We hypothesized that inactivity effects on diaphragm muscle contractile and morphometric properties are attenuated if phrenic motoneurons are also inactive. Three models of rat diaphragm inactivity were compared: 1) spinal isolation; 2) tetrodotoxin (TTX) nerve blockade; and 3) denervation (Dnv). Motoneuron and muscle fiber inactivities were matched only in spinal isolated animals. After 2 wk, maximum tetanic force decreased in all three groups compared with control group but to a greater extent in TTX and Dnv animals. Fatigue resistance improved, and maximum unloaded shortening velocity slowed only in TTX and Dnv groups. Type IIa fiber proportions decreased in all three groups, and type IIx fiber proportions increased in TTX and Dnv animals. Type I fiber cross-sectional area increased in all three groups but to a greater extent in TTX and Dnv animals. Type IIa fibers hypertrophied, whereas type IIx and IIb fibers atrophied only in TTX and Dnv groups. These results support the hypothesis that muscle adaptations to prolonged inactivity are attenuated when muscle fiber and motoneuron inactivities are matched.

We hypothesized that metabolic adaptations to muscle inactivity are most pronounced when neurotrophic influence is disrupted. In rat diaphragm muscle (Dia(m)), 2 wk of unilateral denervation or tetrodotoxin nerve blockade resulted in a reduction in succinate dehydrogenase (SDH) activity of type I, IIa, and IIx fibers (approximately 50, 70, and 24%, respectively) and a decrease in SDH variability among fibers (approximately 63%). In contrast, inactivity induced by spinal cord hemisection at C2 (ST) resulted in much less change in SDH activity of type I and IIa fibers (approximately 27 and 24%, respectively) and only an approximately 30% reduction in SDH variability among fibers. Actomyosin adenosinetriphosphatase (ATPase) activities of type I, IIx, and IIb fibers in denervated and tetrodotoxin-treated Dia(m) were reduced by approximately 20, 45, and 60%, respectively, and actomyosin ATPase variability among fibers was approximately 60% lower. In contrast, only actomyosin ATPase activity of type IIb fibers was reduced (approximately 20%) in ST Dia(m). These results suggest that disruption of neurotrophic influence has a greater impact on muscle fiber metabolic properties than inactivity per se.

In the rat diaphragm muscle, the histochemical classification of type I, IIa, IIb, or IIx fibers was correlated with myosin heavy chain (MHC) immunoreactivity. Expression of MHC isoforms in single dissected fibers was also assessed electrophoretically. Most fibers (approximately 86%) expressed a single MHC isoform, and when present, coexpression of MHC-2X and MHC-2B isoforms was most prevalent. Type I and IIa fibers were the smallest, type IIb fibers were the largest, and type IIx fibers were intermediate. Succinate dehydrogenase (SDH) and calcium-activated myosin adenosinetriphosphatase (actomyosin ATPase) activities were measured with quantitative histochemical procedures. Type I and IIa fibers had the highest SDH activities, followed in rank order by type IIx and IIb fibers. Type I fibers had the lowest actomyosin ATPase activity, followed in rank order by type IIa, IIx, and IIb fibers. Across all fibers, there was an inverse relationship between fiber SDH activity and cross-sectional area and a positive correlation between fiber actomyosin ATPase activity and cross-sectional area. The SDH and actomyosin ATPase activities of muscle fibers were also inversely correlated. These phenotypic differences in SDH and ATPase activities may be important in determining the contractile and fatigue properties of different fiber types in the rat diaphragm muscle.

Using real-time confocal microscopy, we examined the dynamic intracellular Ca2+ concentration ([Ca2+]i) response of porcine tracheal smooth muscle (TSM) cells to acetylcholine (ACh). Exposure to ACh caused regenerative, propagating [Ca2+]i oscillations. The amplitude and fall time of the [Ca2+]i oscillations were inversely correlated to basal [Ca2+]i, whereas the frequency and rise time were directly correlated to basal [Ca2+]i. ACh-induced [Ca2+]i oscillations were initiated in the absence of extracellular Ca2+ and after membrane depolarization with KCl, suggesting that 1) [Ca2+]i oscillations primarily arise by release from internal stores such as the sarcoplasmic reticulum (SR), and 2) Ca2+ influx is necessary for maintenance of oscillations. Exposure to both caffeine and ryanodine inhibited ongoing ACh-induced [Ca2+]i oscillations, suggesting a role for caffeine-sensitive ryanodine receptor (RyR) SR Ca2+ channels. Inhibition of SR Ca2+ reuptake by thapsigargin increased basal [Ca2+]i and decreased [Ca2+]i oscillation amplitude, suggesting that Ca2+ reuptake is also essential. The present results suggest that [Ca2+]i oscillations in porcine TSM cells involve repetitive Ca2+ release and reuptake from RyR channels, perhaps through a Ca2+ -induced Ca2+ release mechanism.