Skeletal muscle fibers in suspension: a new approach to the study of oxidative and glycolytic metabolism in differentiated muscle

A preparation of suspended fibers from m. flexor digitorum brevis of the rat was characterized with respect to morphological features, and its relevance for the study of muscular metabolism investigated. The activities of oxidative (palmitate and pyruvate oxidation) and glycolytic (lactate formation) pathways were enhanced in myofiber suspensions when compared to intact whole muscle. The rate of glycolysis was stimulated about two-fold by insulin in both the myofiber suspensions and intact muscle. Parameters of oxidative metabolism responded similarly to metabolic effectors in the myofiber suspensions and in intact muscle. It is concluded that the myofiber suspensions have distinct advantages over intact muscle for biochemical and pharmacological studies.     

The KATP channel Kir6.2 subunit content is higher in glycolytic than oxidative skeletal muscle fibers

It has long been suggested that in skeletal muscle, the ATP-sensitive K(+) channel (K(ATP)) channel is important in protecting energy levels and that abolishing its activity causes fiber damage and severely impairs function. The responses to a lack of K(ATP) channel activity vary between muscles and fibers, with the severity of the impairment being the highest in the most glycolytic muscle fibers. Furthermore, glycolytic muscle fibers are also expected to face metabolic stress more often than oxidative ones. The objective of this study was to determine whether the t-tubular K(ATP) channel content differs between muscles and fiber types. K(ATP) channel content was estimated using a semiquantitative immunofluorescence approach by staining cross sections from soleus, extensor digitorum longus (EDL), and flexor digitorum brevis (FDB) muscles with anti-Kir6.2 antibody. Fiber types were determined using serial cross sections stained with specific antimyosin I, IIA, IIB, and IIX antibodies. Changes in Kir6.2 content were compared with changes in CaV1.1 content, as this Ca(2+) channel is responsible for triggering Ca(2+) release from sarcoplasmic reticulum. The Kir6.2 content was the lowest in the oxidative soleus and the highest in the glycolytic EDL and FDB. At the individual fiber level, the Kir6.2 content within a muscle was in the order of type IIB > IIX > IIA ≥ I. Interestingly, the Kir6.2 content for a given fiber type was significantly different between soleus, EDL, and FDB, and highest in FDB. Correlations of relative fluorescence intensities from the Kir6.2 and CaV1.1 antibodies were significant for all three muscles. However, the variability in content between the three muscles or individual fibers was much greater for Kir6.2 than for CaV1.1. It is suggested that the t-tubular K(ATP) channel content increases as the glycolytic capacity increases and as the oxidative capacity decreases and that the expression of K(ATP) channels may be linked to how often muscles/fibers face metabolic stress.     

Downhill running preferentially increases CGRP in fast glycolytic muscle fibers.

Calcitonin gene-related peptide (CGRP) is present in some spinal cord motoneurons and at neuromuscular junctions in skeletal muscle. We previously reported increased numbers of CGRP-positive (CGRP+) motoneurons supplying hindlimb extensors after downhill exercise (Homonko DA and Theriault E, Inter J Sport Med 18: 1-7, 1997). The present study identifies the responding population with respect to muscle and motoneuron pool and correlates changes in CGRP with muscle fiber type-identified end plates. Twenty seven rats were divided into the following groups: control and 72 h and 2 wk postexercise. FluoroGold was injected into the soleus, lateral gastrocnemius, and the proximal (mixed fiber type) or distal (fast-twitch glycolytic) regions of the medial gastrocnemius (MG). Untrained animals ran downhill on a treadmill for 30 min. The number of FluoroGold/CGRP+ motoneurons within proximal and distal MG increased by 72 h postexercise (P<0.05). No significant changes were observed in soleus or lateral gastrocnemius motoneurons postexercise. The number of alpha-bungarotoxin/CGRP+ motor end plates in the MG increased exclusively at fast-twitch glycolytic muscle fibers 72 h and 2 wk postexercise (P<0.05). One interpretation of these results is that unaccustomed exercise preferentially activates fast-twitch glycolytic muscle fibers in the MG.     

Mitochondrial dynamics: Shaping and remodeling an organelle network

Mitochondria form networks that continually remodel and adapt to carry out their cellular function. The mitochondrial network is remodeled through changes in mitochondrial morphology, number, and distribution within the cell. Mitochondrial dynamics depend directly on fission, fusion, shape transition, and transport or tethering along the cytoskeleton. Over the past several years, many of the mechanisms underlying these processes have been uncovered. It has become clear that each process is precisely and contextually regulated within the cell. Here, we discuss the mechanisms regulating each aspect of mitochondrial dynamics, which together shape the network as a whole.     

Altered glycolytic and oxidative capacities of skeletal muscle contribute to insulin resistance in NIDDM

Simoneau, Jean-Aimé, and David E. Kelley. Alteredglycolytic and oxidative capacities of skeletal muscle contribute toinsulin resistance in NIDDM. J. Appl.Physiol. 83(1): 166-171, 1997.The insulinresistance of skeletal muscle in glucose-tolerant obese individuals isassociated with reduced activity of oxidative enzymes and adisproportionate increase in activity of glycolytic enzymes. Becausenon-insulin-dependent diabetes mellitus (NIDDM) is a disordercharacterized by even more severe insulin resistance of skeletal muscleand because many individuals with NIDDM are obese, the present studywas undertaken to examine whether decreased oxidative and increasedglycolytic enzyme activities are also present in NIDDM. Percutaneousbiopsy of vatus lateralis muscle was obtained in eight lean (L) andeight obese (O) nondiabetic subjects and in eight obese NIDDM subjectsand was assayed for marker enzymes of the glycolytic[phosphofructokinase, glyceraldehyde phosphate dehydrogenase,hexokinase (HK)] and oxidative pathways [citrate synthase(CS), cytochrome-c oxidase], aswell as for a glycogenolytic enzyme (glycogen phosphorylase) and amarker of anaerobic ATP resynthesis (creatine kinase). Insulinsensitivity was measured by using the euglycemic clamp technique.Activity for glycolytic enzymes (phosphofructokinase, glyceraldehyephosphate dehydrogenase, HK) was highest in subjects with subjects with NIDDM, following the order of NIDDM > O > L, whereas maximumvelocity for oxidative enzymes (CS,cytochrome-c oxidase) was lowest in subjects with NIDDM. The ratio between glycolytic andoxidative enzyme activities within skeletal muscle correlatednegatively with insulin sensitivity. The HK/CS ratio had the strongestcorrelation (r = 0.60, P < 0.01) with insulinsensitivity. In summary, an imbalance between glycolytic and oxidativeenzyme capacities is present in NIDDM subjects and is more severe thanin obese or lean glucose-tolerant subjects. The altered ratio betweenglycolytic and oxidative enzyme activities found in skeletal muscle ofindividuals with NIDDM suggests that a dysregulation betweenmitochondrial oxidative capacity and capacity for glycolysis is animportant component of the expression of insulin resistance.

     

Calmodulin supports the force-generating function in desensitized muscle fibers

Externally added calmodulin (CaM) restored Ca2+ regulation for the tension development by skeletal muscle fibers of hamster and rabbit desensitized by the troponin C (TnC) extraction treatment. CaM produced this action by combining with the TnC-denuded sites in the fiber. However, the binding properties differed strikingly from TnC: unlike TnC, CaM binding required the continued presence of Ca2+ and the bound portion was completely released with EGTA in the physiological milieu. The maximal uptake was 1.7 g of CaM/kg of muscle in the present study. The apparent Ca2+ sensitivity for force development with 200 micrograms/ml CaM in the solution was lower than in the native fiber or in the TnC-loaded fiber. The apparent association constant for CaM binding to the TnC-denuded sites was found as 4.9 x 10(5) M-1, and the extrapolated maximum force (Fmax) with CaM was close to PO. The intrinsic CaM level in intact muscle was also measured and was 18.6 mg/kg, amounting to about 1% of the total TnC or the CaM uptake by TnC-denuded fibers. The intrinsic CaM was not dislodged by EDTA treatment, indicating tight binding and suggesting that it exists in a separate pool from the vacated TnC sites adsorbing externally added CaM. The stringent Ca+ dependence of the CaM adsorption to TnC sites in the regulatory complex in the fiber supports the view that the evolutionary replacement of residues in the amino terminus helix portion of the "EF-hand" motif of site IV may be critical for the functional specialization by TnC.     

Mitochondrial superoxide production in skeletal muscle fibers of the rat and decreased fiber excitability

Mammalian skeletal muscles generate marked amounts of superoxide (O₂·^–) at 37°C, but it is not well understood which is the main source of O₂·^– production in the muscle fibers and how this interferes with muscle function. To answer these questions, O₂·^– production and twitch force responses were measured at 37°C in mechanically skinned muscle fibers of rat extensor digitorum longus (EDL) muscle. In mechanically skinned fibers, the sarcolemma is removed avoiding potential sources of O₂·^– production that are not intrinsically part of the muscle fibers, such as nerve terminals, blood cells, capillaries and other blood vessels in the whole muscle. O₂·^– production was also measured in split single EDL muscle fibers, where part of the sarcolemma remained attached, and small bundles of intact isolated EDL muscle fibers at rest, in the presence and absence of modifiers of mitochondrial function. The results lead to the conclusion that mitochondrial production of O₂·^– accounts for most of the O₂·^– measured intracellularly or extracellularly in skeletal muscle fibers at rest and at 37°C. Muscle fiber excitability at 37°C was greatly improved in the presence of a membrane permeant O₂·^– dismutase mimetic (Tempol), demonstrating a direct link between O₂·^– production in the mitochondria and muscle fiber performance. This implicates mitochondrial O₂·^– production in the down-regulation of skeletal muscle function, thus providing a feedback pathway for communication between mitochondria and plasma membranes that is not directly related to the main function of mitochondria as the power plant of the mammalian muscle cell. excitation-contraction coupling; mechanically skinned fiber; physiological temperature     

Flight muscle function in Drosophila requires colocalization of glycolytic enzymes.

Structural relationships between the myofibrillar contractile apparatus and the enzymes that generate ATP for muscle contraction are not well understood. We explored whether glycolytic enzymes are localized in Drosophila flight muscle and whether localization is required for function. We find that glycerol-3-phosphate dehydrogenase (GPDH) is localized at Z-discs and M-lines. The glycolytic enzymes aldolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are also localized along the sarcomere with a periodic pattern that is indistinguishable from that of GPDH localization. Furthermore, localization of aldolase and GAPDH requires simultaneous localization of GPDH, because aldolase and GAPDH are not localized along the sarcomere in muscles of strains that carry Gpdh null alleles. In an attempt to understand the process of glycolytic enzyme colocalization, we have explored in more detail the mechanism of GPDH localization. In flight muscle, there is only one GPDH isoform, GPDH-1, which is distinguished from isoforms found in other tissues by having three C-terminal amino acids: glutamine, asparagine, and leucine. Transgenic flies that can produce only GPDH-1 display enzyme colocalization similar to wild-type flies. However, transgenic flies that synthesize only GPDH-3, lacking the C-terminal tripeptide, do not show the periodic banding pattern of localization at Z-discs and M-lines for GPDH. In addition, neither GAPDH nor aldolase colocalize at Z-discs and M-lines in the sarcomeres of muscles from GPDH-3 transgenic flies. Failure of the glycolytic enzymes to colocalize in the sarcomere results in the inability to fly, even though the full complement of active glycolytic enzymes is present in flight muscles. Therefore, the presence of active enzymes in the cell is not sufficient for muscle function; colocalization of the enzymes is required. These results indicate that the mechanisms by which ATP is supplied to the myosin ATPase, for muscle contraction, requires a highly organized cellular system.     

Myokinase and contractile function of glycerinated muscle fibers

Glycerinated rabbit psoas muscle fibers containing native CPK, ATPase, and myokinase activities were used and isometric contraction and relaxation responses to either ADP or ATP + CP or to ATP alone in the presence and absence of P1, P5-di(adenosine-5'-pentaphosphate), a myokinase inhibitor, were compared. In previous (14) work it was shown that CP generated more efficient and faster contraction and relaxation of glycerinated muscle fibers than ATP. The present work deals with the role of myokinase in the differential response of fibers to CP and ATP. Inhibition of the myokinase activity of these fibers caused slight diminution of the rate of contraction at physiological concentrations of ATP. Uninhibited fibers were not able to reach maximum contraction, because the tension began to drop gradually even in the presence of Ca2+. Addition of Ap5A permitted maximum contraction and the ability to stay at the contracted state. In the case of CP + adenosine nucleotides (ATP or ADP), myokinase activity decreased the rate of tension development which was statistically significant after 5-7 sec of contraction. Thus, a higher tension was obtainable when myokinase was inhibited. At high concentration of adenine nucleotides (greater than 2 mM) and in the absence of Ap5A, not only the maximum tension never was reached, but a spontaneous drop in tension was observed before addition of EGTA, as was seen with ATP alone. Relaxation was faster and more complete in the presence of uninhibited myokinase activity except that the ADP was low (125 mM). These observations provide further evidence for a close functional interaction of these three enzymes in the mechanism of contraction and relaxation, giving further support to the notion of the creatine-phosphocreatine energy shuttle.     

Deviant nucleosomes: the functional specialization of chromatin

Regulatory proteins exist with strong sequence and structural similarities to the bistone proteins. Molecular genetic and cell biological analyses suggest that these proteins are localized at particular sites within the chromosome. Their assembly into nucleosomal structures confers specialized functions to individual chromosomal domains.     

Absence of regional differences in the size and oxidative capacity of diaphragm muscle fibers

The oxidative capacity and cross-sectional area of muscle fibers were compared between the costal and crural regions of the cat diaphragm and across the abdominal-thoracic extent of the muscle. Succinate dehydrogenase (SDH) activity of individual fibers was quantified using a microphotometric procedure implemented on an image-processing system. In both costal and crural regions, population distributions of SDH activities were unimodal for both type I and II fibers. The continuous distribution of SDH activities for type II fibers indicated that no clear threshold exists for the subclassification of fibers based on differences in oxidative capacity (e.g., the classification of fast-twitch glycolytic and fast-twitch oxidative glycolytic fiber types). No differences in either SDH activity or cross-sectional area were noted between fiber populations of the costal and crural regions. Differences in SDH activity and cross-sectional area were noted, however, between fiber populations located on the abdominal and thoracic sides of the costal region. Both type I and II fibers on the abdominal side of the costal diaphragm were larger and more oxidative than comparable fibers on the thoracic side.