Regulatory elements governing transcription in specialized myofiber subtypes

Skeletal myofibers of vertebrates acquire specialized metabolic and physiological properties as a consequence of developmental cues in the embryo and different patterns of contractile activity in the adult. The myoglobin gene is regulated stringently in muscle fibers, such that high myoglobin expression is observed in mitochondria-rich, oxidative myofibers (Types I and IIa) compared with glycolytic fibers (Type IIb). Using germ-line transgenesis and somatic cell gene transfer methods, we defined discrete regions of the murine and human genes encoding myoglobin that are sufficient to confer muscle- and fiber type-specific expression to reporter genes. Mutational analysis confirms the importance of A/T-rich, MEF2-binding motifs in myoglobin gene regulation, as suggested by previous studies using different experimental approaches. In addition, we demonstrated a previously unsuspected role for an intragenic E-box motif as a negative regulatory element contributing to the tightly regulated variation in myoglobin gene expression among particular myofiber subtypes.     

Calcineurin is necessary for the maintenance but not embryonic development of slow muscle fibers

Skeletal muscles are a mosaic of slow and fast twitch myofibers. During embryogenesis, patterns of fiber type composition are initiated that change postnatally to meet physiological demand. To examine the role of the protein phosphatase calcineurin in the initiation and maintenance of muscle fiber types, we used a "Flox-ON" approach to obtain muscle-specific overexpression of the modulatory calcineurin-interacting protein 1 (MCIP1/DSCR1), an inhibitor of calcineurin. Myo-Cre transgenic mice with early skeletal muscle-specific expression of Cre recombinase were used to activate the Flox-MCIP1 transgene. Contractile components unique to type 1 slow fibers were absent from skeletal muscle of adult Myo-Cre/Flox-MCIP1 mice, whereas oxidative capacity, myoglobin content, and mitochondrial abundance were unaltered. The soleus muscles of Myo-Cre/Flox-MCIP1 mice fatigued more rapidly than the wild type as a consequence of the replacement of the slow myosin heavy chain MyHC-1 with a fast isoform, MyHC-2A. MyHC-1 expression in Myo-Cre/Flox-MCIP1 embryos and early neonates was normal. These results demonstrate that developmental patterning of slow fibers is independent of calcineurin, while the maintenance of the slow-fiber phenotype in the adult requires calcineurin activity.     

Skeletal muscle Nur77 expression enhances oxidative metabolism and substrate utilization

Mitochondrial dysfunction has been implicated in the pathogenesis of type 2 diabetes. Identifying novel regulators of mitochondrial bioenergetics will broaden our understanding of regulatory checkpoints that coordinate complex metabolic pathways. We previously showed that Nur77, an orphan nuclear receptor of the NR4A family, regulates the expression of genes linked to glucose utilization. Here we demonstrate that expression of Nur77 in skeletal muscle also enhances mitochondrial function. We generated MCK-Nur77 transgenic mice that express wild-type Nur77 specifically in skeletal muscle. Nur77-overexpressing muscle had increased abundance of oxidative muscle fibers and mitochondrial DNA content. Transgenic muscle also exhibited enhanced oxidative metabolism, suggestive of increased mitochondrial activity. Metabolomic analysis confirmed that Nur77 transgenic muscle favored fatty acid oxidation over glucose oxidation, mimicking the metabolic profile of fasting. Nur77 expression also improved the intrinsic respiratory capacity of isolated mitochondria, likely due to the increased abundance of complex I of the electron transport chain. These changes in mitochondrial metabolism translated to improved muscle contractile function ex vivo and improved cold tolerance in vivo. Our studies outline a novel role for Nur77 in the regulation of oxidative metabolism and mitochondrial activity in skeletal muscle.     

Developmentally regulated expression of three slow isoforms of myosin heavy chain: diversity among the first fibers to form in avian muscle.

At least three slow myosin heavy chain (MHC) isoforms were expressed in skeletal muscles of the developing chicken hindlimb, and differential expression of these slow MHC isoforms produced distinct fiber types from the outset of skeletal muscle myogenesis. Immunohistochemistry with isoform-specific monoclonal antibodies demonstrated differences in MHC content among the fibers of the dorsal and ventral premuscle masses and distinctions among fibers before splitting of the premuscle masses into individual muscles (Hamburger and Hamilton Stage 25). Immunoblot analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of myosin extracted from the hindlimb demonstrated the presence throughout development of different mobility classes of MHCs with epitopes associated with slow MHC isoforms. Immunopeptide mapping showed that one of the MHCs expressed in the embryonic limb was the same slow MHC isoform, slow MHC1 (SMHC1), that is expressed in adult slow muscles. SMHC1 was expressed in the dorsal and ventral premuscle masses, embryonic, fetal, and some neonatal and adult hindlimb muscles. In the embryo and fetus SMHC1 was expressed in future fast, as well as future slow muscles, whereas in the adult only the slow muscles retained expression of SMHC1. Those embryonic muscles destined in the adult to contain slow fibers or mixed fast/slow fibers not only expressed SMHC1, but also an additional slow MHC not previously described, designated as slow MHC3 (SMHC3). Slow MHC3 was shown by immunopeptide mapping to contain a slow MHC epitope (reactive with mAb S58) and to be structurally similar to a MHC expressed in the atria of the adult chicken heart. SMHC3 was designated as a slow MHC isoform because (i) it was expressed only in those muscles destined to be of the slow type in the adult, (ii) it was expressed only in primary fibers of muscles that subsequently are of the slow type, and (iii) it had an epitope demonstrated to be present on other slow, but not fast, isoforms of avian MHC. This study demonstrates that a difference in phenotype between fibers is established very early in the chicken embryo and is based on the fiber type-specific expression of three slow MHC isoforms.     

Deletion of Kinin B2 Receptor Alters Muscle Metabolism and Exercise Performance

Metabolic syndrome is a cluster of metabolic risk factors such as obesity, diabetes and cardiovascular diseases. Mitochondria is the main site of ATP production and its dysfunction leads to decreased oxidative phosphorylation, resulting in lipid accumulation and insulin resistance. Our group has demonstrated that kinins can modulate glucose and lipid metabolism as well as skeletal muscle mass. By using B2 receptor knockout mice (B2R^-/-) we investigated whether kinin action affects weight gain and physical performance of the animals. Our results show that B2R^-/- mice are resistant to high fat diet-induced obesity, have higher glucose tolerance as well as increased mitochondrial mass. These features are accompanied by higher energy expenditure and a lower feed efficiency associated with an increase in the proportion of type I fibers and intermediary fibers characterized by higher mitochondrial content and increased expression of genes related to oxidative metabolism. Additionally, the increased percentage of oxidative skeletal muscle fibers and mitochondrial apparatus in B2R^-/- mice is coupled with a higher aerobic exercise performance. Taken together, our data give support to the involvement of kinins in skeletal muscle fiber type distribution and muscle metabolism, which ultimately protects against fat-induced obesity and improves aerobic exercise performance.     

Selective expression of the type 3 isoform of ryanodine receptor Ca2+ release channel (RyR3) in a subset of slow fibers in diaphragm and cephalic muscles of adult rabbits

The expression pattern of the RyR3 isoform of Ca2+ release channels was analysed by Western blot in neonatal and adult rabbit skeletal muscles. The results obtained show that the expression of the RyR3 isoform is developmentally regulated. In fact, RyR3 expression was detected in all muscles analysed at 2 and 15 days after birth while, in adult animals, it was restricted to a subset of muscles that includes diaphragm, masseter, pterygoideus, digastricus, and tongue. Interestingly, all of these muscles share a common embryonic origin being derived from the somitomeres or from the cephalic region of the embryo. Immunofluorescence analysis of rabbit skeletal muscle cross-sections showed that RyR3 staining was detected in all fibers of neonatal muscles. In contrast, in those adult muscles expressing RyR3 only a fraction of fibers was labelled. Staining of these muscles with antibodies against fast and slow myosins revealed a close correlation between expression of RyR3 and fibers expressing slow myosin isoform.     

Expression of unique and developmental myosin heavy chain isoforms in adult human digastric muscle.

Digastric muscle (DGM) is a powerful jaw-opening muscle that participates in chewing, swallowing, breathing, and speech. For better understanding of its contractile properties, five pairs of adult human DGMs were obtained from autopsies and processed with immunocytochemistry and/or immunoblotting. Monoclonal antibodies against alpha-cardiac, slow tonic, neonatal, and embryonic myosin heavy chain (MHC) isoforms were employed to determine whether the DGM fibers contain these MHC isoforms, which have previously been demonstrated in restricted specialized craniocervical skeletal muscles but have not been reported in normal adult human trunk and limb muscles. The results showed expression of all these MHC isoforms in adult human DGMs. About half of the fibers reacted positively to the antibody specific for the alpha-cardiac MHC isoform in DGMs, and the number of these fibers decreased with age. Slow tonic MHC isoform containing fibers accounted for 19% of the total fiber population. Both the alpha-cardiac and slow tonic MHC isoforms were found to coexist mainly with the slow twitch MHC isoform in a fiber. A few DGM fibers expressed the embryonic or neonatal MHC isoform. The findings suggest that human DGM fibers may be specialized to facilitate performance of complex motor behaviors in the upper airway and digestive tract.     

Mitochondrial gene expression in mammalian striated muscle. Evidence that variation in gene dosage is the major regulatory event

The oxidative capacity of mammalian striated muscles can vary markedly over a nearly 10-fold range, reflecting major differences in the expression of genes that encode enzymes of oxidative metabolism, including genes located exclusively within mitochondrial DNA. To clarify the regulatory events that govern expression of mitochondrial genes in striated muscle, nucleic acid hybridization procedures employing cloned segments of mitochondrial DNA as probes were utilized to determine the concentrations of mitochondrial DNA, mitochondrial ribosomal RNA, and cytochrome b mRNA (a mitochondrial gene product) in rabbit striated muscles of markedly different oxidative capacities. When cardiac muscle and Type I (red, oxidative) skeletal muscle were compared to Type II (white, glycolytic) skeletal muscle, mitochondrial DNA, mitochondrial ribosomal RNA, and cytochrome b mRNA, each increased in direct proportion to increases in oxidative capacity. Furthermore, when the phenotypic characteristics of Type II skeletal muscle were altered by electrical stimulation in vivo, mitochondrial DNA, mitochondrial rRNA, and cytochrome b mRNA also increased proportionately with increases in oxidative capacity. These results indicate that the expression of mitochondrial genes in mammalian striated muscle is proportionate to their copy number, and support the hypothesis that amplification of the mitochondrial genome relative to chromosomal DNA is an important feature underlying enhanced expression of mitochondrial genes in highly oxidative tissues.     

Ultrastructural localization of lectin receptors on the bone-marrow sinusoidal endothelium of the rat

The purpose of this study was to estimate the absolute and relative masses of the three types of skeletal muscle fibers in the total hindlimb of the male Sprague-Dawley rat (Rattus norvegicus). For six rats, total body mass was recorded and the following weights taken from dissection of one hindlimb: 32 individual major muscles or muscle parts, remaining skeletal musculature (small hip muscles and intrinsic foot muscles), bone, inguinal fat pad, and skin. The fibers from the 32 muscles or muscle parts (which constituted 98% of the hindlimb skeletal muscle mass) were classified from histochemistry as fast-twitch oxidative glycolytic (FOG), fast-twitch glycolytic (FG), or slow-twitch oxidative (SO), and their populations were determined. Fiber cross-sectional areas from the same muscles were measured with a digitizer. Mass of each of the fiber types within muscles and in the total hindlimb was then calculated from fiber-type population, fiber-type area, and muscle-mass data. Skeletal muscle made up 71% of the total hindlimb mass. Of this, 76% was occupied by FG fibers, 19% by FOG fibers, and 5% by SO fibers. Thus, the FG fiber type is clearly the predominant fiber type in the rat hindlimb in terms of muscle mass. Fiber-type mass data are compared with physiological (blood flow) and biochemical (succinate dehydrogenase activities) data for the muscles taken from previous studies, and it is demonstrated that these functional properties are closely related to the proportions of muscle mass composed of the various fiber types.     

Development of muscle fiber types in the prenatal rat hindlimb

Immunohistochemistry was used to examine the expression of embryonic, slow, and neonatal isoforms of myosin heavy chain in muscle fibers of the embryonic rat hindlimb. While the embryonic isoform is present in every fiber throughout prenatal development, by the time of birth the expression of the slow and neonatal isoforms occurs, for the most part, in separate, complementary populations of fibers. The pattern of slow and neonatal expression is highly stereotyped in individual muscles and mirrors the distribution of slow and fast fibers found in the adult. This pattern is not present at the early stages of myogenesis but unfolds gradually as different generations of fibers are added. As has been noted by previous investigators (e.g., Narusawa et al., 1987, J. Cell Biol. 104, 447-459), all of the earliest generation (primary) muscle fibers initially express the slow isoform but some of these primary fibers later lose this expression. In this study we show that loss of slow myosin in these fibers is accompanied by the expression of neonatal myosin. This switch in isoform expression occurs in all primary fibers located in specific regions of particular muscles. However, in other muscles primary fibers which retain their slow expression are extensively intermixed with those that switch to neonatal expression. Later generated (secondary) muscle fibers, which are interspersed among the primary fibers, express neonatal myosin, although a few of them in stereotyped locations later switch from neonatal to slow myosin expression. Many of the observed changes in myosin expression occur coincidentally with the arrival of axons in the limb or the invasion of axons into individual muscles. Thus, although both fiber birth date and intramuscular position are grossly predictive of fiber fate, neither factor is sufficient to account for the final pattern of fiber types seen in the rat hindlimb. The possibility that fiber diversification is dependent upon innervation is tested in the accompanying paper (K. Condon, L. Silberstein, H.M. Blau, and W.J. Thompson, 1990, Dev. Biol. 138, 275-295).     

Stimulation of slow skeletal muscle fiber gene expression by calcineurin in vivo

Adult skeletal muscle fibers can be categorized into fast and slow twitch subtypes based on specialized contractile and metabolic properties and on distinctive patterns of muscle gene expression. Muscle fiber-type characteristics are dependent on the frequency of motor nerve stimulation and are thought to be controlled by calcium-dependent signaling. The calcium, calmodulin-dependent protein phosphatase, calcineurin, stimulates slow fiber-specific gene promoters in cultured skeletal muscle cells, and the calcineurin inhibitor, cyclosporin A, inhibits slow fiber gene expression in vivo, suggesting a key role of calcineurin in activation of the slow muscle fiber phenotype. Calcineurin has also been shown to induce hypertrophy of cardiac muscle and to mediate the hypertrophic effects of insulin-like growth factor-1 on skeletal myocytes in vitro. To determine whether activated calcineurin was sufficient to induce slow fiber gene expression and hypertrophy in adult skeletal muscle in vivo, we created transgenic mice that expressed activated calcineurin under control of the muscle creatine kinase enhancer. These mice exhibited an increase in slow muscle fibers, but no evidence for skeletal muscle hypertrophy. These results demonstrate that calcineurin activation is sufficient to induce the slow fiber gene regulatory program in vivo and suggest that additional signals are required for skeletal muscle hypertrophy.     

Effects of Resveratrol on the Recovery of Muscle Mass Following Disuse in the Plantaris Muscle of Aged Rats

Aging is associated with poor skeletal muscle regenerative ability following extended periods of hospitalization and other forms of muscular disuse. Resveratrol (3,5,4’-trihydroxystilbene) is a natural phytoalexin which has been shown in skeletal muscle to improve oxidative stress levels in muscles of aged rats. As muscle disuse and reloading after disuse increases oxidative stress, we hypothesized that resveratrol supplementation would improve muscle regeneration after disuse. A total of thirty-six male Fisher 344 × Brown Norway rats (32 mo.) were treated with either a water vehicle or resveratrol via oral gavage. The animals received hindlimb suspension for 14 days. Thereafter, they were either sacrificed or allowed an additional 14 day period of cage ambulation during reloading. A total of six rats from the vehicle and the resveratrol treated groups were used for the hindlimb suspension and recovery protocols. Furthermore, two groups of 6 vehicle treated animals maintained normal ambulation throughout the experiment, and were used as control animals for the hindlimb suspension and reloading groups. The data show that resveratrol supplementation was unable to attenuate the decreases in plantaris muscle wet weight during hindlimb suspension but it improved muscle mass during reloading after hindlimb suspension. Although resveratrol did not prevent fiber atrophy during the period of disuse, it increased the fiber cross sectional area of type IIA and IIB fibers in response to reloading after hindlimb suspension. There was a modest enhancement of myogenic precursor cell proliferation in resveratrol-treated muscles after reloading, but this failed to reach statistical significance. The resveratrol-associated improvement in type II fiber size and muscle mass recovery after disuse may have been due to decreases in the abundance of pro-apoptotic proteins Bax, cleaved caspase 3 and cleaved caspase 9 in reloaded muscles. Resveratrol appears to have modest therapeutic benefits for improving muscle mass after disuse in aging.     

Mitochondrial coupling in vivo in mouse skeletal muscle

The coupling of mitochondrial ATP synthesis and oxygen consumption (ratio of ATP and oxygen fluxes, P/O) plays a central role in cellular bioenergetics. Reduced P/O values are associated with mitochondrial pathologies that can lead to reduced capacity for ATP synthesis and tissue degeneration. Previous work found a wide range of values for P/O in normal mitochondria. To measure mitochondrial coupling under physiological conditions, we have developed a procedure for determining the P/O of skeletal muscle in vivo. This technique measures ATPase and oxygen consumption rates during ischemia with ³¹P magnetic resonance and optical spectroscopy, respectively. This novel approach allows the independent quantitative measurement of ATPase and oxygen flux rates in intact tissue. The quantitative measurement of oxygen consumption is made possible by our ability to independently measure the saturations of hemoglobin (Hb) and myoglobin (Mb) from optical spectra. Our results indicate that the P/O in skeletal muscle of the mouse hindlimb measured in vivo is 2.16 ± 0.24. The theoretical P/O for resting muscle is 2.33. Systemic treatment with 2,4-dinitrophenol to partially uncouple mitochondria does not affect the ATPase rate in the mouse hindlimb but nearly doubles the rate of oxygen consumption, reducing in vivo P/O to 1.37 ± 0.22. These results indicate that only a small fraction of the oxygen consumption in resting mouse skeletal muscle is nonphosphorylating under physiological conditions, suggesting that mitochondria are more tightly coupled than previously thought. P/O; oxidative phosphorylation; proton leak; optical spectroscopy     

The unique mitochondrial form and function of Antarctic channichthyid icefishes

Antarctic icefishes of the family Channichthyidae are the only vertebrate animals that as adults do not express the circulating oxygen-binding protein hemoglobin (Hb). Six of the 16 family members also lack the intracellular oxygen-binding protein myoglobin (Mb) in the ventricle of their hearts and all lack Mb in oxidative skeletal muscle. The loss of Hb has led to substantial remodeling in the cardiovascular system of icefishes to facilitate adequate oxygenation of tissues. One of the more curious adaptations to the loss of Hb and Mb is an increase in mitochondrial density in cardiac myocytes and oxidative skeletal muscle fibers. The proliferation of mitochondria in the aerobic musculature of icefishes does not arise through a canonical pathway of mitochondrial biogenesis. Rather, the biosynthesis of mitochondrial phospholipids is up-regulated independently of the synthesis of proteins and mitochondrial DNA, and newly-synthesized phospholipids are targeted primarily to the outer-mitochondrial membrane. Consequently, icefish mitochondria have a higher lipid-to-protein ratio compared to those from red-blooded species. Elevated levels of nitric oxide in the blood plasma of icefishes, compared to red-blooded notothenioids, may mediate alterations in mitochondrial density and architecture. Modifications in mitochondrial structure minimally impact state III respiration rates but may significantly enhance intracellular diffusion of oxygen. The rate of oxygen diffusion is greater within the hydrocarbon core of membrane lipids compared to the aqueous cytosol and impeded only by proteins within the lipid bilayer. Thus, the proliferation of icefish's mitochondrial membranes provides an optimal conduit for the intracellular diffusion of oxygen and compensates for the loss of Hb and Mb. Currently little is known about how mitochondrial phospholipid synthesis is regulated and integrated into mitochondrial biogenesis. The unique architecture of the oxidative muscle cells of icefishes highlights the need for further studies in this area.     

Differential expression of the fast skeletal muscle proteome following chronic low-frequency stimulation

Physiological and biochemical responses of skeletal muscle fibres to enhanced neuromuscular activity under conditions of maximum activation can be studied experimentally by chronic low-frequency stimulation of fast muscles. Stimulation-induced changes in the expression pattern of the rabbit fast skeletal muscle proteome were evaluated by two-dimensional gel electrophoresis and compared to the altered isoform expression profile of established transformation markers such as the Ca2+-ATPase, calsequestrin and the myosin heavy chain. Sixteen muscle proteins exhibited a marked change in their expression level. This included albumin with a 4-fold increase in abundance. In contrast, glycolytic enzymes, such as enolase and aldolase, showed a decreased expression. Concomitant changes were observed with marker elements of the contractile apparatus. While the fast isoforms of troponin T and myosin light chain 2 were drastically down-regulated, their slow counterparts exhibited increased expression. Interestingly, mitochondrial creatine kinase expression increased while the cytosolic isoform of this key muscle enzyme decreased. The expression of the small heat shock protein HSP-B5/alphaB-crystallin and the oxygen carrier protein myoglobin were both increased 2-fold following stimulation. The observed changes indicate that the conversion into fatigue-resistant red fibres depends on: (i) the optimum utilization of free fatty acids via albumin transportation, (ii) a rearrangement of the creatine kinase isozyme pattern for enhanced mitochondrial activity, (iii) an increased availability of oxygen for aerobic metabolism via myoglobin transport, (iv) the conversion of the contractile apparatus to isoforms with slower twitch characteristics and (v) the up-regulation of chaperone-like proteins for stabilising myofibrillar components during the fast-to-slow transition process.     

Hypoxia differentially regulates muscle oxidative fiber type and metabolism in a HIF-1α-dependent manner

Loss of skeletal muscle oxidative fiber types and mitochondrial capacity is a hallmark of chronic obstructive pulmonary disease and chronic heart failure. Based on in vivo human and animal studies, tissue hypoxia has been hypothesized as determinant, but the direct effect of hypoxia on muscle oxidative phenotype remains to be established. Hence, we determined the effect of hypoxia on in vitro cultured muscle cells, including gene and protein expression levels of mitochondrial components, myosin isoforms (reflecting slow-oxidative versus fast-glycolytic fibers), and the involvement of the regulatory PPAR/PGC-1α pathway. We found that hypoxia inhibits the PPAR/PGC-1α pathway and the expression of mitochondrial components through HIF-1α. However, in contrast to our hypothesis, hypoxia stimulated the expression of slow-oxidative type I myosin via HIF-1α. Collectively, this study shows that hypoxia differentially regulates contractile and metabolic components of muscle oxidative phenotype in a HIF-1α-dependent manner.