INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

DURING INSPIRATION, the diaphragm (Dia) muscle shortens, thereby generating negative intrathoracic pressure, airflow, and an increase in lung volume. As with other striated muscles, these functions can be characterized mechanically as the ability to generate force, shorten, produce power, and perform work. Clinically, an inability of the Dia muscle to generate power and perform physiological work may contribute to ventilatory failure in certain experimental or disease states (9, 14, 18) and has been reported to occur with exercise-induced fatigue in healthy humans (11). Previous in vitro studies have demonstrated a decrement in work performance or power output of Dia muscle strips during repetitive activation (15-17, 24). However, power output of muscle depends on both the force generated and load-specific shortening velocity. Therefore, decrements in maximum power output (_max) of the Dia muscle with repetitive activation may reflect either a reduced force and/or a reduced shortening velocity.

Previous studies examining fatigue in rat Dia muscle have determined decrements in isometric force with repeated activation. A major limitation to this approach is that isometric force is a relatively poor index of in vivo Dia muscle function, providing no information concerning the ability of the Dia muscle to shorten and generate power. Other studies making simple comparisons between fatigue due to repetitive isometric and isotonic contractions are limited because of the differences in energetics between those contraction types (1). Furthermore, many studies that have assessed effects of fatigue on maximal force and power production have relied on postfatigue contractions, which may be confounded by recovery (1, 4, 5, 8, 15). Thus these prior approaches have the inherent limitations that 1) some recovery could occur at the conclusion of trial, possibly obscuring the fatigue-induced effects on power capacity, and 2) maximal force and power production could not be assessed simultaneously over time during fatigue.

Thus the major purpose of the present study was to introduce a novel method to evaluate decrements in both maximum specific force (P_o) and _max during repeated activation of the Dia muscle. This approach allows some insight into fatigue as a dynamic process, because the critical variables can be monitored over time, as opposed to a pre- and posttrial evaluation. We hypothesized that reductions in _max during repeated activation would be the result of a disproportionate reduction in P_o, compared with an ability to shorten. The results clearly indicate that the major determinant of power loss with repetitive contractions of the Dia muscle is a decrement in force capacity and suggest that cross-bridge cycling kinetics, as reflected by velocity, are less altered as power capacity declines.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The Animal Care and Use Facility of the Mayo Clinic approved all animal procedures. Adult male Sprague-Dawley rats (~300 g) were anesthetized with pentobarbital sodium (50 mg/kg ip), and the Dia muscle was rapidly excised en bloc with a portion of central tendon and ribs intact. Muscle segments (2-3 mm wide) were dissected from the right midcostal region and immersed in aerated (95% O₂-5% CO₂) modified Rees-Simpson saline solution with the following composition (in mM): 135 Na⁺, 5 K⁺, 2 Ca²⁺, 1 Mg²⁺, 120 Cl, 25 HCO₃, 11 glucose, 0.3 glutamic acid, 0.4 glutamine, 5 N,N'-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid buffer, and 0.012 d-tubocurarine, at 26°C and pH 7.39.

Measurement of Dia muscle contractile properties. The methods for in vitro measurements of isometric and isotonic contractile properties of the rat Dia muscle have been previously described (16, 17, 24). Briefly, aluminum foil was adhered to the central tendon end of the muscle segment by using cyanoacrylate and was secured to a Cambridge dual-mode length-force servo controller (model 300B) via a stainless steel wire (0.0025 in.). The costal end of the segment was attached to a micromanipulator for muscle length adjustment. The muscle segment was stimulated directly (rectangular 0.5-ms duration pulses) by using platinum electrodes connected to a current amplifier (Mayo Section of Engineering). Stimulus intensity was increased until maximum isometric twitch force was achieved and then set to 125% of this value (~250 mA). Muscle length was adjusted until maximum twitch force was achieved [optimal muscle length (L_o) = 20 ± 1 mm]. P_o was obtained at a stimulation rate of 75 Hz (1 s). Forces were normalized for muscle cross-sectional area, which was estimated based on the following calculation (3): cross-sectional area (cm²) = muscle weight (g)/L_o (cm) × 1.056 (g/cm³).

Force-velocity and force-power relationships. The force-velocity relationship of the Dia muscle was determined in one group of rats (n = 8) by using a load-clamp technique with loads ranging from 3 to 100% of P_o (16, 17, 24). At each load clamp level, the muscle segment was stimulated at 75 Hz (600-ms-duration train), and the velocity of shortening was measured over a 30-ms period beginning 10 ms after the initiation of muscle shortening (to eliminate the contribution of series elastance). The force-velocity measurements were least squares fitted to a hyperbolic curve using the Hill equation (10). V_max (expressed as L_o/s) was determined by extrapolation to zero load and gave an indication of the overall average shortening velocity of the Dia muscle segment. _max was subsequently determined from force-velocity data (24). Power output of the Dia muscle at each load was calculated as the product of isotonic load and shortening velocity. The _max of the Dia muscle corresponded to a load of ~30% P_o and a shortening velocity of ~30% V_max, in agreement with prior studies in rat muscle (6, 24). These results served as the basis for selecting a constant velocity of 30% V_max in the fatigue protocol, as described below.

Maximum unloaded shortening velocity. In another group (n = 7), the maximum unloaded shortening velocity (V_o) of the Dia muscle was determined by using the slack test (7, 13). The slack test primarily provides information about the shortening velocity of the fastest fibers within the muscle (2) and is useful in determining their capacity in relation to fatigue. In this procedure, the muscle was first maximally activated under isometric conditions and then rapidly released (<2 ms) to a shorter length by steps of 6, 8, 10, and 12% L_o. During the rapid release, force was dropped to zero, and the muscle was shortened against zero external load. At each length step, the delay between the change in length and the redevelopment of force was measured. The slope of the line relating length change and time for force redevelopment was used to determine V_o.

Fatigue protocol. A different group of rats (n = 8) was used in experiments with a fatigue protocol that was designed such that decrements in P_o and _max could be continuously followed during repetitive activation of the Dia muscle. In this protocol, Dia muscle segments were activated at 75 Hz in 400-ms-duration trains repeated each second (40% duty cycle) for a 2-min period. The 75-Hz stimulation frequency was chosen because it was the frequency at which P_o was achieved. During the initial 360 ms of each train, the muscle was isometrically activated at L_o to allow evaluation of changes in P_o. During the subsequent 40 ms of each train, the muscle was allowed to shorten at a constant velocity of 30% V_max, corresponding to _max. Figure 1 shows a representative example of length and force changes during the last 40 ms of a stimulus train. During the shortening activation, length changes were linear (~7% L_o). However, changes in force were curvilinear, being especially curved within the first portion of the response and less so toward the end. Therefore, power output and work performed were calculated during the last 20 ms of each train, providing continuous assessment of these variables during fatigue. Power was calculated as the product of the force times the isovelocity at which the muscle shortened during each contraction. Work was calculated as the product of that same force multiplied by the distance shortened with each isovelocity contraction. Both power and work variables were expressed as values per cross-sectional area of muscle. Fatigue of the Dia muscle was characterized as the decrement in P_o (measured during the first 360 ms of each train), as well as the decrement in _max (measured during the last 20 ms of the isovelocity shortening period of each train). V_max and V_o were remeasured within 30 s after completion of the fatigue protocol.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Representative example of length (A) and force (B) changes during the isovelocity shortening period. Vertical dotted lines on each plot show the time-sampling window used for collection of velocity and force data in the determination of power output and work for each contraction of the fatigue trial. Lo, optimal muscle length.

Statistical analysis. Values are reported as means ± SE. Changes in P_o and _max during the fatigue protocol were compared by using an ANOVA with repeated-measures design. Pre- and postfatigue values of V_o and V_max were compared by using a paired t-test. Results were considered statistically significant at P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Force fatigue. During the fatigue protocol, the maximum isometric force generated by the Dia muscle progressively decreased (Fig. 2) such that, by the end of the 2-min period, maximum isometric force declined by 75% (Table 1). The force generated by the Dia muscle during constant-velocity shortening at 30% V_max also decreased progressively during the 2-min fatigue protocol. During the first stimulus train, the force generated at 30% V_max was 6.8 ± 0.6 N/cm². After 2 min of repetitive activation, only 1.8 ± 0.2 N/cm² force was generated during constant-velocity shortening (74% decrease).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Force decline of diaphragm (Dia) muscle during repetitive isometric/isovelocity contractions. Isovelocity contraction was performed at a velocity of 30% Vmax. The tetanic force of Dia muscle was expressed as N/cm2 (A) and as a percentage of initial force (B).

Velocity fatigue. The V_max of the Dia muscle was calculated based on extrapolation of the force-velocity relationship to zero load (Fig. 3A). After 2 min of repetitive activation, the force-velocity relationship of the Dia muscle was shifted leftward, and V_max slowed by 43% (Fig. 3A; Table 1). The V_o of the Dia muscle, determined by the slack test (Fig. 4), was 27% faster than V_max (Table 1). After 2 min of repetitive activation, V_o of the Dia muscle was also reduced, but to a lesser extent than was V_max (P < 0.05).

View larger version (18K): [in this window] [in a new window]   Fig. 3.   A: force-velocity relationship of Dia muscle (pre- vs. postfatigue protocol). Vmax was estimated by extrapolating the force-velocity curve to zero load. B: force-power relationship of Dia muscle (pre- vs. postfatigue protocol). Maximum prefatigue power output was generated at ~30% maximum specific force and ~30% Vmax.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Determination of maximum unloaded shortening velocity (Vo) of Dia muscle using the slack test. A: decrement in Vo with fatigue, as assessed by plot of force redevelopment time vs. length steps. Individual responses to length steps in same muscle prefatigue (B) and postfatigue (C) are shown. Examples show the linear relationship between the muscle length step (6-12% Lo) and the time for force redevelopment.

Power fatigue. The reduction in curvature and leftward shift of the normalized force-velocity relationship as a consequence of fatigue resulted in a reduction and leftward shift in the force-power relationship (Fig. 3B). Throughout the period of repetitive activation, power output of the Dia muscle progressively declined, decreasing by 73% after 2 min (Fig. 5, Table 1). After 2 min of repetitive activation, the work capacity of the Dia muscle decreased by a similar magnitude.

View larger version (10K): [in this window] [in a new window]   Fig. 5.   Power decline of Dia muscle during repetitive isometric/isovelocity contractions, expressed as percentage of initial power.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

The results of the present study indicate that the decrease in _max during repetitive activation of the rat Dia muscle is due primarily to declines in force, and that velocity decrements play only a secondary role. With repetitive activation, decrements in _max and P_o are comparable (73-75%), whereas the slowing of shortening velocity is proportionately less (V_max 43% and V_o 22%). Consequently, the decrement in _max of the rat Dia muscle with repetitive activation appears to reflect primarily either impaired cross-bridge recruitment or reduced force per cross bridge. A slowing of cross-bridge cycling rate, although significant, is of secondary importance in the decline in _max. Based on smaller reductions in V_o compared with V_max, we also conclude that fatigue of the Dia muscle cannot simply be attributed to an inactivation of fast, fatigable fiber types.

The major advantage of our fatigue protocol is that it allowed continuous observation and quantification of maximal force and power production of the Dia muscle during a repetitive contraction trial. This is important because the Dia muscle must shorten, produce power, and perform work to generate negative intrathoracic pressure, airflow, and increases in tidal volume in vivo. Furthermore, it allowed us to avoid confounding effects of recovery in the assessment of the effects of fatigue on power production. This contrasts with the use of repetitive isometric contractions, which are of limited relevance to the function of this muscle, especially given that, clinically, it is the inability of the Dia muscle to shorten, generate pressure, and perform work that may lead to respiratory failure.

The results of the present study, which clearly indicates that the decrement in _max is primarily attributed to a decrement in force generation, suggest intracellular mechanisms of fatigue. Force generation reflects cross-bridge recruitment, which is dependent on elevation of intracellular calcium concentration ([Ca²⁺]_i). Therefore, a decrease in force during repetitive activation may reflect either a decrease in [Ca²⁺]_i or a reduction in force generated at any level of [Ca²⁺]_i (reduced Ca²⁺ sensitivity).

Metabolites produced as a result of repetitive activations may have influenced Ca²⁺ release and force generation in the present study. For example, during repetitive activation of muscle fibers, ATP hydrolysis results in the accumulation of hydrogen ions (H⁺) and P_i. Westerblad and Allen reported that intracellular acidification (21) and injection of P_i into the myoplasm of intact mouse skeletal muscle fibers decreased both maximum force production and Ca²⁺ sensitivity (20). Thus the force fatigue observed in the present study is consistent with increases in H⁺ and P_i concentrations. Unfortunately, the effect on shortening velocity of P_i injection or intracellular acidification was not assessed in these studies (20, 21). Consequently, it is impossible to make firm conclusions regarding the influence of these intracellular mechanisms on the force and velocity fatigue observed in the present study.

In studies in which Ca²⁺-sensitive fluorescent dyes have been used to measure [Ca²⁺]_i in intact skeletal muscle fibers, it has been demonstrated that repetitive activation causes an eventual reduction in sarcoplasmic reticulum (SR) Ca²⁺ release (12, 19). The reduction in [Ca²⁺]_i appears to be due to reduced SR Ca²⁺ release secondary to impairments in SR Ca²⁺ uptake, or to a reduced availability of intraluminal Ca²⁺ from within the SR. In this last regard, the injection of P_i decreased the [Ca²⁺]_i of intact mouse skeletal muscle fibers, suggesting that P_i may impair Ca²⁺ release from the SR. Thus declines in Ca²⁺ release over time during repetitive stimulation may have contributed to the force fatigue observed. Reductions in [Ca²⁺]_i have been reported to have no effect on reducing V_o of intact skeletal muscle fibers. For example, Edman (7) and Westerblad et al. (22) have provided evidence that V_o of intact skeletal muscle fibers from the frog and mouse, respectively, is not reduced by the Ca²⁺ release antagonist dantrolene sodium, suggesting that [Ca²⁺]_i does not influence V_o in living fibers. Thus a reduction in Ca²⁺ release, although possibly an important determinant of force loss, would not necessarily affect velocity. This mechanism may explain the large drop in maximal force and relatively smaller drop in maximal velocity observed in the present study.