Creatine loading elevates the intracellular phosphorylation potential and alters adaptive responses of rat fast-twitch muscle to chronic low-frequency stimulation.

This study tested the hypothesis that elevating the intracellular phosphorylation potential (IPP = [ATP]/[ADP]free) within rat fast-twitch tibialis anterior muscles by creatine (Cr) loading would prevent fast-to-slow fibre transitions induced by chronic low-frequency electrical stimulation (CLFS, 10 Hz, 12 h/day). Creatine-control and creatine-CLFS groups drank a solution of 1% Cr + 5% dextrose, ad libitum, for 10 days before and during 10 days of CLFS; dextrose-control and dextrose-CLFS groups drank 5% dextrose. Cr loading increased total Cr (P < 0.025), phosphocreatine (PCr) (P < 0.003), and the IPP (P < 0.0008) by 34%, 45%, and 64%, respectively. PCr and IPP were 46% (P < 0.002) and 76% (P < 0.02) greater in creatine-CLFS than in dextrose-CLFS. Higher IPP was confirmed by a 58% reduction in phospho-AMP-activated protein kinase α (Thr172) (P < 0.006). In dextrose-CLFS, myosin heavy chain (MyHC) I and IIa transcripts increased 32- and 38-fold (P < 0.006), respectively, whereas MyHC-IIb mRNA decreased by 75% (P < 0.03); the corresponding MyHC-I and MyHC-IIa protein contents increased by 2.0- (P < 0.03) and 2.7-fold (P < 0.05), respectively, and MyHC-IIb decreased by 30% (P < 0.03). In contrast, within creatine-CLFS, MyHC-I and MyHC-IIa mRNA were unchanged and MyHC-IIb mRNA decreased by 75% (P < 0.003); the corresponding MyHC isoform contents were not altered. Oxidative reference enzymes were similarly elevated (P < 0.01) in dextrose-CLFS and creatine-CLFS, but reciprocal reductions in glycolytic reference enzymes occurred only in dextrose-CLFS (P < 0.02). Preservation of the glycolytic potential and greater SERCA2 and parvalbumin contents in creatine-CLFS coincided with prolonged time to peak tension and half-rise time (P < 0.01). These results highlight the IPP as an important physiological regulator of muscle fibre plasticity and demonstrate that training-induced changes typically associated with improvements in muscular endurance or increased power output are not mutually exclusive in Cr-loaded muscles.

Nitric oxide synthase inhibition prevents activity-induced calcineurin-NFATc1 signalling and fast-to-slow skeletal muscle fibre type conversions.

The calcineurin­NFAT (nuclear factor of activated T-cells) signalling pathway is involved in the regulation of activity-dependent skeletal muscle myosin heavy chain (MHC) isoform type expression. Emerging evidence indicates that nitric oxide (NO) may play a critical role in this regulatory pathway. Thus, the purpose of this study was to investigate the role of NO in activity-induced calcineurin­NFATc1 signalling leading to skeletal muscle faster-to-slower fibre type transformations in vivo. Endogenous NO production was blocked by administering L-NAME (0.75 mg ml(−1)) in drinking water throughout 0, 1, 2, 5 or 10 days of chronic low-frequency stimulation (CLFS; 10 Hz, 12 h day(−1)) of rat fast-twitch muscles (L+Stim; n = 30) and outcomes were compared with control rats receiving only CLFS (Stim; n = 30). Western blot and immunofluorescence analyses revealed that CLFS induced an increase in NFATc1 dephosphorylation and nuclear localisation, sustained by glycogen synthase kinase (GSK)-3ß phosphorylation in Stim, which were all abolished in L+Stim. Moreover, real-time RT-PCR revealed that CLFS induced an increased expression of MHC-I, -IIa and -IId(x) mRNAs in Stim that was abolished in L+Stim. SDS-PAGE and immunohistochemical analyses revealed that CLFS induced faster-to-slower MHC protein and fibre type transformations, respectively, within the fast fibre population of both Stim and L+Stim groups. The final fast type IIA to slow type I transformation, however, was prevented in L+Stim. It is concluded that NO regulates activity-induced MHC-based faster-to-slower fibre type transformations at the transcriptional level via inhibitory GSK-3ß-induced facilitation of calcineurin­NFATc1 nuclear accumulation in vivo, whereas transformations within the fast fibre population may also involve translational control mechanisms independent of NO signalling.

Alpha-catalytic subunits of 5'AMP-activated protein kinase display fiber-specific expression and are upregulated by chronic low-frequency stimulation in rat muscle.

5'-AMP-activated protein kinase (AMPK) signaling initiates adaptive changes in skeletal muscle fibers that restore homeostatic energy balance. The purpose of this investigation was to examine, in rats, the fiber-type protein expression patterns of the alpha-catalytic subunit isoforms in various skeletal muscles, and changes in their respective contents within the tibialis anterior (TA) after chronic low-frequency electrical stimulation (CLFS; 10 Hz, 10 h daily), applied for 4 +/- 1.2 or 25 +/- 4.8 days. Immunocytochemical staining of soleus (SOL) and medial gastrocnemius (MG) showed that 86 +/- 4.1 to 97 +/- 1.4% of type IIA fibers stained for both the alpha1- and alpha2-isoforms progressively decreased to 63 +/- 12.2% of type IID/X and 9 +/- 2.4% of IIB fibers. 39 +/- 11.4% of IID/X and 83 +/- 7.9% of IIB fibers expressed only the alpha2 isoform in the MG, much of which was localized within nuclei. alpha1 and alpha2 contents, assessed by immunoblot, were lowest in the white gastrocnemius [WG; 80% myosin heavy chain (MHC) IIb; 20% MHCIId/x]. Compared with the WG, alpha1 content was 1.6 +/- 0.08 (P < 0.001) and 1.8 +/- 0.04 (P < 0.0001)-fold greater in the red gastrocnemius (RG: 13%, MHCIIa) and SOL (21%, MHCIIa), respectively, and increased in proportion to MHCIIa content. Similarly, alpha2 content was 1.4 +/- 0.10 (P < 0.02) and 1.5 +/- 0.07 (P < 0.001)-fold greater in RG and SOL compared with WG. CLFS induced 1.43 +/- 0.13 (P < 0.007) and 1.33 +/- 0.08 (P < 0.009)-fold increases in the alpha1 and alpha2 contents of the TA and coincided with the transition of faster type IIB and IID/X fibers toward IIA fibers. These findings indicate that fiber types differ with regard to their capacity for AMPK signaling and that this potential is increased by CLFS.

Effect of satellite cell ablation on low-frequency-stimulated fast-to-slow fibre-type transitions in rat skeletal muscle.

The purpose of this study was to determine whether satellite cell ablation within rat fast-twitch muscles exposed to chronic low-frequency stimulation (CLFS) would limit fast-to-slow fibre-type transitions. Twenty-nine male Wistar rats were randomly assigned to one of three groups. Satellite cells of the left tibialis anterior were ablated by weekly exposure to a 25 Gy dose of gamma-irradiation during 21 days of CLFS (IRR-Stim), whilst a second group received only 21 days of CLFS (Stim). A third group received weekly doses of gamma-irradiation (IRR). Non-irradiated right legs served as internal controls. Continuous infusion of 5-bromo-2'-deoxyuridine (BrdU) revealed that CLFS induced an 8.0-fold increase in satellite cell proliferation over control (mean +/-s.e.m.: 23.9 +/- 1.7 versus 3.0 +/- 0.5 mm(-2), P < 0.0001) that was abolished by gamma-irradiation. M-cadherin and myogenin staining were also elevated 7.7- and 3.8-fold (P < 0.0001), respectively, in Stim compared with control, indicating increases in quiescent and terminally differentiating satellite cells; these increases were abolished by gamma-irradiation. Myonuclear content was elevated 3.3-fold (P < 0.0001) in Stim, but remained unchanged in IRR-Stim. Immunohistochemical analyses revealed attenuation of fast-to-slow fibre-type transitions in IRR-Stim compared with Stim. Comparable changes were observed at the protein level by SDS-PAGE. It is concluded that although considerable adaptive potential exists within myonuclei, satellite cells play a role in facilitating fast-to-slow fibre-type transitions.

Chronic low-frequency stimulation upregulates uncoupling protein-3 in transforming rat fast-twitch skeletal muscle.

The purpose of this investigation was to examine the temporal changes in uncoupling protein (UCP)-3 expression, as well as related adaptive changes in mitochondrial density and fast-to-slow fiber type transitions during chronically enhanced contractile activity. We examined the effects of 1-42 days of chronic low-frequency electrical stimulation (CLFS), applied to rat tibialis anterior (TA) for 10 h/day, on the expression of UCP-3 and concomitant changes in myosin heavy chain (MHC) protein expression and increases in oxidative capacity. UCP-3 protein content increased from 1 to 12 days, reaching 1.5-fold over control (P < 0.0005); it remained elevated for up to 42 days. In contrast, UCP-3 mRNA decreased in response to CLFS, reaching a level that was threefold lower than control (P < 0.0007). The activities of the mitochondrial reference enzymes citrate synthase (EC 4.1.3.7) and 3-hydroxyacyl-CoA-dehydrogenase (EC 1.1.1.35), which are known to increase in proportion to mitochondrial density, progressively increased up to an average of 2.3-fold (P < 0.00001). These changes were accompanied by fast-to-slow fiber type transitions, characterized by a shift in the pattern of MHC expression (P <0.0002): MHCI and MHCIIa expression increased by 1.7- and 4-fold, whereas MHCIIb displayed a 2.4-fold reduction. We conclude that absolute increases in UCP-3 protein content in the early adaptive phase were associated with the genesis of mitochondria containing a normal complement of UCP-3. However, during exposure to long-term CLFS, mitochondria were generated with a lower complement of UCP-3 and coincided with the emergence of a growing population of oxidative type IIA fibers.

Effects of strength, endurance and combined training on myosin heavy chain content and fibre-type distribution in humans.

This study investigated the effect of strength training, endurance training, and combined strength plus endurance training on fibre-type transitions, fibre cross-sectional area (CSA) and MHC isoform content of the vastus lateralis muscle. Forty volunteers (24 males and 16 females) were randomly assigned to one of four groups: control (C), endurance training (E), strength training (S), or concurrent strength and endurance training (SE). The S and E groups each trained three times a week for 12 weeks; the SE group performed the same S and E training on alternate days. The development of knee extensor muscle strength was S>SE>E ( P<0.05) and has been reported elsewhere. The reduction in knee extensor strength development in SE as compared to S corresponded to a 6% increase in MHCIIa content ( P<0.05) in SE at the expense of the faster MHCIId(x) isoform ( P<0.05), as determined by electrophoretic analyses; reductions in MHCIId/x content after S or E training were attenuated by comparison. Both S and SE induced three- to fourfold reductions ( P<0.05) in the proportion of type IIA/IID(X) hybrid fibres. S also induced fourfold increases in the proportion of type I/IIA hybrid fibres within both genders, and in a population of fibres expressing a type I/IID(X) hybrid phenotype within the male subjects. Type I/IIA hybrid fibres were not detected after SE. Both S and SE training paradigms induced similar increases (16-19%, P<0.05) in the CSA of type IIA fibres. In contrast, the increase in CSA of type I fibres was 2.9-fold greater ( P<0.05) in S as compared to SE after 12 weeks. We conclude that the interference of knee extensor strength development in SE versus S was related to greater fast-to-slow fibre-type transitions and attenuated hypertrophy of type I fibres. Data are given as mean (SEM) unless otherwise stated.

Effects of spaceflight on myosin heavy-chain content, fibre morphology and succinate dehydrogenase activity in rat diaphragm.

The present study examined the effect of 14 days of exposure to microgravity during the Spacelab Life Sciences-2 (SLS-2) space shuttle mission on the myosin heavy-chain (MHC) content, fibre size and type distributions and metabolic properties of rat diaphragm. Five adult male Sprague-Dawley rats were exposed to 14 days of microgravity (SF, spaceflight) and compared to five ground-based controls (C). Immunohistochemical analyses using isoform-specific anti-MHC monoclonal antibodies revealed that 14 days of SF did not alter the proportions of type-I, -IIA, -IID/X or -IIB fibres within the crural, sternal or lateral costal regions of the diaphragm; the electrophoretically quantified MHC-isoform contents also remained unchanged. In contrast, the medial gastrocnemius (MG) and tibialis anterior (TA) muscles displayed slow-to-fast fibre type transitions: within the MG the proportion of type-IID/X fibres was reduced by 59% ( P<0.04) and corresponded to a 51% increase ( P<0.03) in type-IIB fibres. Within the TA, the sum of type-IID/X+IIB fibres was elevated by 24% ( P<0.02) at the expense of the slower type-IIA fibres, which decreased by 33% ( P<0.04). Electrophoretic analyses yielded qualitatively similar patterns of transformation. SF did not induce atrophic changes within the diaphragm, MG or TA. Succinate dehydrogenase activity remained unchanged in the crural diaphragm ( P>0.96) but was 34% lower ( P<0.0001) in the TA. We conclude that 14 days of SF did not alter structural or metabolic factors that are known to underlie functional properties of the diaphragm. The findings of the present study show that 14 days of SF does not induce deleterious adaptive changes in the rat diaphragm that occur in hindlimb muscles.

AMPK activation increases uncoupling protein-3 expression and mitochondrial enzyme activities in rat muscle without fibre type transitions.

The present study examined the effect of chronic activation of 5'-AMP-activated protein kinase (AMPK) on the metabolic profile, including uncoupling protein-3 (UCP-3) and myosin heavy chain (MHC)-based fibre phenotype of rodent fast-twitch tibialis anterior muscle. Sprague-Dawley rats were given daily injections of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), a known activator of AMPK, or vehicle (control) for 28 days. After AICAR treatment, UCP-3 expression at the mRNA level was elevated 1.6 +/- 0.1-fold (P < 0.006) and corresponded to a 3.3 +/- 0.2-fold increase in UCP-3 protein content (P < 0.0001). In addition, the activities of the mitochondrial reference enzymes citrate synthase (EC 4.1.3.7) and 3-hydroxyacyl-CoA-dehydrogenase (EC 1.1.1.35), which are known to increase in proportion to mitochondrial volume density, were elevated 1.6-fold (P < 0.006), while the activity of lactate dehydrogenase (EC 1.1.1.27) was reduced to 80 % of control (P < 0.02). No differences were detected after AICAR treatment in the activities of the glycolytic reference enzymes glyceraldehydephosphate dehydrogenase (EC 1.2.1.12) or phosphofructokinase (EC 2.7.1.11), nor were MHC-based fibre-type transitions observed, using immunohistochemical or electrophoretic analytical methods. These changes could not be attributed to variations in inter-organ signalling by metabolic substrates or insulin. We conclude that an AMPK-dependent pathway of signal transduction does mimic some of the metabolic changes associated with chronic exercise training, but does not affect expression of the MHC-based structural phenotype. Thus, the metabolic and MHC-based fibre types do not appear to be regulated in a co-ordinated way, but may be independently modified by different signalling pathways.

Effects of chronic AICAR administration on the metabolic and contractile phenotypes of rat slow- and fast-twitch skeletal muscles.

The present study examined the effects of chronic activation of 5'-AMP-activated protein kinase (AMPK) on the oxidative capacity and myosin heavy chain (MHC) based fibre phenotype of rodent fast- and slow-twitch muscles. Sprague-Dawley rats received daily injections for 4 weeks of the known AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) or vehicle (control). The AICAR group displayed increases in hexokinase-II (HXK-II) activity, expression, and phosphorylation in fast-twitch muscles (P<0.001) but not in the slow-twitch soleus (SOL). In the AICAR group, citrate synthase (EC 4.1.3.7) and 3-hydroxyacyl-CoA-dehydrogenase (EC 1.1.1.35) were elevated 1.6- and 2.1-fold (P<0.05), respectively, in fast-twitch medial gastrocnemius (MG), and by 1.2- and 1.4-fold (P<0.05) in the slower-twitch plantaris (PLANT). No changes were observed in the slow-twitch SOL. In contrast, the activity of glyceraldehyde phosphate dehydrogenase (EC 1.2.1.12) remained unchanged in all muscles. AICAR treatment did not alter the MHC-based fibre type composition in fast- or slow-twitch muscles, as determined by immunohistochemical and electrophoretic analytical methods or by RT-PCR. We conclude that chronic activation of AMPK mimics the metabolic changes associated with chronic exercise training (increased oxidative capacity) in the fast-twitch MG and PLANT, but does not coordinately alter MHC isoform content or mRNA expression.