David L. Allen

Hat its inhibition can significantly attenuate the progression of obesity and diabetes. Although at least part of these effects on metabolism can be attributable to myostatin's influence over skeletal muscle growth and therefore on the total volume of metabolically active lean body mass, there is mounting evidence that myostatin affects the growth and metabolic state of other tissues, including the adipose and the liver. In addition, recent work has explored the role of myostatin in substrate mobilization, uptake, and/or utilization of muscle independent of its effects on body composition. Finally, the effects of both endurance and resistance exercise on myostatin expression, as well as the potential role of myostatin in the beneficial metabolic adaptations occurring in response to exercise, have also begun to be delineated in greater detail. The purpose of this review was to summarize the work to date on the expression and function of myostatin in obesity, diabetes, and exercise adaptation....

Matrix metalloproteinases (MMPs) play a major role in the degradation of the extracellular matrix (ECM) of skeletal muscle, and the inducible gelatinase MMP-9 in particular appears to be critical for the remodeling of muscle ECM during growth and repair. Here we determined the effects of MMP-9 gene inactivation on fiber type and size in the tibialis anterior (TA), gastrocnemius (GAST), and soleus (SOL) muscles in female mice. In the TA, the cross-sectional area (CSA) of the myosin heavy chain (MyHC) IIb-expressing fibers was significantly smaller in MMP-9 null mice while in the GAST, CSA of all three fast fiber types was decreased. In the SOL, MyHC type I-expressing fibers were significantly smaller in the MMP-9 null mice. The percentage of MyHC type IIb-expressing fibers was significantly increased in the TA and GAST of MMP-9 null mice, while the percentage of MyHC IId-expressing fibers significantly decreased in the GAST of MMP-9 null mice. Fiber percentages in the SOL were not significantly different between the two lines. Despite these changes in fiber size and type, in vivo hindlimb force production was not changed in MMP-9 null mice. Meanwhile, neither expression of the constitutive gelatinase MMP-2 nor immunohistochemical staining for type IV collagen was significantly altered by MMP-9 inactivation in any muscles examined. The present study demonstrates that MMP-9 inactivation results in changes in fiber size and type in adult mouse hindlimb muscles that may depend on indirect mechanisms involving reduced bone growth or nerve changes in response to MMP-9 inactivation.

Expression of the antigrowth factor myostatin (MSTN) differs between fast and slow skeletal muscles and is increased in nearly every form of muscle atrophy, but the contribution of transcriptional vs. posttranscriptional mechanisms to its differing expression in these states has not been defined. We show here that levels of mature MSTN mRNA were sixfold greater in fast vs. slow muscle and were increased twofold in fast muscle in response to dexamethasone (Dex) injection in vivo and in C₂C₁₂ myotubes following Dex treatment in vitro, but that levels of MSTN pre-mRNA, a readout of transcription, only minimally and nonsignificantly differed in these states. Moreover, Dex treatment with or without cotransfection with a glucocorticoid receptor expression construct had only modest effects on mouse MSTN promoter activity in C₂C₁₂ myotubes. We therefore explored the potential contribution of posttranscriptional mechanisms, and the role of the microRNAs miR-27a and b in particular, on MSTN expression. The MSTN 3′-untranslated region (UTR) contains a putative recognition sequence for miR-27a and b that is conserved across a wide range of vertebrate species. Cotransfection of a MSTN 3′-UTR-luciferase construct with a miR-27b expression construct significantly attenuated by approximately half while mutation of the miR-27 recognition sequence significantly increased by approximately twofold the activity of a MSTN 3′-UTR construct and decreased mRNA degradation of a luciferase reporter construct in C₂C₁₂ myotubes. Expression of miR-27a and b was almost sixfold greater in slow-twitch than in fast-twitch muscle in vivo, and miR-27a expression was significantly decreased by nearly half by glucocorticoid treatment in vitro. Finally, the miR-27a and b promoters were activated by cotransfection with the slow-specific signaling molecules calcineurin and peroxisome proliferator-activated receptor-γ coactivator-1α. The present data represent the first demonstration that posttranscriptional mechanisms involving miR-27a and b may contribute to fast-specific and glucocorticoid-dependent myostatin expression in muscle.

We recently demonstrated that mRNA levels of three members of the CCAAT/enhancer binding factor (C/EBP) family of transcription factors are increased in skeletal muscle following 12 days of spaceflight. In the present study, we further explored the expression of C/EBP-δ in atrophying fast skeletal muscle by examining its expression in muscle from food-deprived (FD) mice, and investigated its role in regulating the expression of the secreted antigrowth factor myostatin. C/EBP-δ mRNA and protein levels were significantly increased by 2 days of food deprivation in the tibialis anterior (TA) muscle, and expression of both myostatin and C/EBP-δ mRNA during food deprivation was attenuated by injection with the glucocorticoid inhibitor RU486. The increase in myostatin mRNA levels with food deprivation appears to be at least partially transcriptionally driven, since levels of myostatin pre-mRNA were significantly increased in the TA muscle. C/EBP-δ mRNA levels and promoter activity were significantly increased by transfection of C₂C₁₂ myotubes with a glucocorticoid receptor construct and 24 h of treatment with the synthetic glucocorticoid dexamethasone. Furthermore, activity of the C/EBP-δ promoter was significantly increased with as little as 1 h of dexamethasone treatment, while activity of the mouse myostatin promoter was only significantly increased with longer treatment periods of 24 h or more. Activity of the myostatin promoter-reporter construct was significantly increased in C₂C₁₂ myotubes by cotransfection with expression constructs for C/EBP-α, -β, and -δ, with C/EBP-δ having the greatest effect. The myostatin promoter contains two potential C/EBP binding sequences, a CCAAT box, and a C/EBP binding element (CBE). Mutation of the CCAAT box attenuated basal myostatin promoter activity but potentiated C/EBP-δ-activated myostatin promoter activity in C₂C₁₂ myotubes in vitro, while mutation of the CBE abolished glucocorticoid receptor and C/EBP-δ responsiveness. The present results support a model in which glucocorticoid-induced increases in C/EBP-δ expression may contribute to myostatin transcription during atrophic states.

Psychological stress is known to attenuate body size and lean body mass. We tested the effects of 1, 3, or 7 days of two different models of psychological stress, 1 h of daily restraint stress (RS) or daily cage-switching stress (CS), on skeletal muscle size and atrophy-associated gene expression in mice. Thymus weights decreased in both RS and CS mice compared with unstressed controls, suggesting that both models activated the hypothalamic-pituitary-adrenal axis. Body mass was significantly decreased at all time points for both models of stress but was greater for RS than CS. Mass of the tibialis anterior (TA) and soleus (SOL) muscles was significantly decreased after 3 and 7 days of RS, but CS only significantly decreased SOL mass after 7 days. TA mRNA levels of the atrophy-associated genes myostatin (MSTN), atrogin-1, and the phosphatidylinositol 3-kinase inhibitory subunit p85α were all significantly increased relative to unstressed mice after 1 and 3 days of RS, and expression of MSTN and p85α mRNA remained elevated after 7 days of RS. Expression of muscle ring finger 1 was increased after 1 day of RS but returned to baseline at 3 and 7 days of RS. MSTN, atrogin-1, and p85α mRNA levels also significantly increased after 1 and 3 days of CS but atrogen-1 mRNA levels had resolved back to normal levels by 3 days and p85α with 7 days of CS. p21CIP mRNA levels were significantly decreased by 3 days of CS or RS. Finally, body mass was minimally affected, and muscle mass was completely unaffected by 3 days of RS in mice null for the MSTN gene, and MSTN inactivation attenuated the increase in atrogin-1 mRNA levels with 4 days of RS compared with wild-type mice. Together these data suggest that acute daily psychological stress induces atrophic gene expression and loss of muscle mass that appears to be MSTN dependent.

During food deprivation (FD), skeletal muscle protein is broken down to produce amino acids for hepatic gluconeogenesis to maintain blood glucose levels. However, it is unclear what role, if any, the secreted antigrowth factor myostatin (MSTN) plays in the muscle atrophy induced by FD. We therefore examined expression and function of MSTN in FD in mice. Two days of FD significantly decreased muscle mass and protein content and increased mRNA levels of ubiquitin ligases MuRF-1 and atrogin-1 in fast-twitch tibialis anterior (TA) muscle but not slow-twitch soleus (Sol) muscle, while 2 days of refeeding returned these to fed values in TA. MSTN mRNA levels were significantly increased approximately threefold by 2 days, but not 1 day, of FD and returned to fed levels with 2 days of refeeding in TA but were not significantly affected by FD or refeeding in Sol. TA mass decreased to a similar amount after 1 day of FD in wild-type mice and mice null for the MSTN gene but was decreased to a greater amount in wild-type than MSTN-null mice by 2 days of FD. In addition, blood glucose levels decreased and corticosterone levels increased to a greater extent in MSTN-null mice after 2 days of FD, but surprisingly muscle MuRF-1 and atrogin-1 mRNA levels were not affected by the lack of MSTN during FD. Similarly, changes in hepatic enzyme expression in response to FD were identical between wild-type and MSTN-null mice. Our data are consistent with the hypothesis that MSTN is dispensable for the initial atrophy occurring in response to FD but attenuates the decrease in fast-twitch muscle mass during prolonged FD.

Expression of the cytokine interleukin-6 (IL-6) by skeletal muscle is hugely increased in response to a single bout of endurance exercise, and this appears to be mediated by increases in intracellular calcium. We examined the effects of endurance exercise on IL-6 mRNA levels and promoter activity in skeletal muscle in vivo, and the role of the calcium-activated calcineurin signaling pathway on muscle IL-6 expression in vivo and in vitro. IL-6 mRNA levels in the mouse tibialis anterior (TA) were increased 2–10-fold by a single bout of treadmill exercise or by 3 days of voluntary wheel running. Moreover, an IL-6 promoter-driven luciferase transgene was activated in TA by both treadmill and wheel-running exercise and by injection with a calcineurin plasmid. Exercise also increased muscle mRNA expression of the calcineurin regulatory gene MCIP1, as did treatment of C₂C₁₂myotubes with the calcium ionophore A23187. Cotransfection of C₂C₁₂myotubes with a constitutively active calcineurin construct significantly increased while cotransfection with the calcineurin inhibitor CAIN inhibited activity of a mouse IL-6 promoter-reporter construct. Cotransfection with a myocyte enhancer-factor-2 (MEF-2) expression construct increased basal IL-6 promoter activity and augmented the effects of calcineurin cotransfection, while cotransfection with the MEF-2 antagonist MITR repressed calcineurin-activated IL-6 promoter activity in vitro. Surprisingly, cotransfection with a dominant-negative form of another calcineurin-activated transcription factor, nuclear factor activator of T cells (NFAT), greatly potentiated both basal and calcineurin-stimulated IL-6 promoter activity in C₂C₁₂myotubes. Mutation of the MEF-2 DNA binding sites attenuated, while mutation of the NFAT DNA binding sites potentiated basal and calcineurin-activated IL-6 promoter activity. Finally, CREB and C/EBP were necessary for basal IL-6 promoter activity and sufficient to increase IL-6 promoter activity but had minimal roles in calcineurin-activated IL-6 promoter activity. Together, these results suggest that IL-6 transcription in skeletal muscle cells can be activated by a calcineurin-MEF-2 axis which is antagonized by NFAT.

For over 40 years, our primary understanding of how eukaryotic gene transcription is regulated has centered on the promoter concept. In this model, transcriptional activators bind to consensus sequences in the proximal upstream regulatory region of a gene, altering chromatin structure to allow recruitment of the RNA polymerase complex to a promoter sequence, which then creates a complementary transcript of the negative strand ([Fig. 1 A ][1]). However, many genes in eukaryotes exist in gene clusters formed via gene duplication, and a key question centers on how coordinated regulation of expression of genes within these clusters occurs. Specifically, gene isoform switching within a given cell or tissue in response to developmental, physiological, or pathological stimuli often requires sequential temporal and/or spatial expression of adjacent genes; thus, expression of two or more genes must be coordinated, which requires an additional level of integration. Locus control regions (LCRs), or groups of regulatory elements upstream of the gene cluster that regulate the sequential activation of the genes in the cluster, represent one mechanism by which this occurs ([Fig. 1 B ][1]). LCRs have been identified for several key gene clusters, including the globin and interleukin genes ([5][2]). Rinaldi et al. ([14][3]) provide further evidence for another mechanism regulating isoform switching in a set of clustered genes, the striated muscle myosin heavy chain (MHC) genes. Eight MHC genes are expressed in cardiac and skeletal muscle and are clustered in two groups: the predominantly cardiac-expressed β- and α-MHC genes are found in this order on chromosome 14 in the mouse and humans and chromosome 15 in the rat, while six skeletal-specific isoforms are found clustered on chromosome 10 in the rat, chromosome 11 in the mouse, and chromosome 17 in humans ([18][4]). Differential expression of these isoforms in both heart and skeletal muscle occurs in response to a number of developmental, neural, hormonal, and mechanical stimuli, but the mechanisms coordinating this transcriptional switching have remained obscure. In previous work ([7][5]), this group demonstrated that switches between the predominantly cardiac-expressed β- and α-MHC genes occur via an intergenic bidirectional promoter ([Fig. 1 C ][1]) that simultaneously produces a sense α-transcript, as well as an antisense β-transcript that decreases β-MHC expression. Previous investigators had identified the presence of antisense MHC transcripts in the heart ([4][6], [10][7]–[12][8]), but it was not clear what, if any, function these had. The paper by Haddad et al. ([7][5]) demonstrated that increased activity of this bidirectional promoter increases expression of α- while decreasing expression of β-MHC by increasing the sense and antisense transcripts for these loci, respectively. These authors have subsequently shown that this mechanism contributes to the α- and β-MHC switching occurring in the heart in response to a wide variety of physiological and pathological conditions, including development ([9][9]), hypo- and hyperthyroidism ([6][10], [7][5], [9][9]), pressure overload ([8][11]), and diabetes ([6][10], [7][5]). The present work extends this work by examining whether a bidirectional intergenic sense/antisense promoter exists between the IIx and IIb skeletal muscle MHC genes. Three “fast” MHC isoforms, IIa, IIx and IIb, are expressed in adult skeletal muscle in the rodent. Switching between these three MHC isoforms can occur in response to changes in activity levels, with increased activity favoring a switch toward type IIa and reduced activity favoring a switch toward type IIb; these shifts always occur sequentially through the three isoforms in the order IIa-IIx-IIb, which is the order of these genes in the cluster. In a previous paper, this group showed that an intergenic bidirectional promoter exists between the IIa and IIx MHC genes that inactivates IIa and activates IIx MHC expression during conditions of muscle atrophy in response to decreased usage ([13][12]). The present paper expands upon this previous work in two ways: it examines whether a second bidirectional intergenic promoter exists between the IIx and IIb MHC genes, and if so, whether it can account for the shift from IIb to IIx occurring in response to models of increased muscle activity. Using a model of muscle stimulation simulating a 4-day regimen of resistance exercise training in rats, Rinaldi et al. ([14][3]) show that levels of IIx pre-mRNA and mature mRNA are increased while levels of IIb pre-mRNA and mature mRNA are decreased. Moreover, using strand-specific primers, they demonstrate that an antisense RNA spanning all of the IIx MHC gene and extending into the 3′ end of the IIa MHC gene is present in control rats but is suppressed following resistance training. Sequence analysis identified an evolutionarily conserved region between the IIx and IIb MHC genes that contains several putative consensus binding sequences that may function as the sense/antisense promoter for producing these two transcripts ([14][3]). Thus this work supports the hypothesis that intergenic bidirectional promoter activity is a strong contributor to regulation of the skeletal MHC gene cluster as a whole in response to both increased and decreased muscle activity and may be another mechanism for regulating gene switching among clusters in general. Other examples of antisense RNA production or “opposite strand transcription” have been reported in other systems ([17][13]), including the hox genes ([16][14]), and thus, this may represent a fairly common mechanism for regulating expression of genes and/or switching between adjacent isoform genes. However, several questions have yet to be addressed. First, the mechanisms by which the antisense RNA interferes with gene expression have yet to be...