tetrodotoxin and Muscular-Atrophy

The expression of five myosin heavy chain (MHC) isoforms was analyzed in the rat soleus (Sol) and the deep and superficial medial gastrocnemius (dGM, sGM) muscle after 2 and 4 wk of TTX paralysis by using immunohistochemical techniques. In Sol, after 4 wk of paralysis, fibers containing type I MHC were either pure type I (14%) or also contained developmental (D; 76%), IIa (26%), or IIx (18%) MHC. Values for corresponding fibers in dGM were 8.5, 65, 38, and 22%. Also, by 4 wk an increase was seen in the proportions of fibers expressing IIa MHC in Sol (from 16 to 38%) and dGM (from 24 to 74%). In a region of sGM in control muscles containing pure IIb fibers, a major proportion (86%) remained pure after 4 wk of paralysis, with the remainder coexpressing IIb and IIx. The results indicate that TTX-induced muscle paralysis results in an increase in fibers containing multiple MHC isoforms and that the D isoform appears in a major proportion of these hybrid fibers.

Topics: Animals; Body Weight; Female; Immunohistochemistry; Muscle Fibers, Skeletal; Muscle, Skeletal; Muscular Atrophy; Myosin Heavy Chains; Organ Size; Paralysis; Protein Isoforms; Rats; Rats, Sprague-Dawley; Tetrodotoxin; Time Factors

Topics: Adenosine; Adenosine-5'-(N-ethylcarboxamide); Animals; Arterioles; Blood Flow Velocity; Capillaries; Male; Muscle, Skeletal; Muscular Atrophy; Rats; Rats, Inbred F344; Tetrodotoxin; Vasodilator Agents

In disuse atrophied skeletal muscle, the staircase response is virtually absent and light chain phosphorylation does not occur. The purpose of the present study was to determine if staircase could be restored in atrophied muscle with continued absence of myosin light chain phosphorylation, by reducing what appears to be an otherwise enhanced calcium release. Control (untreated) and sham-operated female Sprague-Dawley rats were compared with animals after 2 weeks of complete inactivity induced by tetrodotoxin (TTX) application to the left sciatic nerve. In situ isometric contractile responses of rat gastrocnemius muscle were analyzed before and after administration of dantrolene sodium (DS), a drug which is known to inhibit Ca2+ release in skeletal muscle. Twitch active force (AF) was attenuated by DS from 2.2 +/- 0.2 N, 2.7 +/- 0.1 N and 2.4 +/- 0.2 N to 0.77 +/- 0.2 N, 1.05 +/- 0.1 N and 1.01 +/- 0.2 N in TTX (N = 5), sham (N = 11) and control (N = 7) muscles, respectively. Following dantrolene treatment, 10 s of 10-Hz stimulation increased AF to 1.32 +/- 0.2 N, 1.52 +/- 0.1 N and 1.45 +/- 0.2 N for the TTX, sham and control groups, respectively, demonstrating a positive staircase response. Regulatory light chain (R-LC) phosphorylation was lower for TTX-treated (5.5 +/- 5.5%) than for control (26.1 +/- 5.3%) and sham (20.0 +/- 5%) groups. There was no significant change from resting levels for any of the groups after DS treatment (P = 0.88). This study shows that treatment with dantrolene permits staircase in atrophied muscle as well as control muscle, by a mechanism which appears to be independent of R-LC phosphorylation.

Topics: Animals; Dantrolene; Female; Muscle Contraction; Muscle Relaxants, Central; Muscle, Skeletal; Muscular Atrophy; Myosin Light Chains; Phosphorylation; Propylene Glycol; Rats; Rats, Sprague-Dawley; Tetrodotoxin

The aim of the study was to address discrepant findings in the literature regarding coupling between decreased functional demand during disuse and reduced capillarity. We previously reported [K. Tyml, O. Mathieu-Costello, and E. Noble. Microvasc. Res. 49: 17-32, 1995] that severe disuse of rat extensor digitorum longus (EDL) muscle caused by a 2-wk application of tetrodotoxin (TTX) on the sciatic nerve is not accompanied by capillary loss. Using the same animal model, the present study examined whether this absence of coupling could be explained in terms of 1) too short a duration of disuse and 2) muscle-specific response to disuse. Fischer 344 rats were exposed to either no treatment (control) or to 2- or 8-wk TTX applications. Fiber size, capillary density per fiber cross-sectional area, and capillary-to-fiber (C/F) ratio were determined by morphometry in the EDL muscle (control, 2- and 8-wk groups) and in the superficial portion of medial gastrocnemius (Gas) muscle (control, 2 wk). In both muscles, microvascular blood flow was evaluated by intravital microscopy [red blood cell velocity in capillaries (V(RBC))] and by laser Doppler flowmetry (LDF). Regardless of duration of TTX application or muscle type, TTX-induced disuse resulted in a significant reduction of fiber area (44-71%). However, capillary density increased in EDL muscle (both at 2 and 8 wk) but not in Gas muscle. C/F ratio decreased in EDL muscle at 8 wk (18%) and in Gas muscle (39%). This indicates that the effect on capillarity depended on duration of disuse and on muscle type. V(RBC) and LDF signal were significantly larger in EDL than in Gas muscle. Analysis of change in capillarity vs. V(RBC) suggested that the outcome of disuse may be modulated by blood flow. We conclude that the duration of skeletal muscle disuse per se does not dictate capillary loss, and we hypothesize that discrepant findings of coupling between functional demand and capillarity could be due to the presence/absence of flow-related angiogenesis superimposed on the capillary removal process during disuse.

Topics: Animals; Blood Volume; Body Weight; Capillaries; Hemodynamics; Hindlimb; Ischemia; Male; Microcirculation; Muscle, Skeletal; Muscular Atrophy; Rats; Rats, Inbred F344; Sciatic Nerve; Tetrodotoxin; Time Factors

Contractile parameters of a twitch contraction and changes in these parameters during repetitive stimulation are modified by muscle atrophy induced by tetrodotoxin (TTX). These altered parameters included developed tension (DT), contraction time (tC), half-relaxation time (tR, 1/2), average rate of force development (DT tC-1) and peak rate of relaxation (DTdtmin-1). These modifications may be related to different Ca2+ concentration transients in the myoplasm during muscle stimulation. We have used dantrolene sodium (DS) in TTX-treated rat gastrocnemius muscle to test this hypothesis. In situ isometric contractile responses of rat gastrocnemius muscle during repetitive stimulation at 10 Hz were analysed before and after administration of DS. After DS administration, twitch amplitude, tC, tR, 1/2 and DT tC-1 decreased and DTdtmin-1 relative to DT increased in atrophied muscle. During repetitive stimulation, a progressive enhancement developed tension (staircase) was absent in atrophied muscle, but DT increased to 171 +/- 4%, presenting a staircase response after DS treatment. This potentiation was accompanied by an increase in DT tC-1 to 175.6 +/- 7%. Inhibition of Ca2+ release in atrophied muscle resulted in twitch contractile parameters and contractile responses to 10 Hz stimulation that were similar, in many respects, to those responses in control (non-atrophied) muscles.

Topics: Animals; Calcium; Dantrolene; Electric Stimulation; Female; Muscle Contraction; Muscle Relaxants, Central; Muscle Relaxation; Muscle, Skeletal; Muscular Atrophy; Rats; Rats, Sprague-Dawley; Sarcoplasmic Reticulum; Tetrodotoxin; Time Factors

Staircase potentiation does not occur in atrophied rat gastrocnemius muscle subjected to repetitive stimulation. Since twitch potentiation is strongly correlated with phosphorylation of the regulatory light chains (R-LC) of myosin, our purpose was to determine if R-LC phosphorylation with repetitive stimulation is also diminished in atrophied gastrocnemius muscle. Following 2 weeks of disuse, induced by delivery of tetrodotoxin (TTX) to the sciatic nerve, phosphorylation and twitch developed tension were measured at 10 s of 10-Hz stimulation in situ in control, sham, and TTX-treated rats. Phosphorylation was significantly diminished in TTX-treated animals (9.3 +/- 3.7%), compared with control (57.0 +/- 3.4%) and sham-treated (45.5 +/- 5.6%) animals. Concomitantly, at 10 s of 10-Hz stimulation, potentiation was absent in atrophied muscles (74.4 +/- 9.4% of initial twitch), whereas control (188.4 +/- 10.3%) and sham-treated (151.7 +/- 10.3%) muscles demonstrated a great deal of potentiation. The twitch contraction of the atrophied muscles had a decreased rate of tension development and peak rate of relaxation, and an increased contraction time and half-relaxation time. The observation that the light chains are present, but not phosphorylated, suggests that the process of phosphorylation is impaired. The association of diminished phosphorylation with the absence of staircase supports the theory that R-LC phosphorylation may be the principal mechanism for twitch potentiation but does not rule out other contributing factors.

Topics: Animals; Electric Stimulation; Female; Muscle Contraction; Muscle Relaxation; Muscle, Skeletal; Muscular Atrophy; Myosin Light Chains; Phosphorylation; Rats; Rats, Sprague-Dawley; Tetrodotoxin

It has previously been demonstrated that muscle atrophy associated with aging and disuse is accompanied by changes in microvascular function including absolute loss of capillaries, increased mean red blood cell velocity (VRBC), and absence of reactive hyperemia. The purpose of the present study was to determine whether disuse could account for these changes. The right extensor digitorum longus muscle in male Fisher 344 rats was subjected to 15 days of disuse through the neural application of tetrodotoxin (TTX). Microvascular function, as assessed using intravital microscopy, was compared for muscles from control (n = 8) and TTX-treated (n = 5) animals. The TTX-induced disuse was associated with a 40.5% decrease in muscle weight, a 51.6% decrease in fiber cross-sectional area, a 62% decrease in mitochondrial volume density, and increased capillary damage (TTX, 11% control, 1.1%). Although capillary density in the disused muscle increased (by 139%), when corrected for muscle atrophy, the absolute number of capillaries was maintained. With TTX disuse, VRBC heterogeneity was not different from that in the control rats while the mean velocity increased 3.18x. TTX disuse did not alter the pattern of reactive hyperemia following 30 min of complete ischemia. These results suggest that short-term TTX-induced atrophy affects both microvascular structure and resting state blood flow in rat skeletal muscle, but it does not affect the vascular responsiveness following a metabolic challenge.

Topics: Animals; Blood Flow Velocity; Endothelium, Vascular; Ischemia; Male; Microcirculation; Muscle, Skeletal; Muscular Atrophy; Rats; Rats, Inbred F344; Tetrodotoxin

The roles of innervation, muscle electrical activity, and muscle contraction in regulating the formation and survival of primary and secondary myotubes during embryonic and fetal development of skeletal muscle were studied using the mouse mutants peroneal muscular atrophy (pma) and muscular dysgenesis (mdg). The pma phenotype includes the absence of the peroneal division of the sciatic nerve, so muscles in the anterior compartment of the lower hindlimb are aneural throughout development. Muscles in mdg mice are paralyzed due to the absence of excitation-contraction coupling and hyperinnervated due to suppression of motoneuron death in consequence of their paralysis, but otherwise are electrically excitable and receive synaptic transmission. In a quantitative comparison between control and mutant extensor digitorum longus (EDL) muscles at E15, primary myotube numbers were depressed by 20-30% in both mutants and in paralyzed or denervated muscles from control strain animals. The number of secondary myotubes, however, was normal in pma mutants and two and a half times greater than normal in the hyperinnervated mdg EDL muscles, so that the ratio of secondary to primary myotubes was increased by 300% in the mutant with respect to heterozygous or -/- littermates. Chronic paralysis with tetrodotoxin (TTX) caused no further depression of primary myotube numbers in aneural pma muscles, but secondary myotube numbers were reduced by 40%, reducing the ratio of secondary to primary myotubes by 35%. We conclude that during normal development the generation of secondary myotubes depends on neurally evoked electrical activity in primary myotubes, which stimulates mitosis of secondary myoblasts. The effect of TTX shows that aneural pma primary myotubes discharge spontaneous myogenic action potentials, while mdg muscles may receive greater than normal electrical activation due to their hyperinnervation, explaining the presence and numbers of secondary myotubes in the mutant mouse muscles.

Topics: Action Potentials; Animals; Cell Count; Mice; Mice, Mutant Strains; Muscles; Muscular Atrophy; Muscular Dystrophies; Neurons; Tetrodotoxin

The extent to which the remaining active or passive components of muscle mechanical stress not associated with weightbearing are involved in preserving muscle morphological and functional characteristics in the rodent hindlimb suspension model is not known. Such information would be relevant to the construction of appropriate countermeasures for the disuse atrophy associated with muscle unloading. This question was addressed by superimposing 2 weeks of hindlimb suspension and neuromuscular quiescence, achieved by the chronic neural application of the sodium channel blocker tetrodotoxin. A major portion of the muscle size characteristics of the fast anti-gravity gastrocnemius and plantaris, and the functional characteristics of the plantaris, were maintained by the full range voluntary activity remaining after suspension. Muscle mass of the slow soleus was compromised regardless of this residual activity. Indeed, for fast ankle extensors, hindlimb unloading resembles more closely a model of normal usage than of disuse, but for slow extensors this condition appears to be extremely detrimental.

Topics: Action Potentials; Animals; Body Weight; Electromyography; Female; Hindlimb; Models, Biological; Muscle Contraction; Muscle Denervation; Muscles; Muscular Atrophy; Neuromuscular Junction; Rats; Rats, Inbred Strains; Tetrodotoxin; Weightlessness

The extent of recovery in rat gastrocnemius muscles which have undergone atrophic changes due to 4 weeks of inactivity caused by sciatic nerve superfusion of tetrodotoxin, was investigated. Control and tetrodotoxin-treated female Sprague-Dawley rats were subjected to either a daily program of grid-climbing or swim-training for 4 weeks, or benefited from cage activity only. In situ contractile properties of the gastrocnemius were then measured. Twenty-eight days of recovery was insufficient to allow a complete reversal of the changes produced by 28 days of inactivity. The gastrocnemius remained atrophied (25%) and tetanically weaker than normal in the recovered animals. The maximal rate of rise of the twitch was partially recovered whereas that developed during a maximal contraction was normal. Maximal rate of rise expressed relative to the tension output was elevated, however, for both twitch (%Pt/ms) and maximal contraction (%Po/ms). Swimming did not influence the recovery of muscle size or strength. Grid-climbing, on the other hand, produced heavier and stronger muscles and a faster recovery of %Pt/ms. This study clearly illustrates the importance of muscle load in regulating muscle size and strength.

Topics: Animals; Female; Humans; Muscle Contraction; Muscles; Muscular Atrophy; Physical Exertion; Rats; Rats, Inbred Strains; Swimming; Tetrodotoxin

The effect of disuse on the functional properties of fast-twitch mammalian muscle is controversial, perhaps because the various disuse models reduce activity to different degrees, and may introduce factors other than reduced activity per se. Our goal was to compare the effects of 14 days of disuse produced by neurally applied tetrodotoxin and joint fixation (knee and ankle) on several morphologic and functional characteristics of the rat gastrocnemius. Joint fixation produced a decrease in muscle wet weight and absolute tetanic tension measured in situ, and a preferential atrophy of slow-twitch fibers. The degree of atrophy was more severe with TTX-disuse and affected all fiber types to the same extent. In further contrast to joint fixation, TTX-disuse caused a preferential loss of myofibrillar protein and a decrease in tetanic tension per unit muscle wet weight. In addition, TTX-disuse resulted in an elevation of twitch:tetanic ratio, a prolonged twitch, and generated a relatively higher proportion of tetanic force at 50 Hz. The normalized maximal rate of tetanic tension development (% Po/ms) was highest in the TTX group. The fatigue index was unaffected by either intervention. The data suggest that complete disuse of mammalian fast-twitch muscle causes atrophy, prolongation of the twitch, and a loss in contractile strength per gram of tissue, and are consistent with qualitative or quantitative changes in the sarcoplasmic reticulum and a decrease in myofibrillar protein concentration with disuse.

Topics: Animals; Ankle Joint; Female; Knee Joint; Models, Biological; Muscle Contraction; Muscular Atrophy; Rats; Rats, Inbred Strains; Tetrodotoxin