Power fatigue of the rat diaphragm muscle.

We hypothesized that decrements in maximum power output (W(max)) of the rat diaphragm (Dia) muscle with repetitive activation are due to a disproportionate reduction in force (force fatigue) compared with a slowing of shortening velocity (velocity fatigue). Segments of midcostal Dia muscle were mounted in vitro (26 degrees C) and stimulated directly at 75 Hz in 400-ms-duration trains repeated each second (duty cycle = 0.4) for 120 s. A novel technique was used to monitor instantaneous reductions in maximum specific force (P(o)) and W(max) during fatigue. During each stimulus train, activation was isometric for the initial 360 ms during which P(o) was measured; the muscle was then allowed to shorten at a constant velocity (30% V(max)) for the final 40 ms, and W(max) was determined. Compared with initial values, after 120 s of repetitive activation, P(o) and W(max) decreased by 75 and 73%, respectively. Maximum shortening velocity was measured in two ways: by extrapolation of the force-velocity relationship (V(max)) and using the slack test [maximum unloaded shortening velocity (V(o))]. After 120 s of repetitive activation, V(max) slowed by 44%, whereas V(o) slowed by 22%. Thus the decrease in W(max) with repetitive activation was dominated by force fatigue, with velocity fatigue playing a secondary role. On the basis of a greater slowing of V(max) vs. V(o), we also conclude that force and power fatigue cannot be attributed simply to the total inactivation of the most fatigable fiber types.     

Isotonic contractile and fatigue properties of developing rat diaphragm muscle

Postnatal transitions in myosin heavy chain (MHC) isoformexpression were found to be associated with changes in both isometric and isotonic contractile properties of rat diaphragm muscle(Dia_m). Expression of MHC_neo predominated inneonatal Dia_m fibers but was usually coexpressed withMHC_slow or MHC_2A isoforms. Expression ofMHC_neo disappeared by day 28. Expression ofMHC_2X and MHC_2B emerged at day 14 andincreased thereafter. Associated with these MHC transitions in theDia_m, maximum isometric tetanic force (P_o), maximum shortening velocity, and maximum power output progressively increased during early postnatal development. Maximum power output ofthe Dia_m occurred at ~40% P_o at days0 and 7 and at ~30% P_o in older animals.Susceptibility to isometric and isotonic fatigue, defined as a declinein force and power output during repetitive activation, respectively,increased with maturation. Isotonic endurance time, defined as the timefor maximum power output to decline to zero, progressively decreasedwith maturation. In contrast, isometric endurance time, defined as thetime for force to decline to 30-40% P_o, remained>300 s until after day 28. We speculate that with thepostnatal transition to MHC_2X and MHC_2Bexpression energy requirements for contraction increase, especiallyduring isotonic shortening, leading to a greater imbalance betweenenergy supply and demand.

     

Denervation alters myosin heavy chain expression and contractility of developing rat diaphragm muscle.

We hypothesized that unilateral denervation (DNV) of the rat diaphragm muscle (Dia(m)) in neonates at postnatal day 7 (D-7) alters normal transitions of myosin heavy chain (MHC) isoform expression and thereby affects postnatal changes in maximum specific force (P(o)) and maximum unloaded shortening velocity (V(o)). The relative expression of different MHC isoforms was analyzed electrophoretically. With DNV at D-7, expression of MHC(neo) in the Dia(m) persisted, and emergence of MHC(2X) and MHC(2B) was delayed. By D-21 and D-28, relative expression of MHC(2A) and MHC(2B) was reduced in DNV compared with control (CTL) animals. Expression of MHC(neo) also reappeared in adult Dia(m) by 2-3 wk after DNV, and relative expression of MHC(2B) was reduced. At each age, P(o) was reduced and V(o) was slowed by DNV, compared with CTL. In CTL Dia(m), postnatal changes in P(o) and V(o) were associated with an increase in fast MHC isoform expression. In DNV Dia(m), no such association existed. We conclude that, in the Dia(m), DNV induces alterations in both MHC isoform expression and contractile properties, which are not necessarily causally linked.     

Effect of nutritional deprivation on diaphragm contractility and muscle fiber size

The influence of nutritional deprivation on the contractile and fatigue properties of the diaphragm was studied in adult rats. Food access was restricted to one-third of normal daily intake until the body weight of nutritionally deprived (ND) animals was approximately 50% of controls (CTL). Isometric contractile properties were studied in an in vitro nerve muscle strip preparation. Both twitch (Pt) and tetanic (Po) tensions of diaphragms from the ND animals were markedly reduced compared with CTL; however, Pt/Po was higher for the ND group. The shape of the force-frequency curve (normalized to Po) was generally similar between the two groups, except at 5 and 10 pulses/s stimulation, where greater relative tensions were produced in diaphragms from the ND animals. Diaphragm fatigue was induced by repetitive stimulation at either 20 or 100 pulses/s. Endurance time (defined as the time required for tension to fall to 50% of initial) of diaphragms from ND animals was prolonged at both 20 and 100 pulses/s. Immediately after induction of fatigue, force-frequency curves for both ND and CTL diaphragms were shifted to the right. However, this rightward shift was attenuated in the ND group compared with CTL. Nutritional deprivation had no effect on the proportions of different fiber types within the diaphragm but did result in a significant decrease in the cross-sectional area of both fast-and slow-twitch fibers. This decrease in cross-sectional area was significantly greater for fast-twitch fibers. We conclude that these changes in diaphragm contractile and fatigue properties occur as a result of the influence of malnutrition on muscle fiber cross-sectional area.     

Respiratory sensation and pattern of respiratory muscle activation during diaphragm fatigue

We have examined the relationship between respiratory effort sensation (modified Borg scale) and amplitude of the integrated surface electromyogram of the diaphragm (Edi, esophageal electrode), rib cage muscles (Erc), and sternomastoid muscle (Esm) during the development of diaphragm fatigue in five normal subjects. Three conditions were studied: run A: transdiaphragmatic pressure (Pdi), 65% Pdimax; esophageal pressure (Pes), 60% Pesmax; run B: Pdi, 50% Pdimax; Pes, 60% Pesmax; and run C: Pdi, 50% Pdimax; Pes, 20% Pesmax. During all runs there was a progressive rise in sensation, which was greater in runs A and B than in run C (P less than 0.05, analysis of variance). There was no difference between runs A and B. At the end of run C subjects did not report a maximal Borg score despite their inability to generate the target Pdi. The increase in sensory score with fatigue correlated highly with Esm/Esmmax and with Erc/Ercmax. There was no correlation between sensory score and Edi/Edimax. We conclude that the increase in respiratory effort sensation that accompanies diaphragm fatigue is not due to perception of increased diaphragmatic activation. It may reflect increased overall respiratory motor output not directed to the diaphragm.     

Theophylline, fatigue, and diaphragm contractility: cellular levels of 45Ca and cAMP.

The relationship between variations in diaphragmatic contractility and corresponding changes in total tissue levels of 45Ca and adenosine 3',5'-cyclic monophosphate (cAMP) was examined. The contractile performance of perfused contracting rat diaphragms was manipulated with theophylline (10(-4) M), induced fatigue, or both. The increased contractility associated with theophylline was related to significant increases in 45Ca levels without changes in cAMP levels. Fatigue-diminished contractility was associated with increases in both 45Ca and cAMP levels. The increased 45Ca and cAMP levels associated with fatigue persisted, even in the presence of theophylline. Calcium channel blockade with 10(-4) M verapamil blocked the positive inotropic influence of theophylline as well as the theophylline-associated increase in 45Ca levels. Verapamil had no effect on either the fatigue-associated decreases in contractility or the fatigue-enhanced 45Ca uptake. The results of this study strongly suggest that the enhanced contractility associated with theophylline is related to its influence on cellular calcium metabolism. The elevated level of isotopic calcium measured in fatigued muscle probably represents calcium sequestered in the sarcoplasmic reticulum, the result of cAMP-enhanced Ca-adenosine triphosphatase activity.     

Effects of respiratory muscle unloading on exercise-induced diaphragm fatigue.

We previously compared the effects of increased respiratory muscle work during whole body exercise and at rest on diaphragmatic fatigue and showed that the amount of diaphragmatic force output required to cause fatigue was reduced significantly during exercise (Babcock et al., J Appl Physiol 78: 1710, 1995). In this study, we use positive-pressure proportional assist ventilation (PAV) to unload the respiratory muscles during exercise to determine the effects of respiratory muscle work, per se, on exercise-induced diaphragmatic fatigue. After 8-13 min of exercise to exhaustion under control conditions at 80-85% maximal oxygen consumption, bilateral phrenic nerve stimulation using single-twitch stimuli (1 Hz) and paired stimuli (10-100 Hz) showed that diaphragmatic pressure was reduced by 20-30% for up to 60 min after exercise. Usage of PAV during heavy exercise reduced the work of breathing by 40-50% and oxygen consumption by 10-15% below control. PAV prevented exercise-induced diaphragmatic fatigue as determined by bilateral phrenic nerve stimulation at all frequencies and times postexercise. Our study has confirmed that high- and low-frequency diaphragmatic fatigue result from heavy-intensity whole body exercise to exhaustion; furthermore, the data show that the workload endured by the respiratory muscles is a critical determinant of this exercise-induced diaphragmatic fatigue.     

Effects of chronic nicotine exposure on electrogenic activity of the Na+, K(+)-ATPase and contractility in the rat diaphragm muscle

Rats were chronically treated with nicotine via subcutaneous injections up to a dose 6 mg/kg/day during 2-3 weeks. After this period, resting membrane potential and action potentials of muscle fibres as well as isometric twitch and tetanic (20 s(-1) and 50(-1)) contractions of isolated rat diaphragm were studied. To estimate electrogenic contribution of the alpha2 isoform of the Na+, K(+)-ATPase ouabain in concentration 1 microM was used. Chronic nicotine exposure induced depolarization of resting membrane potential of 2.2 +/- 0.6 mV (p < 0.01). In rats chronically exposed to nicotine, electrogenic contribution of the Na+, K(+)-ATPase alpha2 isoform was twofold lesser than in control animals (3.7 +/- 0.6 mV and 6.4 +/- 0.6 mV, respectively, p < 0.01). Chronic nicotine exposure did not affect force of twitch and tetanic contractions in response to direct or indirect stimulation. A decrease in the twitch contraction time as well as in the rise time of tetanic contractions was observed. Fatigue dynamics was unchanged. The results suggest that chronic nicotine exposure leads to decrease of the Na+, K(+)-ATPase alpha2 isoform electrogenic activity, and as a consequence to damage of the rat diaphragm muscle electogenesis.     

Comparison of some metabolic parameters in the perfused and the incubated rat diaphragm muscle with diaphragm muscle in vivo

1. A method is described for perfusing the rat diaphragm muscle. 2. The following parameters were compared in both perfused and non-perfused incubated preparations: water content, sorbitol space, rate of lactate production, and the concentrations of tissue glucose, pyruvate, lactate, hexose phosphate intermediates, ATP and AMP. No significant differences were found. 3. Significant differences, however, were found on comparison of the tissue kept in vitro with the tissue in vivo. Immediately after removal of the tissue from the animal, the concentrations of the hexose phosphates and ATP were found to be much higher than after incubation or perfusion, and the concentrations of free glucose and of AMP were much lower, possibly indicating that the capacity for oxidative phosphorylation of glucose is impaired in vitro because of hypoxia.     

Temperature dependence of mammalian muscle contractions and ATPase activities

Isolated rat and mouse extensor digitorum longus (EDL) and soleus muscles were studied under isometric and isotonic conditions at temperatures from approximately 8 degrees -38 degrees C. The rate constant for the exponential rise of tension during an isometric tetanus had a Q10 of approximately 2.5 for all muscles (corresponding to an enthalpy of activation, delta H = 66 kJ/mol, if the rate was determined by a single chemical reaction). The half-contraction time, contraction time, and maximum rate of rise for tension in an isometric twitch and the maximum shortening velocity in an isotonic contraction all had a similar temperature dependence (i.e., delta H approximately 66 kJ/mol). The Mg++ ATPase rates of myofibrils prepared from rat EDL and soleus muscles had a steeper temperature dependence (delta H = 130 kJ/mol), but absolute rates at 20 degrees C were lower than the rate of rise of tension. This suggests that the Mg++ ATPase cycle rate is not limiting for force generation. A substantial fraction of cross-bridges may exist in a resting state that converts to the force-producing state at a rate faster than required to complete the cycle and repopulate the resting state. The temperature dependence for the rate constant of the exponential decay of tension during an isometric twitch or short tetanus (and the half-fall time of a twitch) had a break point at approximately 20 degrees C, with apparent enthalpy values of delta H = 117 kJ/mol below 20 degrees C and delta H = 70 kJ/mol above 20 degrees C. The break point and the values of delta H at high and low temperatures agree closely with published values for the delta H of the sarcoplasmic reticulum (SR) Ca++ ATPase. Thus, the temperature dependence for the relaxation rate of a twitch or a short tetanus is consistent with that for the reabsorption rate of Ca++ into the SR.     

Salbutamol enhances isotonic contractile properties of rat diaphragm muscle

The effects of the₂-adrenoceptor agonistsalbutamol (Slb) on isometric and isotonic contractile properties ofthe rat diaphragm muscle(Dia_mus) were examined. Aloading dose of 25 µg/kg Slb was administered intracardially beforeDia_mus excision to ensure adequatediffusion. Studies were then performed with 0.05 µMSlb in the in vitro tissue chamber. cAMP levels were determined by radioimmunoassay. Compared with controls (Ctl), cAMP levels were elevated after Slb treatment. In Slb-treated rats, isometric twitch andmaximum tetanic force were increased by ~40 and ~20%,respectively. Maximum shortening velocity increased by ~15% afterSlb treatment, and maximum power output increased by ~25%. Duringrepeated isotonic activation, the rate of fatigue was faster in theSlb-treated Dia_mus, but bothSlb-treated and Ctl Dia_musfatigued to the same maximum power output. Still, endurance time duringrepetitive isotonic contractions was ~10% shorter in the Slb-treatedDia_mus. These results areconsistent with the hypothesis that -adrenoceptor stimulation by Slbenhances Dia_mus contractility andthat these effects of Slb are likely mediated, at least in part, byelevated cAMP.

     

Effects of modulation of nitric oxide on rat diaphragm isotonic contractility during hypoxia.

Nitric oxide (NO) is essential for optimal myofilament function of the rat diaphragm in vitro during active shortening. Little is known about the role of NO in muscle contraction under hypoxic conditions. Hypoxia might increase the NO synthase (NOS) activity within the rat diaphragm. We hypothesized that NO plays a protective role in isotonic contractile and fatigue properties during hypoxia in vitro. The effects of the NOS inhibitor N(G)-monomethyl-l-arginine (l-NMMA), the NO scavenger hemoglobin, and the NO donor spermine NONOate on shortening velocity, power generation, and isotonic fatigability during hypoxia were evaluated (Po(2) approximately 7 kPa). l-NMMA and hemoglobin slowed the shortening velocity, depressed power generation, and increased isotonic fatigability during hypoxia. The effects of l-NMMA were prevented by coadministration with the NOS substrate l-arginine. Spermine NONOate did not alter isotonic contractile and fatigue properties during hypoxia. These results indicate that endogenous NO is needed for optimal muscle contraction of the rat diaphragm in vitro during hypoxia.