fold). These data collectively suggest that with age, MuRF1 plays an important role in the control of skeletal muscle mass and growth capacity through the regulation of cellular stress.

Muscle hypertrophy is associated with increases in proteasome activity that is independent of MuRF1 and MAFbx expression

The regulation of skeletal muscle mass depends on the balance between protein synthesis and degradation. The role of protein degradation and in particular, the ubiquitin proteasome system, and increased expression of the E3 ubiquitin ligases, MuRF1 and MAFbx/atrogin-1, in the regulation of muscle size in response to growth stimuli is unclear. Thus, the aim of this study was to measure both proteasome activity and protein synthesis in mice over a 14-day period of chronic loading using the functional overload (FO) model. Further, the importance of MuRF1 and MAFbx expression in regulating muscle hypertrophy was examined by measuring muscle growth in response to FO in mice with a null deletion (KO) of either MuRF1 or MAFbx. In wild type (WT) mice, the increase in muscle mass correlated with significant increases (2-fold) in protein synthesis at 7 and 14 days. Interestingly, proteasome activity significantly increased in WT mice after one day, and continued to increase, peaking at 7 days following FO. The increase in proteasome activity was correlated with increases in the expression of the Forkhead transcription factors, FOXO1 and FOXO3a, which increased after both MuRF1 and MAFbx increased and returned to baseline. As in WT mice, hypertrophy in the MuRF1 and MAFbx KO mice was associated with significant increases in proteasome activity after 14 days of FO. The increase in plantaris mass was similar between the WT and MuRF1 KO mice following FO, however, muscle growth was significantly reduced in female MAFbx KO mice. Collectively, these results indicate that muscle hypertrophy is associated with increases in both protein synthesis and degradation. Further, MuRF1 or MAFbx expression is not required to increase proteasome activity following increased loading, however, MAFbx expression may be required for proper growth/remodeling of muscle in response to increase loading.

Acute resistance exercise activates rapamycin‐sensitive and insensitive mechanisms that control translational activity and capacity in skeletal muscle

Ribosome biogenesis is the primary determinant of translational capacity, but its regulation in skeletal muscle following acute resistance exercise is poorly understood. Resistance exercise increases muscle protein synthesis acutely, and muscle mass with training, but the role of translational capacity in these processes is unclear. Here, we show that acute resistance exercise activated pathways controlling translational activity and capacity through both rapamycin‐sensitive and ‐insensitive mechanisms. Transcription factor c‐Myc and its downstream targets, which are known to regulate ribosome biogenesis in other cell types, were upregulated after resistance exercise in a rapamycin‐independent manner and may play a role in determining translational capacity in skeletal muscle. Local inhibition of myostatin was also not affected by rapamycin and may contribute to the rapamycin‐independent effects of resistance exercise.

Altered gene expression patterns in muscle ring finger 1 null mice during denervation- and dexamethasone-induced muscle atrophy

Muscle atrophy can result from inactivity or unloading on one hand or the induction of a catabolic state on the other. Muscle-specific ring finger 1 (MuRF1), a member of the tripartite motif family of E3 ubiquitin ligases, is an essential mediator of multiple conditions inducing muscle atrophy. While most studies have focused on the role of MuRF1 in protein degradation, the protein may have other roles in regulating skeletal muscle mass and metabolism. We therefore systematically evaluated the effect of MuRF1 on gene expression during denervation and dexamethasone-induced atrophy. We find that the lack of MuRF1 leads to few differences in control animals, but there were several significant differences in specific sets of genes upon denervation- and dexamethasone-induced atrophy. For example, during denervation, MuRF1 knockout mice showed delayed repression of metabolic and structural genes and blunted induction of genes associated with the neuromuscular junction. In the latter case, this pattern correlates with blunted HDAC4 and myogenin upregulation. Lack of MuRF1 caused fewer changes in the dexamethasone-induced atrophy program, but certain genes involved in fat metabolism and intracellular signaling were affected. Our results demonstrate a new role for MuRF1 in influencing gene expression in two important models of muscle atrophy.

Role of contraction duration in inducing fast‐to‐slow contractile and metabolic protein and functional changes in engineered muscle

The role of factors such as frequency, contraction duration and active time in the adaptation to chronic low‐frequency electrical stimulation (CLFS) is widely disputed. In this study we explore the ability of contraction duration (0.6, 6, 60, and 600 sec) to induce a fast‐to‐slow shift in engineered muscle while using a stimulation frequency of 10 Hz and keeping active time constant at 60%. We found that all contraction durations induced similar slowing of time‐to‐peak tension. Despite similar increases in total myosin heavy (MHC) levels with stimulation, increasing contraction duration resulted in progressive decreases in total fast myosin. With contraction durations of 60 and 600 sec, MHC IIx levels decreased and MHC IIa levels increased. All contraction durations resulted in fast‐to‐slow shifts in TnT and TnC but increased both fast and slow TnI levels. Half‐relaxation slowed to a greater extent with contraction durations of 60 and 600 sec despite similar changes in the calcium sequestering proteins calsequestrin and parvalbumin and the calcium uptake protein SERCA. All CLFS groups resulted in greater fatigue resistance than control. Similar increases in GLUT4, mitochondrial enzymes (SDH and ATPsynthase), the fatty acid transporter CPT‐1, and the metabolic regulators PGC‐1α and MEF2 were found with all contraction durations. However, the mitochondrial enzymes cytochrome C and citrate synthase were increased to greater levels with contraction durations of 60 and 600 sec. These results demonstrate that contraction duration plays a pivotal role in dictating the level of CLFS‐induced contractile and metabolic adaptations in tissue‐engineered skeletal muscle. J. Cell. Physiol. 230: 2489–2497, 2015.

Age-related Differences in Dystrophin: Impact on Force Transfer Proteins, Membrane Integrity, and Neuromuscular Junction Stability

The loss of muscle strength with age has been studied from the perspective of a decline in muscle mass and neuromuscular junction (NMJ) stability. A third potential factor is force transmission. The purpose of this study was to determine the changes in the force transfer apparatus within aging muscle and the impact on membrane integrity and NMJ stability. We measured an age-related loss of dystrophin protein that was greatest in the flexor muscles. The loss of dystrophin protein occurred despite a twofold increase in dystrophin mRNA. Importantly, this disparity could be explained by the four- to fivefold upregulation of the dystromir miR-31. To compensate for the loss of dystrophin protein, aged muscle contained increased α-sarcoglycan, syntrophin, sarcospan, laminin, β1-integrin, desmuslin, and the Z-line proteins α-actinin and desmin. In spite of the adaptive increase in other force transfer proteins, over the 48 hours following lengthening contractions, the old muscles showed more signs of impaired membrane integrity (fourfold increase in immunoglobulin G-positive fibers and 70% greater dysferlin mRNA) and NMJ instability (14- to 96-fold increases in Runx1, AchRδ, and myogenin mRNA). Overall, these data suggest that age-dependent alterations in dystrophin leave the muscle membrane and NMJ more susceptible to contraction-induced damage even before changes in muscle mass are obvious.