This study determined whether susceptibility to opening of the permeability transition pore (PTP) varies according to muscle phenotype represented by the slow oxidative soleus (Sol) and superficial white gastrocnemius (WG). Threshold for Ca2+-induced mitochondrial Ca2+ release following PTP opening was determined with a novel approach using permeabilized ghost myofibers. Threshold values for PTP opening were approximately threefold higher in fibers from WG compared with those from Sol (124+/-47 vs. 30.4+/-6.8 pmol Ca2+/mU citrate synthase). A similar phenomenon was also observed in isolated mitochondria (threshold: 121+/-60 vs. 40+/-10 nmol Ca2+/mg protein in WG and Sol), indicating that this was linked to differences in mitochondrial factors between the two muscles. The resistance of WG fibers to PTP opening was not related to the expression of putative protein modulators (cyclophilin D, adenylate nucleotide translocator-1, and voltage-dependent anion channels) or to difference in respiratory properties and occurred despite the fact that production of reactive oxygen species, which promote pore opening, was higher than in the Sol. However, endogenous matrix Ca2+ measured in mitochondria isolated under resting baseline conditions was approximately twofold lower in the WG than in the Sol (56+/-4 vs. 111+/-11 nmol/mg protein), which significantly accounted for the resistance of WG. Together, these results reveal fiber type differences in the sensitivity to Ca2+-induced PTP opening, which may constitute a physiological mechanism to adapt mitochondria to the differences in Ca2+ dynamics between fiber types.

We sought to determine whether decreased neuromuscular use in the form of hindlimb unweighting (HU) would affect the properties of innervating motoneurones. Hindlimb weight-bearing was removed in rats for a period of 2 weeks via hindlimb suspension by the tail. Following this the electrophysiological properties of tibial motoneurones were recorded under anaesthesia in situ. After HU, motoneurones had significantly (P < 0.05) elevated rheobase currents, lower antidromic spike amplitudes, lower afterhyperpolarization (AHP) amplitudes, faster membrane time constants, lower cell capacitances, and depolarized spike thresholds. Frequency-current (f -I) relationships were shifted significantly to the right (i.e. more current required to obtain a given firing frequency), although there was no change in f -I slopes. 'Slow' motoneurones (AHP half-decay times, > 20 ms) were unchanged in proportions in HU compared to weight-bearing rats. Slow motoneurones had significantly lower minimum firing frequencies and minimum currents necessary for rhythmic firing than 'fast' motoneurones in weight-bearing rats; these differences were lost in HU rats, where slow motoneurones resembled fast motoneurones in these properties. In a five-compartment motoneurone model with ion conductances incorporated to resemble firing behaviour in vivo, most of the changes in passive and rhythmic firing properties could be reproduced by reducing sodium conductance by 25% and 15% in the initial segment and soma, respectively, or by increasing potassium conductance by 55% and 42%, respectively. This supports previous conclusions that changes in chronic neuromuscular activity, either an increase or decrease, may result in physiological adaptations in motoneurones due to chronic changes in ion conductances. The rat model of hindlimb unloading (HU), also known as hindlimb suspension, has proven to be a valuable ground-based model of the weightlessness that occurs during space flight (Morey-Holton & Globus, 2002). Despite the wealth of information from studies using HU on changes that occur in muscle following the removal of weight-bearing, very little is known about changes in the electrophysiological properties of the innervating motoneurones. Reported changes in voluntary recruitment and locomotor patterns following space flight and HU are consistent with possible changes in the excitability of motoneurones (Layne et al. 1997;Canu & Falempin, 1997Anderson et al. 1999;Edgerton et al. 2000;Recktenwald et al. 2000;Hodgson et al. 2000;Canu et al. 2001). Changes have been reported in hindlimb Hoffman reflex gain following 3 weeks of HU; however, it is unknown to what extent motoneurone excitability versus synaptic efficacy were involved in this adaptation (Anderson et al. 1999). During various models of decreased neuromuscular activity, including joint immobilization, bed rest, 'dry' water immersion and space flight, the ability to maximally activate muscles voluntarily is compromised, and there is EMG evidence that motor control is significantly altered (Duch...

Loss of neural input to skeletal muscle fibres induces atrophy and degeneration with evidence of mitochondria-mediated cell death. However, the effect of denervation on the permeability transition pore (PTP), a mitochondrial protein complex implicated in cell death, is uncertain. In the present study, the impact of 21 days of denervation on the sensitivity of the PTP to Ca 2+ -induced opening was studied in isolated muscle mitochondria. Muscle denervation increased the sensitivity to Ca 2+ -induced opening of the PTP, as indicated by a significant decrease in calcium retention capacity (CRC: 111 ± 12 versus 475 ± 33 nmol (mg protein) -1 for denervated and sham, respectively). This phenomenon was partly attributable to in vivo mitochondrial and whole muscle Ca 2+ overload. Cyclosporin A, which inhibits PTP opening by binding to cyclophilin D (CypD), was significantly more potent in mitochondria from denervated muscle and restored CRC to the level observed in mitochondria from sham-operated muscles. In contrast, the CypD independent inhibitor trifluoperazine was equally effective at inhibiting PTP opening in sham and denervated animals and did not correct the difference in CRC between groups. This phenomenon was associated with a significant increase in the content of the PTP regulating protein CypD relative to several mitochondrial marker proteins. Together, these results indicate that Ca 2+ overload in vivo and an altered expression of CypD could predispose mitochondria to permeability transition in denervated muscles.

Muscle phenotype is regulated by mechanical forces. However, it is not well understood how these forces are translated into intracellular signalling that influences gene expression. The purpose of this study was to test the hypothesis that muscles displaying a wide range of metabolic profiles and fibre-type composition exhibit differences in the detection and transmission of mechanical stimuli. A mechanical challenge in the form of passive stretch normalized to 3 N/g muscle weight was applied to the rat extensor digitorum longus (EDL), soleus (SOL), and plantaris (PLN) in situ for 5 min, following which activities of the mechanically-responsive p54 c-jun NH(2)-terminal kinase (JNK) and extracellular-regulated kinase (ERK) 1/2 were measured. EDL, SOL, and PLN were not different in their stretch-induced JNK (4.5, 5.2 and 6-fold baseline, respectively) or ERK (2.2, 2.2 and 1.9-fold baseline, respectively) responses, in spite of differing fibre-type compositions. The medial gastrocnemius (MG), a compartmentalized muscle with red (MGr) and white (MGw) regions, was subjected to the same normalized mechanical stretch protocol. The resulting JNK and ERK activities were significantly higher in MGr (13 and 4.5-fold baseline, respectively) than in MGw (5 and 1.2-fold baseline, respectively) and all other muscles. In contrast to stimulation by passive stretch, stimulation of the MG by isometric contractile activity did not result in a heterogeneous response between compartments. This study demonstrates an absence of difference among muscles of varying phenotype in their ability to transmit mechanical stimuli to the mitogen-activated protein kinase signalling pathways, and hence in their mechanosensitivity. Furthermore, the results highlight the importance of considering aspects of the functional organization of different muscles, such as compartmentalization and architecture, when studying mechanical signalling in vivo.

Mechanical loading is thought to be an important stimulus regulating muscle mass. However, the responsiveness of a muscle atrophied by a period of mechanical unloading to a subsequently imposed mechanical challenge is not well understood. This study examined the phosphorylation of the mechanically sensitive p54 c-jun NH(2)-terminal kinase (JNK) signaling protein in atrophied rat soleus muscle in response to a mechanical challenge in situ (isometric contractions; 100 Hz, 150 ms, once every 1 s for 5 min). Rats underwent either 7 or 14 days of hindlimb suspension (HLS) following which phosphorylation of JNK was measured biochemically. Immunofluorescence analysis revealed that phosphorylated JNK was localized in myonuclei. Baseline JNK phosphorylation measured in non-stimulated soleus muscles of 7- and 14-day HLS groups was 3.0- and 2.8-fold, respectively, the baseline phosphorylation measured in muscle of weight-bearing control animals (CTL). Following a mechanical challenge, JNK phosphorylation in stimulated CTL and 7-day HLS groups was significantly increased by 3.2- and 1.8-fold at the non-stimulated baseline levels, respectively. In stimulated muscle of 14-day HLS, JNK phosphorylation levels did not significantly differ from the baseline levels suggesting that the ability to elicit a mechanically induced phosphorylation of the JNK signaling protein gradually decreases with unweighting and is attenuated after 14-day HLS. Changes in the responsiveness of mechanically sensitive intracellular signaling pathways in atrophic muscle may contribute to the functional impairment experienced by muscle in the absence of weight bearing for prolonged periods.