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J Appl Physiol 94: 612-620, 2003. First published October 18, 2002; doi:10.1152/japplphysiol.00441.2002
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Vol. 94, Issue 2, 612-620, February 2003

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

Xiaoping Zhu1,2, Leo M. A. Heunks1, Herwin A. Machiels1, Leo Ennen1, and P. N. Richard Dekhuijzen1

1 Department of Pulmonary Diseases, University Medical Centre Nijmegen, 6500 HB Nijmegen, The Netherlands; and 2 Department of Pulmonary Diseases, NingXia Medical College Hospital, 750004 Yinchuan, NingXia, China


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 NG-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 (PO2 ~ 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.

NG-monomethyl-L-arginine; hemoglobin; spermine NONOate


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

SKELETAL MUSCLE, INCLUDING the diaphragm, continuously produces nitric oxide (NO) (3, 26). NO is produced in biological systems via the enzymatic action of NO synthase (NOS) (32). NO generation by the constitutive NOS isoforms (neuronal NOS and endothelial NOS) is calcium dependent (16). NO production within skeletal muscle is enhanced during contractile activity (5).

NO has been shown to modulate contractile properties of skeletal muscle in vitro. The overall effect depends on the experimental paradigm. Inhibition of NOS increases twitch and submaximal tetanic force of the rat diaphragm. These alterations are reversed by NO donors (26), suggesting that NO inhibits excitation-contraction coupling in unfatigued muscle. On the other hand, NO is essential for optimal myofilament function in the rat diaphragm during active shortening in vitro (36). Inhibition of NOS decreases the shortening velocity and power generation of the rat diaphragm under hyperoxic conditions (36). These findings of NOS inhibition on force generation and velocity of shortening indicate multiple possible targets for NO in skeletal muscle. In fatiguing contractions, supplementation with exogenous NO slowed the decline of maximal force in mouse soleus muscle (38). This finding suggests that NO preserves skeletal muscle function in vitro during strenuous contractile activity.

Hypoxia impairs force generation and accelerates skeletal muscle fatigue in vitro (21, 48). To date, no study has investigated the role of NO in shortening velocity or isotonic fatigability during hypoxia. This is of particular interest because NO affects excitation-contraction coupling in striated muscle (26), and O2 tension is an important factor in regulating NOS in an in vitro system (19). Furthermore, the isotonic contractile properties better reflect diaphragm muscle performance in vivo (46). Hypoxia impairs the sarcoplasmic reticulum (SR) Ca2+ reuptake from the intracellular space (12, 52). This could result in an increase of intracellular Ca2+ concentration levels ([Ca2+]i) and may, in turn, potentiate NO production by Ca2+-dependent NOS (19). Based on previous studies showing that NO facilitates the shortening velocity and preserves muscle function (36, 38), we hypothesized that NO has a protective role in hypoxia-mediated modulation of muscle contraction in the rat diaphragm in vitro. Accordingly, the aim of the present study was to investigate the effects of the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA), the NO scavenger hemoglobin, and the NO donor spermine NONOate (Sp-NO) on the shortening velocity, power generation, and fatigue endurance of rat diaphragm during hypoxia in vitro.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

General Procedures

Adult male outbred Wistar rats with a mean body weight of 310 ± 10 (SE) g were used. The rats were anesthetized with pentobarbital sodium (70 mg/kg body wt ip). Diaphragm bundles were prepared as previous described (21). Briefly, a tracheotomy was performed, and a polyethylene cannula was inserted. The animals were mechanically ventilated with 100% oxygen. The diaphragm and adherent lower ribs were quickly excised and were immediately submersed in cooled oxygenated (95% O2-5% CO2) Krebs solution at pH ~ 7.4. This Krebs solution consisted of 137 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 1 mM KH2PO4, 24 mM NaHCO3, 7 mM glucose, and 25 µM d-tubocurarine (Sigma, Bornem, Belgium). One rectangular strip was dissected from the central costal region of the hemidiaphragm. Silk sutures were tied firmly to both ends.

Contractile Measurements

Contractile properties were measured as described previously (21). Briefly, the insertion of the muscle bundles at the costal margin was attached to a metal clamp. The suture attached to the central tendon was connected to the lever arm (model 308B, Cambridge Technologies). The Cambridge system was controlled by using the software program Poly 5.0 (Inspektor Research Systems, Amsterdam, The Netherlands). Length and force outputs were digitized by using a data-acquisition board (DASH 1602, Keithley) at a sampling frequency of 2.0 kHz. The muscle was stimulated directly with platinum plate electrodes. Rectangular current pulses (0.5 ms) were generated by a stimulator (ID-electronics, University of Nijmegen) activated by a personal computer. The strip was stimulated 1.25 times the current needed for maximal activation (~200-250 mA). Muscle preload force was adjusted until the optimal fiber length (Lo) for maximal twitch force (Pt) was achieved.

The Cambridge system was first set for length control (isometric mode). After 15 min of thermoequilibration during hyperoxia (95% O2 and 5% CO2; 26°C), Pt and maximal tetanic force at 100 Hz (Po) were measured twice with a 2-min interval. Subsequently, the perfusion of the tissue bath was either maintained with hyperoxia or switched to hypoxia (95% N2 and 5% CO2; 26°C); the Krebs solution was changed to the experimental conditions (see treatment groups below). After either 60 or 30 min of incubation, Pt and Po were remeasured. The Cambridge system was then set for force control (isotonic mode). The muscle was simulated at 100 Hz (330-ms train duration) while force was clamped at different levels ranging from 1 to 100% of Po. There was a 2-min interval between each force clamp level. The muscle shortening velocity at each load clamp is expressed as muscle lengths per second (Lo/s). To determine isotonic fatigue, the load clamp was set for maximal power output (~33.3% Po), and the muscle was stimulated at 100 Hz (330-ms train duration) every 2 s. Stimulations continued until no muscle shortening could be observed, and this period was defined as the isotonic endurance time.

Effects of L-NMMA, Hemoglobin, L-Arginine, and Sp-NO

The effects of L-NMMA (AcOH, Calbiochem, Breda, The Netherlands), hemoglobin (bovine, Sigma Chemical, Zwijndrecht, the Netherlands), and Sp-NO [N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine; NOC-22; Calbiochem, Breda, The Netherlands] on the isotonic contractile and fatigue properties were determined during hypoxia. Previously, it was shown that 10 µM L-NMMA decreased the release of NO in isolated rat extensor digitorum longus (EDL) muscle by 68% after 1-h incubation (5) and that the NOS activity in the diaphragm is lower than that in EDL muscle (26). Therefore, a final concentration of L-NMMA of 10 µM and an incubation time of 60 min were used in the present study. To exclude the NOS-independent effects of L-NMMA, 30 µM L-arginine, the NOS substrate (Sigma Chemical), were administrated simultaneously to the Krebs solution, because L-arginine in a threefold concentration completely reverses the effect of L-NMMA on endothelium-dependent contractions in vitro (8, 42). To assess the effects of hypoxia on contractile properties, similar measurements were performed under untreated hyperoxic conditions (with 60 min of incubation).

To ascertain that the effects of L-NMMA on contractile properties and isotonic fatigability were indeed mediated by NOS inhibition, similar experiments were performed with hemoglobin. Hemoglobin (150 µM) was administrated based on the previous observation from the study of Kobzik et al. (26). Hemoglobin (50 µM) did not significantly increase the force generation, but 50-250 µM did enhance submaximal force generation in normal diaphragm.

Moreover, the effects of Sp-NO (1 mM) were tested to determine whether the contractile properties were also sensitive to exogenous NO. Sp-NO was added directly to the solution in the tissue bath (45). Compared with other nucleophilic adducts, Sp-NO releases NO slowly (half-life time: 39 min) (31). The action of these agents correlates strongly with the amount of NO that they release in aqueous buffers (35). The maximal vasodilator effect in rabbit aortic rings was achieved 15 min after the addition of Sp-NO. This effect remained constant for 60 min (35). Under physiological conditions, Sp-NO generates ~1.9 mol of NO per mole of adducts (high extent of NO release), but the concentration of Sp-NO to produce 50% relaxation in aortic rings is higher than other nucleophilic adducts (31). Based on these physicochemical properties and previous studies in skeletal muscle (20), 30 min of incubation time and a concentration of 1 mM Sp-NO were used to produce NO in the present study. The effects of L-NMMA, L-arginine, and Sp-NO on isotonic contractile and fatigue properties were compared with that in standard Krebs solution during hypoxia.

Accordingly, seven experimental groups were studied: hyperoxia control (n = 6), hypoxia control for L-NMMA experiment (n = 8), hypoxia plus L-NMMA (n = 8), hypoxia plus L-NMMA plus L-arginine (n = 8), hypoxia control for Sp-NO experiment (n = 8), hypoxia plus Sp-NO (n = 8), and hypoxia plus hemoglobin (n = 7). The diaphragm bundles were randomly allocated to the treatment groups.

To verify the experimental conditions in the present study, PCO2 and PO2 of the Krebs solutions were measured after completion of the contractile experiments. pH was measured at regular intervals throughout the experimental protocol. The system used in the present study was a flow-through chamber that was continuously bubbled with gas mixtures.

Data Treatment and Statistics

After each experiment, muscle bundle length and weight were determined. Cross-sectional area was calculated by dividing diaphragm strip weight (in g) by strip length (in cm) times specific density (1.056). Force is expressed as per cross-sectional area (in N/cm2). The time window for shortening velocity measurements was set to begin 10 ms after the first detectable change in length. Force-velocity characteristics were plotted with respect to force (P)/Po. The data were fitted to the Hill (23) equation: (P + a) (V + b) = (Po + a) b, where Po is maximum isometric force, V is velocity of shortening, and a and b are constants with dimension of force and velocity, respectively. The curvature of the force-velocity relationship, a/Po, can be derived from this equilibration. The constants b and a were calculated by using a computer program (Fig. P for Windows version 2.7; Fig. P Software, Durham, NC) that fitted the data to the Hill equation. Power [force (N/cm2) × shortening velocity (Lo/s)] was calculated for each load clamp release and plotted with respect to load.

Differences in single baseline contractile properties, bundle dimensions, and gas pressure among the experimental groups were analyzed with one-way ANOVA and, if appropriate, Student-Newman-Keuls post hoc testing. Parameters requiring repeated measures over time (e.g., force-velocity, force-power, fatigue) were estimated by using repeated-measures models and, if appropriate, Student-Newman-Keuls post hoc testing. Statistical analysis was performed with the SPSS package version 10.0 (SPSS, Chicago, IL). Data are expressed as means ± SE. Comparisons were considered significant at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Verification of Tissue Bath Hypoxia and Strip Dimensions

Perfusion of the tissue baths with the hypoxic gas mixture reduced PO2 in the Krebs solution to ~7.2 ± 0.2 kPa in all of the hypoxic groups, compared with 84.8 ± 0.9 kPa in the hyperoxic group (P < 0.001). No differences were found in PCO2 and pH among all experimental groups (P > 0.05). pH was 7.38 ± 0.01 in the hypoxic groups and 7.36 ± 0.01 in the hyperoxic group; average PCO2 was 4.7 ± 0.1 kPa in the hypoxic groups and 4.8 ± 0.2 kPa in the hyperoxic group.

Diaphragm muscle strip dimensions were not significantly different among the experimental groups (P > 0.05). Average muscle strip weight was 37.1 ± 0.8 mg, and strip length at Lo was 18.8 ± 0.8 mm.

Isotonic Contractile and Fatigue Properties During Hypoxia

Baseline contractile properties. Baseline Pt and Po were not different among the experimental groups (P > 0.05). Mean values of Pt were 7.2 ± 0.3 N/cm2 in the hypoxic groups and 8.0 ± 0.4 N/cm2 in the hyperoxic group; average baseline values of Po were 23.1 ± 0.8 N/cm2 in the hypoxic groups and 25.3 ± 1.2 N/cm2 in the hyperoxic group. Figure 1A shows that 60 min of hypoxia reduced Pt and Po by 21 and 18%, respectively, compared with hyperoxia control (P < 0.01).


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  		Fig. 1.   Diaphragm maximal twitch tension (Pt) and maximal tetanic tension (Po) after 60 min (A) and 30 min (B) in experimental Krebs solution. Values are means ± SE, expressed as percentage of initial values. A: NG-monomethyl-L-arginine (L-NMMA) tended to elevate Pt during hypoxia (PO2 ~ 7.2 kPa; P = 0.09; n = 6) but did not alter Po. Incubation with L-NMMA plus L-arginine to muscle did not affect Pt and Po. Hypoxia impaired both Pt and Po compared with hyperoxic control (PO2 ~ 85 kPa; P < 0.01). B: spermine NONOate (Sp-NO; 1 mM) reduced Pt but did not affect Po during hypoxia (n = 7). Hemoglobin treatment (n = 7) did not affect Pt or Po. * P < 0.05 compared with hypoxia control.

L-NMMA tended to elevate Pt compared with control during hypoxia (P = 0.09; Fig. 1A) but did not alter Po. Pt and Po were not different between L-NMMA plus L-arginine groups and control groups during hypoxia. Incubated with 1 mM Sp-NO during hypoxia, Pt was lower compared with that in control groups (P < 0.05; Fig. 1B), but Po was not different. Hemoglobin did not significantly affect Pt and Po compared with control groups during hypoxia.

Force-velocity and force-power characteristics during hypoxia. L-NMMA slowed the shortening velocity over a wide range of loads during hypoxia (P < 0.01; Fig. 2A). Vmax of the L-NMMA group was ~28% lower than in the hypoxia control (P < 0.05; Fig. 2B). L-NMMA did not alter the curvature of the force-velocity relationship, as described by a/Po (0.28 ± 0.05 in L-NMMA group and 0.27 ± 0.03 in 60 min of hypoxia control; P > 0.05). L-Arginine abolished the detrimental effects on Vmax caused by L-NMMA (P > 0.05; Fig. 2B). The force-velocity relationship did not differ between control groups and L-NMMA plus L-arginine groups (P > 0.05) and did significantly differ between L-NMMA groups and L-NMMA plus L-arginine groups (P < 0.05). Sp-NO did not alter the shortening velocity relationship compared with hypoxic control (Fig. 3). Hemoglobin slowed the shortening velocity over a wide range of loads during hypoxia (P < 0.05; Fig. 3A). Vmax fell ~22% in hemoglobin-treated muscle compared with hypoxia control (P < 0.05; Fig. 3B). a/Po measured in 30 min of hypoxic control was 0.14 ± 0.01 and was unchanged by hemoglobin (0.18 ± 0.07; P > 0.05). The force-velocity relationship was not different between hyperoxia control and hypoxia control, including Vmax (Fig. 2). a/Po, however, was lower in 60 min of hyperoxia control compared with 60 min of hypoxia control (0.13 ± 0.03 vs. 0.27 ± 0.03).


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  		Fig. 2.   Force-velocity relationship in the rat diaphragm after 60 min of incubation in experimental Krebs solution. A: L-NMMA slowed the shortening velocity over a wide range of loads compared with hypoxia control (n = 8; P < 0.01) but did not alter the curvature during hypoxia. Effects were partly reversed by coadministration with 30 µM L-arginine. Hypoxia did not affect force-velocity relationship compared with hyperoxic control (n = 6). B: bar graph highlights Vmax for the same 4 groups. Vmax was slower in the L-NMMA groups. Effect was abolished by coadministration with L-arginine. Hypoxia did not affect Vmax compared with hyperoxic control. Lo, optimal fiber length. Values are means ± SE. * P < 0.05 compared with hypoxia control.



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  		Fig. 3.   Force-velocity relationship in the rat diaphragm after 30 min of incubation in experimental Krebs solution. A: during hypoxia, Sp-NO did not affect the force-velocity relationship (n = 8), and hemoglobin slowed the shortening velocity over a wide range of loads (P < 0.05; n = 7) but did not alter the curvature of force-velocity relationship. B: bar graph highlights Vmax for the same 3 groups. Sp-NO did not affect Vmax. Vmax was slower in hemoglobin-treated muscle. Values are means ± SE. * P < 0.05 compared with hypoxia control.

L-NMMA depressed the power output over a wide range of loads during hypoxia (P < 0.01; Fig. 4A). This effect was most marked in the region of peak power (51 ± 4 and 83 ± 8 N/cm2 × Lo/s in L-NMMA and hypoxia control, respectively; P < 0.001). Coincubation of muscle bundles with L-arginine prevented the depression of power outputs caused by L-NMMA. Sp-NO did not affect the power output, including the peak power, during hypoxia. Hemoglobin depressed power production over a wide range of loads during hypoxia (P < 0.01; Fig. 4B). There was no significant difference in the force-power relationship between the hypoxia and hyperoxia control groups (Fig. 4A).


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  		Fig. 4.   Force-power relationship from rat diaphragm after 60 min (A) and 30 min (B) of incubation in experimental Krebs solution. A: L-NMMA depressed the power output over a wide range of loads during hypoxia (P < 0.01). Effects were prevented by coincubation with L-arginine. Hypoxia did not affect force-power relationship compared with hyperoxic control. B: Sp-NO did not affect the power output during hypoxia, whereas hemoglobin depressed power production over a wide range of loads (P < 0.01). Values are means ± SE.

Isotonic fatigue properties during hypoxia. With repetitive contractions, power output of the diaphragm progressively declined over time in all experimental groups (Fig. 5). In the L-NMMA groups, the rate of decline in power output was faster and isotonic endurance was less than in control groups (P < 0.05; Figs. 5A and 6A). These effects were prevented by coadministration with L-arginine (P > 0.05 compared with hypoxic control; Figs. 5A and 6A). During hypoxia, Sp-NO did not affect the rate of decline in power output and fatigue endurance compared with hypoxia control (Figs. 5B and 6B). Hemoglobin increased the rate of decline in power production (P < 0.01) and reduced isotonic endurance compared with hypoxic control (P < 0.05; Figs. 5B and 6B). Power output of the diaphragm muscle declined faster in hypoxic control compared with hyperoxic control (P < 0.001; Fig. 5A). Isotonic endurance was 143 ± 11 and 70 ± 2 s in hyperoxic control and hypoxic control, respectively (P < 0.001; Fig. 6A).


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  		Fig. 5.   Power production during repetitive isotonic contractions in the rat diaphragm after 60 min (A) and 30 min (B) of incubation in experimental Krebs solution. A: the rate of decline in power production was faster in the L-NMMA groups compared with control during hypoxia (P < 0.05). Effect was prevented by coincubation with L-arginine. Power output declined faster in hypoxia compared with hyperoxic control (P < 0.001). B: Sp-NO did not affect the rate of decline in power output, whereas power production in hemoglobin-treated muscle was faster compared with hypoxia control. Values are means ± SE.



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  		Fig. 6.   Mean isotonic fatigue endurance in the rat diaphragm after 60 min (A) and 30 min (B) of incubation in experimental Krebs solution. A: endurance time in L-NMMA group is less than that in hypoxic control. The effects were prevented by coadministration with L-arginine. Hypoxia reduced the endurance time compared with hyperoxic control. B: Sp-NO did not prolong isotonic endurance, whereas hemoglobin reduced endurance time during hypoxia. Values are means ± SE. * P < 0.05 compared with hypoxia control. # P < 0.05 compared with hyperoxia control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The main finding of the present study is that either inhibition of NOS by L-NMMA or scavenging NO by hemoglobin slowed velocity of shortening, depressed power generation, and increased the fatigability of the rat diaphragm in vitro during hypoxia. These data indicate that endogenous NO is needed for optimal muscle contraction of the rat diaphragm during hypoxia. Thus endogenous NO has a protective role in hypoxia-mediated modulation of muscle contraction in vitro. Supplementation of exogenous NO with Sp-NO did not affect velocity of shortening, power generation, or fatigability during hypoxia.

L-NMMA, Hemoglobin, and Sp-NO

L-NMMA is an established NOS inhibitor in vitro and is a naturally occurring inhibitor as well (5, 25). It competes with arginine for binding on NOS molecules. It also inhibits L-arginine transport into cultured cells to restrict the availability of NOS substrate (14, 29). During hypoxia, the depressed effects of L-NMMA on Vmax, power generation, and fatigue resistance were prevented by L-arginine. This indicates that the effect of L-NMMA is mediated by NOS modulation. Hemoglobin is known to bind avidly and to nullify rapidly the effects of NO (18). Thus it was used as further evidence that the observed responses were NO mediated. Because hemoglobin does not enter cells and it scavenges NO extracellularly (37), it may scavenge NO generated intracellularly by creating a diffusion gradient of NO out of the cell "sink" (27).

Sp-NO, a nucleophilic type of NO donor, is capable of generating NO extracellularly, by a nonenzymatic process in simple aqueous buffers (24, 31). The rate and extent of NO release depend on the pH and temperature of the solution (34). Specially, this type of NO donor is unique in its ability to generate NO in a predictable manner. Compared with other nucleophilic adducts, Sp-NO generates NO in a long-lasting and constant manner (35). Therefore, it is an ideal vehicle for the delivery of NO in the present study.

Effects of Hypoxia on Isotonic Contractile and Fatigue Properties

The Krebs solution PO2 in the present study was reduced to ~7 kPa. This PO2 would not ordinarily be regarded as severe hypoxia if it were the arterial PO2 (7), because, in vivo, the O2 is carried by the blood delivery of O2 to the muscle cells efficiently. Conversely, there is a larger diffusion distance between bath O2 and muscle cells in vitro (46). Thus it is likely that the degree of tissue hypoxia is "severe" in our experiment (46, 48). Hypoxia impaired Pt and Po and did not affect force-velocity and force-power relationships, which is in line with the data from the previous studies (21, 48). Velocity of shortening is associated with myosin-ATPase activity and determined by the rate of cross-bridge cycling (6); therefore, hypoxia did not reduce myosin ATPase activity of the diaphragm fibers. The different effects of hypoxia on force generation and shortening of velocity are more likely due to the different underlying cellular mechanisms (2). However, hypoxia altered the curvature of the force-velocity relationship. The force-velocity relationship was less curved after 60 min of hypoxia (higher value of a/Po). A high value of a/Po probably indicates a less efficient muscle in terms of energetics (51).

Hypoxia reduced muscle fatigue resistance, as indicated by a rapid decline in power output and less fatigue endurance during repetitive contractions. The rapid decline in power output might reflect the high-energetic demands of dynamic contractions and the impaired ATP-buffing capacity of hypoxic diaphragm, because muscle fatigue results from the imbalance of energetic supply and demand (49). The reduction in power output during repetitive contraction is the result of reduction in velocity, because force was clamped at 33.3% of Po. During hypoxic conditions, the accumulation of intracellular metabolites, i.e., ADP and lactate, could be accentuated under repetitive isotonic contraction compared with hyperoxic conditions (2). The rise of ADP concentration has been shown to reduce the shortening velocity (2). Therefore, the sensitivity of muscle fatigue to hypoxia is greater than that to hyperoxia.

Our control data regarding Po, Vmax, and the curvature of the force-velocity relationship correspond with published values for isolated diaphragm muscle at the same experimental setup (21, 48), but differ from those of Morisson et al. (36) and Metzger et al. (33). Factors that contribute to this variability could include difference in the temperature, animal species, and incubation time. For instance, Vmax was measured at 36°C by Morisson et al., whereas we performed all of our experiments at 26°C. Consequently Vmax was lower in our study. Metzger et al. reported a rise in Vmax and Po of the rat diaphragm with increasing temperature values.

Effects of NO on Isotonic Contractile and Fatigue Properties During Hypoxia

To date, no other study has evaluated the effect of NO on contractile properties in vitro in skeletal muscle during hypoxia, including in diaphragm, soleus, and EDL muscles. We investigated the role of NO in isotonic contractile properties of the rat diaphragm in vitro during hypoxia. L-NMMA slowed the shortening velocity over a wide range of loads during hypoxia. L-NMMA reduced Vmax by 28% of the rat diaphragm in vitro. The effects of L-NMMA were mediated by NO. Similar results were obtained with hemoglobin. Accordingly, both L-NMMA and hemoglobin depressed power generation as well during hypoxia. This finding is of physiological and functional significance, as power is considered as a more physiological estimation of muscle performance in vivo than either force or velocity alone. The effects by L-NMMA and hemoglobin on power generation are mainly due to a reduction of velocity, because power is the product of Po and velocity and L-NMMA and hemoglobin did not affect Po. As mentioned previously, velocity of shortening is determined by the rate of cross-bridge cycling (6); thus these findings indicate that endogenous NO plays a role in cross-bridge cycling during hypoxia, direct or indirect. The different effects of L-NMMA on Pt and shortening of velocity suggest that endogenous NO acts on multiple targets. The fact that the NO donor Sp-NO did not affect velocity of shortening during hypoxia implies that diaphragm muscle might be able to adjust NO generation during active shortening. It has been shown that NO can adjust its own synthase by feedback in restricting NO production (9).

A previous study reported that NOS inhibition depressed velocity of shortening and power generation of the rat diaphragm under hyperoxic conditions in vitro (36). The NOS inhibitor NG-nitro-L-arginine, at a concentration of 10 mM, reduced Vmax and peak power ~16 and ~18%, respectively. But the reduction effect of the NOS inhibitor on isotonic contractility is more pronounced under hypoxic conditions (used in the present study, ~28 and ~39% in Vmax and peak power, respectively) than observed under hyperoxic conditions. A possible explanation is that generation of reactive oxygen species (ROS) is enhanced during hypoxia (13, 39). Therefore, the role of NO as an antioxidant is more important. It is also possible that hypoxia directly affects NOS. Previous studies showed that hypoxia impairs the SR Ca2+ reuptake from the intracellular space (12, 52). This could result in an increase of [Ca2+]i levels (52) and may, in turn, activate NOS via Ca2+-dependent NOS isoforms. Hampl et al. (19) showed that, in pulmonary artery endothelium, 10-min hypoxia (PO2 = 4.9 kPa) increases [Ca2+]i levels and NOS activity. Data regarding the effect of hypoxia on NO production in skeletal muscle are not available.

The present study is the first one to demonstrate the effect of NO on isotonic fatigability in skeletal muscle in vitro. The diaphragm muscles were less able to sustain power generation after incubation with both L-NMMA and hemoglobin. These depression effects were persistent through the period of repetitive contractions. This result indicates that endogenous NO exerts a beneficial effect in the resistance of diaphragm muscle fatigue in vitro during hypoxia. As mentioned previously, either fatiguing stimulation alone or hypoxia alone can enhance the formation of ROS (13, 44). The combination of both conditions may cause more dramatic milieu changes. Data from our laboratory (21) showed that isotonic fatigue properties of the rat diaphragm are inhibited by antioxidants in vitro. This is in line with the observation in the present study. However, our data are in conflict with a previous study (17) using anesthetized ventilated dogs, which found the protection from diaphragmatic fatigue by a NOS inhibitor L-NAME. Difference in the experimental setup and species may explain these discrepancies. In contrast to a significant increase in isotonic fatigability by depletion of NO, the addition of Sp-NO did not change fatigability during hypoxia. This implies that intracellular L-arginine content may be adequate for basal NO production during strenuous contractile activity under hypoxic conditions. It was found that the concentration of L-arginine in the tissue is high and could not be a limiting factor for NOS (30).

The mechanism underlying the beneficial effect of NO on isotonic contractile and fatigue properties in vitro during hypoxia may relate to its antioxidant effect, perhaps either directly or indirectly. Biological activities of NO and ROS are strongly interdependent (28, 43). They and their redox derivatives compete for the same metal centers and thiol groups on the target protein (1, 43). At lower concentrations, NO prevents ROS-mediated oxidation on the Ca2+ release channels [ryanodine receptor 1 (RYR1)] of the SR (1). Moreover, NO can protect cells against toxicity mediated by hydrogen peroxide (H2O2) (50). In addition, the net effect of NO depends not only on its relative concentration but also on the balance between the levels of NO and ROS (11).

A potential issue is whether ROS generated from electrolysis plays a role in the observed effects of NO on isotonic contractile and fatigue properties in vitro during hypoxia. However, it is unlikely that ROS could be generated by electrolysis with the current applied to our system. First, a study (44) using similar stimulus parameters showed that electrolytic superoxide anion radical generation was not detectable after 1 h of repetitive stimulation (~250 mA/s). Second, theoretically, ROS generated by electrolysis, if present, could react with NO released from Sp-NO to form peroxynitrite, which has a strong prooxidant influence and could impair muscle performance (41, 47). However, the fact that the addition of Sp-NO did not change isotonic contractile properties does not support this possibility. Conversely, generation of ROS is enhanced in cardiomyocytes during hypoxia (13); therefore, the fact that Pt was depressed with Sp-NO cannot rule out the possible influence of peroxynitrite formation in force generation during hypoxia.

The mechanisms by which NO mediates its effect on contractile properties remain unclear. In contrast to smooth muscle, in skeletal muscle, the magnitude of cGMP-mediated changes is limited. NO may act directly on modulating regulatory proteins via redox effects (43). Thiol groups of the Ca2+ release channels of the SR are likely sites for such interaction (1, 22). Low concentrations of NO prevent channel opening from oxidation-induced activation, in this way reducing the rate of Ca2+ release. High concentrations promote opening of the RYR1 channels (1). In addition, the effect of NO on the thiol groups of RYR1 could also depend on local PO2 as well (15). The data from our own laboratory (20) also demonstrated that, in permeabilized rabbit psoas muscle fibers, a NO donor reduces Ca2+ sensitivity, which would contribute to a reduction of force. Andrade et al. (4) also observed a reduction of Ca2+ sensitivity in single skeletal muscle from a mouse foot muscle, but force and maximal shortening velocity were largely unchanged. Reactive thiols present on the myosin head are another potential target. This modulation would reduce maximal force generation (40).

In the present study, the NOS activity was not determined in the diaphragm and other skeletal muscle. Previous studies showed that the NOS activity in the rat diaphragm was lower than in EDL muscle, because the activity of neuronal NOS correlated strongly with type II fiber composition and type II composition of the diaphragm is lower compared with EDL muscle (26). Diaphragm muscles contract continuously and have a higher oxidative capacity and higher maximal blood flow. A lower NOS activity might be a match for these special functional and structure properties to downregulate muscle function in stress or to integrate functional characteristics of contrasting fibers in a muscle bundle (10).

Conclusions

In summary, endogenous NO plays a protective role in hypoxia-mediated modulation of muscle contraction in the rat diaphragm in vitro. Depletion of NO reduced velocity of shortening and power generation and increased the fatigability of the rat diaphragm during hypoxia. Supplementation of exogenous NO with Sp-NO did not affect isotonic contractile and fatigue properties during hypoxia. The different effects of the NOS inhibitor on force generation and shortening of velocity suggest that NO acts on multiple targets. The protective effect of NO on isotonic contractile and fatigue properties in vitro may relate to its antioxidant effect under hypoxic conditions.


    ACKNOWLEDGEMENTS

The authors thank Kay Poelen for biotechnical assistance.


    FOOTNOTES

The authors are grateful to The Netherlands University Fund for International Cooperation.

Address for reprint requests and other correspondence: P. N. Richard Dekhuijzen, Dept. of Pulmonary Diseases, Univ. Medical Centre Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands (E-mail: r.dekhuijzen{at}long.umcn.nl).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published October 18, 2002;10.1152/japplphysiol.00441.2002

Received 17 May 2002; accepted in final form 9 October 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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