Mouse Fast-twitch Skeletal Muscle Research Articles

Sex differences in mitochondrial Ca2+ handling in mouse fast-twitch skeletal muscle in vivo.

We investigated sex differences in mitochondrial Ca2+ handling properties in mouse fast-twitch skeletal muscle. Changes in cytoplasmic Ca2+ concentration ([Ca2+]cyto) were measured in vivo using tibialis anterior muscles from male and female mice. The muscles were exposed to increasing concentrations of cyclopiazonic acid [CPA; sarcoplasmic reticulum (SR) Ca2+-ATPase inhibitor] (from 10 to 30 to 50 μM at 10 min intervals). Thirty minutes after treatment, [Ca2+]cyto was increased by 31.6 ± 2.0% and 13.5 ± 4.5% of initial [Ca2+]cyto in male and female muscles, respectively, and there was a significant difference between sexes. However, muscle preincubation for 5 min with 10 μM carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (an inhibitor of mitochondria Ca2+ uptake) eradicated this difference between sexes with respect to the CPA-induced [Ca2+]cyto increase. Both intermyofibrillar mitochondrial number and volume, assessed in longitudinal fiber sections, were higher in females compared with males (mitochondria number: 13.1 ± 1.0 in males vs. 19.9 ± 2.3 in females; mitochondrial volume: 0.034 ± 0.004 μm3/μm3 fiber volume in males vs. 0.066 ± 0.008 μm3/μm3 fiber volume in females, both P < 0.05). There were no sex differences in the content of SR Ca2+-ATPase, mitochondrial Ca2+ uniporter, mitofusin (Mfn) 1, or Mfn2. These results suggest that 1) mitochondrial Ca2+ uptake ability is greater in female than male myocytes, and 2) this superior Ca2+ uptake ability of female myocytes is due, partly, to the higher intermyofibrillar mitochondrial content but not to the expression of mitochondrial proteins related to mitochondrial Ca2+ uptake.NEW & NOTEWORTHY This investigation presents evidence that female versus male fast-twitch muscle exhibits a greater mitochondrial calcium ion uptake capability that is partly conferred by the higher intermyofibrillar mitochondrial volume density.

Long-term, but not short-term high-fat diet induces fiber composition changes and impaired contractile force in mouse fast-twitch skeletal muscle.

In this study, we investigated the effects of a short‐term and long‐term high‐fat diet (HFD) on morphological and functional features of fast‐twitch skeletal muscle. Male C57BL/6J mice were fed a HFD (60% fat) for 4 weeks (4‐week HFD) or 12 weeks (12‐week HFD). Subsequently, the fast‐twitch extensor digitorum longus muscle was isolated, and the composition of muscle fiber type, expression levels of proteins involved in muscle contraction, and force production on electrical stimulation were analyzed. The 12‐week HFD, but not the 4‐week HFD, resulted in a decreased muscle tetanic force on 100 Hz stimulation compared with control (5.1 ± 1.4 N/g in the 12‐week HFD vs. 7.5 ± 1.7 N/g in the control group; P < 0.05), whereas muscle weight and cross‐sectional area were not altered after both HFD protocols. Morphological analysis indicated that the percentage of type IIx myosin heavy chain fibers, mitochondrial oxidative enzyme activity, and intramyocellular lipid levels increased in the 12‐week HFD group, but not in the 4‐week HFD group, compared with controls (P < 0.05). No changes in the expression levels of calcium handling‐related proteins and myofibrillar proteins (myosin heavy chain and actin) were detected in the HFD models, whereas fast‐troponin T‐protein expression was decreased in the 12‐week HFD group, but not in the 4‐week HFD group (P < 0.05). These findings indicate that a long‐term HFD, but not a short‐term HFD, impairs contractile force in fast‐twitch muscle fibers. Given that skeletal muscle strength largely depends on muscle fiber type, the impaired muscle contractile force by a HFD might result from morphological changes of fiber type composition.

Upregulation of RGK Protein Expression in Aging Mouse Fast Twitch Skeletal Muscle

A component of muscle weakness in older individuals is directly attributable to compromised excitation-contraction (EC) coupling. In skeletal muscle, the L-type Ca2+ channel (CaV1.1) serves as the voltage sensor for EC coupling by triggering Ca2+ release from the sarcoplasmic reticulum (SR) in response to plasma membrane depolarization.Although it has been established that impaired EC coupling (termed “EC uncoupling” by O. Delbono and colleagues) in aged individuals is caused by a reduction in the number of L-type channels present in the plasma membrane, the molecular mechanisms responsible for transducing age-dependent cellular signals (e.g., oxidative stress) to decreased CaV1.1 membrane expression remain enigmatic. In this study, we have investigated a role for RGK (Rad, Rem, Rem2, Gem/Kir) family small G proteins in EC uncoupling because: 1) expression of endogenous Rad is enhanced in response to oxidative stress in skeletal muscle, and 2) overexpression of Rem in muscle cells mimics EC uncoupling. We have found that exogenous overexpression of Rad in cultured myotubes also mimics EC uncoupling. Moreover, immunoblotting of fast-twitch tibialis anterior and mixed gastrocnemius muscle lysates revealed progressive age-dependent enhancement of Rad protein levels while Rad expression was largely absent in slow-twitch soleus muscle in all age groups (ranging from 2-36 months). Taken together, our observations raise the possibility that Rad acts as one molecular link between elevated oxidative stress and EC uncoupling in aging fast-twitch muscle. Conversely, the lack of Rad expression in slow twitch muscle suggests that Rad may play a role in the disproportionate retention of slow twitch fibers in aged individuals.

The effect of work cycle frequency on the potentiation of dynamic force in mouse fast twitch skeletal muscle

The purpose of this study was to test the hypothesis that the potentiation of concentric twitch force during work cycles is dependent upon both the speed and direction of length change. Concentric and eccentric forces were elicited by stimulating muscles during the shortening and lengthening phases, respectively, of work cycles. Work cycle frequency was varied in order to vary the speed of muscle shortening and/or lengthening; all forces were measured as the muscle passed though optimal length (L(o)). Both concentric and eccentric force were assessed before (unpotentiated control) and after (potentiated) the application of a tetanic conditioning protocol known to potentiate twitch force output. The influence of the conditioning protocol on relative concentric force was speed dependent, with forces increased to 1.19±0.01, 1.25±0.01 and 1.30±0.01 of controls at 1.5, 3.3 and 6.9 Hz, respectively (all data N=9-10 with P<0.05). In contrast, the conditioning protocol had only a limited effect on eccentric force at these frequencies (range: 1.06±0.01 to 0.96±0.03). The effect of the conditioning protocol on concentric work (force × distance) was also speed dependent, being decreased at 1.5 Hz (0.84±0.01) and increased at 3.3 and 6.9 Hz (1.05±0.01 and 1.39±0.01, respectively). In contrast, eccentric work was not increased at any frequency (range: 0.88±0.02 to 0.99±0.01). Thus, our results reveal a hysteresis-like influence of activity-dependent potentiation such that concentric force and/or work were increased but eccentric force and/or work were not. These outcomes may have implications for skeletal muscle locomotor function in vivo.

Activation of aconitase in mouse fast-twitch skeletal muscle during contraction-mediated oxidative stress

Aconitase is a mitochondrial enzyme that converts citrate to isocitrate in the tricarboxylic acid cycle and is inactivated by reactive oxygen species (ROS). We investigated the effect of exercise/contraction, which is associated with elevated ROS production, on aconitase activity in skeletal muscle. Humans cycled at 75% of maximal workload, followed by six 60-s bouts at 125% of maximum workload. Biopsies were taken from the thigh muscle at rest and after the submaximal and supramaximal workloads. Isolated mouse extensor digitorum longus (EDL; fast twitch) and soleus (slow twitch) muscles were stimulated to perform repeated contractions for 10 min. Muscles were analyzed for enzyme activities and glutathione status. Exercise did not affect aconitase activity in human muscle despite increased oxidative stress, as judged by elevated levels of oxidized glutathione. Similarly, repeated contractions did not alter aconitase activity in soleus muscle. In contrast, repeated contractions significantly increased aconitase activity in EDL muscle by approximately 50%, despite increased ROS production. This increase was not associated with a change in the amount of immunoreactive aconitase (Western blot) but was markedly inhibited by cyclosporin A, an inhibitor of the protein phosphatase calcineurin. Immunoprecipitation experiments demonstrated that aconitase was phosphorylated on serine residues. Aconitase in cell-free extracts was inactivated by the addition of the ROS hydrogen peroxide. In conclusion, the results suggest that aconitase activity can be regulated by at least two mechanisms: oxidation/reduction and phosphorylation/dephosphorylation. During contraction, a ROS-mediated inactivation of aconitase can be overcome, possibly by dephosphorylation of the enzyme. The dual-control system may be important in maintaining aerobic ATP production during muscle contraction.

Reply to Barclay and Loiselle

reply: We appreciate the interest expressed by Drs. Barclay and Loiselle ([2][1]) in our recent paper ([11][2]). We used N -benzyl- p -toluene sulfonamide (BTS) to inhibit cross-bridge force production and myosin ATPase-dependent ATP splitting (measured with analytical biochemistry). BTS has been

Can activation account for 80% of skeletal muscle energy use during isometric contraction?

presence of a submaximal dose of dantrolene (an inhibitor of the skeletal muscle Ca 2 -release channel), tetanic force was reduced to 36% of its “control” value. Nevertheless, activation heat still accounted for only 42% of the total heat liberated. iv) The idea that 20% of isometric energy turnover is due to cross-bridge cycling is incompatible with the known mechanical performance of fast-twitch muscles. If cross bridges account for only 20% of energy use, then the rate of ATP splitting per cross bridge must be one-quarter the value previously assumed. Maintenance of a high isometric force, in any cross-bridge scheme that requires a low rate of turnover, can be achieved only by reducing the rate of cross-bridge detachment. However, a low detachment rate is inconsistent with the rapid force dynamics and the flat force-velocity curve characteristic of EDL muscles (2). Zhang et al. (5) offer no mechanistic explanation for their result but suggest that it differs from previous data due to an effect of species or temperature or as a result of using submaximal activation. Points i and iii above argue against an effect of either species or temperature, and the dantrolene experiments (point iii) counter the idea that activation costs are higher at submaximal levels of activation.

Cross bridges account for only 20% of total ATP consumption during submaximal isometric contraction in mouse fast-twitch skeletal muscle

It is generally believed that cross bridges account for >50% of the total ATP consumed by skeletal muscle during contraction. We investigated the effect of N-benzyl-p-toluene sulfonamide (BTS), an inhibitor of myosin ATPase, on muscle force production and energy metabolism under near-physiological conditions (50-Hz stimulation frequency at 30 degrees C results in 35% of maximal force). Extensor digitorum longus muscles from mice were isolated and stimulated to perform continuous isometric tetanic contractions. Metabolites of energy metabolism were analyzed with fluorometric techniques. ATP turnover was estimated from the changes in phosphocreatine (PCr), ATP, and lactate (-2DeltaATP - DeltaPCr + [1.5Deltalactate]). During contractions (2-10 s), BTS decreased force production to approximately 5% of control. Under these conditions, BTS inhibited ATP turnover by only 18-25%. ATP turnover decreased markedly and similarly with and without BTS as the duration of contraction progressed. In conclusion, cross bridges (i.e., actomyosin ATPase) account for only a small fraction (approximately 20%) of the ATP consumption during contraction in mouse fast-twitch skeletal muscle under near-physiological conditions, suggesting that ion pumping is the major energy-consuming process.

Lipopolysaccharide and proinflammatory cytokines stimulate interleukin-6 expression in C2C12 myoblasts: role of the Jun NH2-terminal kinase.

IL-6 is a major inflammatory cytokine that plays a central role in coordinating the acute-phase response to trauma, injury, and infection in vivo. Although IL-6 is synthesized predominantly by macrophages and lymphocytes, skeletal muscle is a newly recognized source of this cytokine. IL-6 from muscle spills into the circulation, and blood-borne IL-6 can be elevated >100-fold due to exercise and injury. The purpose of the present study was to determine whether inflammatory stimuli, such as LPS, TNF-alpha, and IL-1beta, could increase IL-6 expression in skeletal muscle and C2C12 myoblasts. Second, we investigated the role of mitogen-activated protein (MAP) kinases, and the Jun NH2-terminal kinase (JNK) in particular, as a mediator of this response. Intraperitoneal injection of LPS in mice increased the circulating concentration of IL-6 from undetectable levels to 4 ng/ml. LPS also increased IL-6 mRNA 100-fold in mouse fast-twitch skeletal muscle. Addition of LPS, IL-1beta, or TNF-alpha directly to C2C12 myoblasts increased IL-6 protein (6- to 8-fold) and IL-6 mRNA (5- to 10-fold). The response to all three stimuli was completely blocked by the JNK inhibitor SP-600125 but not as effectively by other MAP kinase inhibitors. SP-600125 blocked LPS-stimulated IL-6 synthesis dose dependently at both the RNA and protein level. SP-600125 was as effective as the synthetic glucocorticoid dexamethasone at inhibiting IL-6 expression. SP-600125 inhibited IL-6 synthesis when added to cells up to 60 min after LPS stimulation, but its inhibitory effect waned with time. LPS stimulated IL-6 mRNA in both myoblasts and myotubes, but myoblasts showed a proportionally greater LPS-induced increase in IL-6 protein expression compared with myotubes. SP-600125 and the proteasomal inhibitor MG-132 blocked LPS-induced degradation of IkappaB-alpha/epsilon and LPS-stimulated expression of IkappaB-alpha mRNA. Yet, only SP-600125 and not MG-132 blocked LPS-induced IL-6 mRNA expression. This suggests that IL-6 gene expression is a downstream target of JNK in C2C12 myoblasts.

Calcium transients and calcium homeostasis in adult mouse fast-twitch skeletal muscle fibers in culture.

Skeletal muscle fibers enzymatically dissociated from adult mouse flexor digitorum brevis muscles were maintained in culture for up to 8 days. After various times in culture, fibers were loaded with fura 2, and Ca2+ transients for trains of 1, 5, and 10 action potentials (100 Hz) triggered by external electrical stimulation were calculated from fluorescence ratio records corrected for noninstantaneous reaction of fura 2 with Ca2+. The decay rate constants of Ca2+ transients decreased with increasing stimulation duration, indicating a slowing of the Ca(2+)-removal properties with increased stimulation duration. After 6 days in culture, Ca2+ decay rate constants decreased dramatically for all stimulation durations and the differences in decay rate constants among 1, 5, and 10 pulses became smaller. Intracellular parvalbumin content measured by single-fiber immunofluorescence decreased with time in culture in parallel with the decrease in the decay rate constant of Ca2+ transients. Our results suggest that there is a correlation between parvalbumin content and the decay rate constant of the Ca2+ transient.

Effect of neonatal denervation on the distribution of fiber types in a mouse fast-twitch skeletal muscle.

This study was designed to assess the changes in fiber-type distribution of the extensor digitorum longus (EDL) muscle of the mouse during the first 21 days of age following neonatal sciatic neurectomy. Denervated and normal muscles were compared at 7, 14, and 21 days of age and the normal EDL was also studied at 1 day of age. Frozen sections of the EDL were treated histochemically to detect NADH-tetrazolium reductase and myosin ATPase reactions. Quantitative assessment included measurements of cross-sectional areas and fiber counting. Denervation resulted in muscle atrophy which was due primarily to a decrease in individual fiber area as opposed to fiber loss. Histochemical maturation of the EDL was severely affected by neonatal denervation during the first three postnatal weeks. By 21 days, two extrafusal fiber types which were both oxidative could be distinguished. One type was highly atrophied and resembled an immature fiber exhibiting myosin ATPase staining at both acid and alkaline preincubation conditions, whereas another type was less atrophied and showed myosin ATPase staining resembling fast-twitch (type IIA) fibers. These findings emphasize the importance of an intact nerve supply in determining the phenotypic expression of skeletal muscle, and point to the early postnatal period as a critical stage in fiber type differentiation.

Chromosomal distribution of genes coding for fast twitch skeletal muscle myosin light chains.

The mouse fast twitch skeletal muscle myosin light chains are encoded by a multigene family which comprises the gene coding for the myosin light chain 2 (Myl2f), and the gene coding for both myosin light chains 1 and 3 (Myl1f/Myl3f). In addition, a Myl1f/Myl3f-related pseudogene is present in the domestic mouse Mus musculus. The members of this gene family were assigned to chromosomes by molecular hybridization, using DNA extracted from a panel of cloned mouse-Chinese hamster somatic hybrid cells and specific DNA probes. The genes coding for the light chains of the myosin molecule are dispersed on several chromosomes, while genes coding for the heavy chain of myosin are located on a single, different chromosome.