Mitochondrial specialization revealed by single muscle fiber proteomics: focus on the Krebs cycle.

We have developed a highly sensitive mass spectrometry-based proteomic workflow to examine the proteome of single muscle fibers. This study revealed significant differences in the mitochondrial proteome of the four major fiber types present in mouse skeletal muscle. Here, we focus on Krebs cycle enzymes and in particular on the differential distribution of the two mitochondrial isocitrate dehydrogenases, IDH2 and IDH3. Type 1/slow fibers contain high levels of IDH2 and relatively low levels of IDH3, whereas fast 2X and 2B fibers show an opposite expression pattern. The findings suggest that in skeletal muscle, IDH2 functions in the forward direction of the Krebs cycle and that substrate flux along the cycle occurs predominantly via IDH2 in type 1 fibers and via IDH3 in 2X and 2B fibers. IDH2-mediated conversion of isocitrate to α-ketoglutarate leads to the generation of NADPH, which is critical to buffering the H2O2 produced by the respiratory chain. Nicotinamide nucleotide transhydrogenase (NNT), the other major mitochondrial enzyme involved in NADPH generation, is also more abundant in type 1 fibers. We suggest that the continuously active type 1 fibers are endowed with a more efficient H2O2 scavenging capacity to cope with the higher levels of reactive oxygen species production.

Resistance vessel remodeling and reparative angiogenesis in the microcirculatory bed of long-term denervated skeletal muscles.

In denervated skeletal muscles, atrophy of muscle fibers and interstitial fibrosis are associated with alterations within the vascular bed. Our study has placed particular emphasis on changes occurring in resistance vessels and the microcirculatory bed of rat hindlimb skeletal muscles that had been denervated for 25 months. We found that the tunica media of the majority of long-term denervated resistance vessels undergoes deterioration. In small intramuscular arteries and arterioles, atrophic vascular smooth muscle cells (vSMCs) enclosed in a thick basal lamina are separated by expanded extracellular space. The remodeling and sclerotic changes in the arterial wall occasionally result in deformation of the lumen. It was also found that the microcirculatory bed undergoes significant alterations. In 25-month denervated extensor digitorum longus muscle, the capillary-to-fiber ratio is only 0.13 +/- 0.01 and the mean number of capillaries per fascicle decreases almost ninefold compared to contralateral control muscle. Ultrastructural findings demonstrate that 24.67 +/- 0.48% of capillaries examined in the chronically denervated fascicles show structural features typical for capillary regeneration. In addition, long cytoplasmic extensions of pericytes might develop a layer completely encircling the capillary endothelium. In pre- and postcapillary segments of the microcirculatory bed, some perivascular cells possess a phenotype that is intermediate between that of pericytes and atrophic vSMCs. RT-PCR and/or Western blot analyses showed that molecules participating in angiogenesis are detected in 25-month denervated skeletal muscle. We hypothesize that despite the fact that the microcirculatory bed of chronically denervated muscle undergoes significant reduction it still sustains the capacity for reparative capillary growth.

Reparative myogenesis in long-term denervated skeletal muscles of adult rats results in a reduction of the satellite cell population.

This study, conducted on 25-month denervated rat hindlimb muscles, was directed toward elucidating the basis for the poor regeneration that is observed in long-term denervated muscles. Despite a approximately 97.6% loss in mean cross-sectional area of muscle fibers, the muscles retained their fascicular arrangement, with the fascicles containing approximately 1.5 times more fibers than age-matched control muscles. At least three distinct types of muscle fibers were observed: degenerating, persisting (original), and newly formed (regenerated) fibers. A majority of newly formed fibers did not appear to undergo complete maturation, and morphologically they resembled myotubes. Sites of former motor end-plates remained identifiable in persisting muscle fibers. Nuclear death was seen in all types of muscle fibers, especially in degenerating fibers. Nevertheless, the severely atrophic skeletal muscles continued to express developmentally and functionally important proteins, such as MyoD, myogenin, adult and embryonic subunits of the nicotinic acetylcholine receptor, and neural-cell adhesion molecule. Despite the prolonged period of denervation, slow and fast types of myosin were found in surviving muscle fibers. The number of satellite cells was significantly reduced in long-term denervated muscles, as compared with age-matched control muscles. In 25-month denervated muscle, satellite cells were only attached to persisting muscle fibers, but were never seen on newly formed fibers. Our data suggest that the absence of satellite cells in a population of immature newly formed muscle fibers that has arisen as a result of continuous reparative myogenesis may be a crucial, although not necessarily the only, factor underlying the poor regenerative ability of long-term denervated muscle.

Regulation of myogenin protein expression in denervated muscles from young and old rats.

Myogenin is a muscle-specific transcription factor participating in denervation-induced increases in nicotinic ACh receptor (nAChR) gene expression. Although myogenin RNA expression in denervated muscle is well documented, surprisingly little is known about myogenin protein expression. Therefore, we assayed myogenin protein and RNA in innervated and denervated muscles from young (4 mo) and old (24-32 mo) rats and compared this expression to that of the nAChR alpha-subunit RNA. These assays revealed increased myogenin protein expression within 1 day of denervation, preceding detectable increases in nAChR RNA. By 3 days of denervation, myogenin and nAChR alpha-subunit RNA were increased 500- and 130-fold, respectively, whereas myogenin protein increased 14-fold. Interestingly, old rats (32 mo) had 6-fold higher myogenin protein and approximately 80-fold higher mRNA levels than young rats. However, after denervation, expression levels were similar for young and old animals. The increased myogenin expression during aging, which tends to localize to small fibers, likely reflects spontaneous denervation and/or regeneration. Our results show that increased myogenin protein in denervated muscles correlates with the upregulation of its mRNA.

Physiology and biochemistry of the kidney vacuolar H+-ATPase.

Vacuolar H+-ATPases have an essential role in renal hydrogen ion secretion in the proximal tubule, collecting duct, and other segments of the nephron. Control of H+ transport is achieved by variations in the intrinsic properties of the renal H+-ATPases and by several cellular regulatory mechanisms, including redistribution of the enzyme both by vesicular traffic and regulated assembly and disassembly, and cytosolic regulatory proteins that interact directly with H+-ATPase. These mechanisms may provide a means for fine control of net acid excretion and for regulating vacuolar H+-ATPases residing on the plasma membrane independently from those in intracellular compartments.

Temporal and spatial appearance of alpha-dystroglycan in differentiated mouse myoblasts in culture.

The dystrophin-glycoprotein complex plays an important role in muscle function. One of the components of the complex, a 156-kDa cell surface glycoprotein (alpha-dystroglycan) binds to laminin, thereby connecting the basal lamina and muscle cells. We have examined the progressive appearance of alpha-dystroglycan and laminin in muscle cells that differentiate in culture. We find that nondifferentiated cultures of C2C12 myoblasts express low amounts of dystroglycan mRNA and, in contrast, this gene is prominently expressed in differentiated myotubes. Immunofluorescence analysis with a monoclonal antibody against alpha-dystroglycan shows its progressive appearance during myoblast differentiation into myotubes. Immunostaining with a monoclonal antibody against laminin shows that it is not present on the surface of undifferentiated myoblasts. Subsequently, laminin becomes apparent on the surface of differentiated myotubes where it codistributes with immunostained alpha-dystroglycan. Immunoblotting of isolated surface membrane proteins of differentiated myotubes with antibodies against alpha-dystroglycan identifies a broad band of about 140-160 kDa, resembling alpha-dystroglycan from rabbit muscle. The composite results indicate that alpha-dystroglycan and laminin appear and become co-distributed on the surface of cultured C2C12 during the progression of differentiation.

Rodent myoblast interactions with laminin require cell surface glycoconjugates but not laminin glycosyl groups.

Laminin glycosyl groups are necessary for the spreading of murine melanoma cells which become attached to this glycoprotein. Laminin has been implicated in myogenesis but the potential role of its glycosyl groups in this process has not been examined. In this study we report the effects of the carbohydrate moieties of laminin on myoblast adhesion, spreading, and differentiation. Unglycosylated laminin from tunicamycin-treated cultures of a mouse cell line, M1536 B3, was used in the experiments. Glycosylated laminin from a murine tumor and from cultures of M1563 B3 cells served as controls. Cell binding experiments with C2C12 mouse myoblasts showed that the cells preferred a laminin-coated surface, compared to the uncoated plastic surface (nontissue culture wells). Myoblasts did not distinguish between glycosylated and unglycosylated laminin substrates. Both glycosylated and unglycosylated forms of laminin promoted myoblast growth and differentiation. In contrast, cells on uncoated plastic surfaces grew very slowly and did not further differentiate. The L6 rat myoblast response to glycosylated and unglycosylated laminin was the same. These results indicate that although rodent myoblasts in culture require a laminin substratum for spreading, growth, and differentiation on a proprietary plastic surface, laminin carbohydrates are not implicated in those cellular responses. In contrast, parallel studies using the lectin, Con A, indicate that cell surface glycoconjugates of myoblasts are implicated in the response of these cells to a laminin substratum.