INTRODUCTION

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IT IS NOW WELL ACCEPTED that NO, a signaling molecule, is involved in many important physiological and pathological functions, including vessel relaxation, neurotransmission, and pathogen suppression (5).

NO is synthesized from L-arginine by NO synthase (NOS). Three major NOS isoforms exist. NOS 1 is a constitutive isoform found in the central and peripheral nervous systems, epithelial cells, and skeletal muscle (11). Neurotransmission, bronchodilation, glucose transport, and regulation of force development are among its functions. NOS 2 (iNOS) is induced by endotoxins and cytokines in most cells (20). This enzyme is believed to release larger amounts of NO_x (nitrogen oxides), which may be important in host defense (20). NOS 3 is a Ca²⁺-calmodulin-dependent constitutive isoform found in vascular endothelial cells, some neuronal populations, platelets, cardiac muscle, and skeletal muscle. Its major activities include relaxation of vascular smooth muscle, inhibition of platelet activation and adhesion, and control of cell respiration (16, 24).

Although many studies have examined the role of NO in ischemia-reperfusion injury of cerebral, myocardial, mesenteric, gastric, and lung tissues (2, 17, 19), the results are paradoxical and no consensus has been reached. In skeletal muscle, the one existing report suggested that NO production during reperfusion injury may be deleterious to survival of muscle tissue (26). However, it has become apparent that the success of replantation and microsurgical reconstructive operations should not be measured by survival alone but also by quantification of subsequent function. The ultimate goal is to regain normal function or, at least, to improve impaired function. To our knowledge, there are no data in the literature pertaining to functional regulation by NO in the reperfused skeletal muscle. The present study was designed to test the hypothesis that NO influences the contractile function of reperfused skeletal muscle.

	MATERIALS AND METHODS

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Sixty-nine male Sprague-Dawley rats with body weights ranging from 90 to 120 g were used. The systemic influence of infusion was studied in 20 rats. The remaining 49 animals were randomly divided into six groups in which the extensor digitorum longus (EDL) muscle was used for evaluating contractile function. The animal grouping and experimental protocol are summarized in Table 1.

Anatomy of the EDL. The EDL is a fusiform muscle located in the deep layer between the peroneus longus muscle and the tibialis anterior muscle. It arises from the lateral epicondyle of the femur and inserts on the base of the third phalanx of digits two to five. In animals of the size used in this study, the EDL has an average length of 23.5 ± 1.2 mm and an average weight of 48 ± 2 mg (4). Ordinarily, there are three nutritional branches entering the EDL arising from the anterior tibial artery at the proximal one-third of the muscle. Branches of the deep peroneal nerve accompany the vessel branches to the muscle.

Surgical procedure. Procedures were based on those of Bolognesi et al. (4). Each animal was anesthetized with intraperitoneal Nembutal (Abbott Laboratories, North Chicago, IL) at a dose of 50 mg/kg body wt. The left femoral artery was cannulated in 20 rats for monitoring systemic influence. Blood pressure, heart rate (HR), and respiratory rate (RR) in response to the infused drugs were monitored by a patient monitor (MR-1300, Mennen Medical, New York, NY) at 10-min intervals for 6.5 h. These 20 rats were not subjected to other surgical procedures or to ischemia-reperfusion.S-nitroso-N-acetylcysteine (SNAC; 100, 1, and 5 µmol/min) and phosphate-buffered saline (PBS) were continuously infused for 6.5 h, five rats for each dosage.

Agent administration. After the left EDL was removed for contractile testing, each animal underwent left external jugular vein catheterization with polyethylene tubing (PE-10, Clay Adams, Parsippany, NJ). The tubing was connected to a 3-ml syringe fixed to a Sage syringe pump (model M362, Analytical Technology, Boston, MA). Groups I and III received SNAC at 100 nmol/min, groups II and IV received 1 µmol/min, group V received 5 µmol/min, and group VI received PBS. The SNAC and PBS were administered through the tubing at a rate of 0.2 ml/h. In muscles not subjected to ischemia-reperfusion (groups I and II), SNAC was continuously infused for 6.5 h. In ischemic-reperfused animals (groups III-VI), SNAC or PBS administration was started 30 min before ischemia and continued throughout the duration of reperfusion, with a total infusion duration of 6.5 h.

Muscle contractile testing. The EDL was suspended in a standard organ chamber system (Radnoti Glass Technology, Monrovia, CA). The proximal end of the muscle was attached by the suture tail to an isometric force transducer (Radnoti model TRN001, Radnoti Glass Technology) which was mounted on the spindle of a nonrotating micrometer head and connected to an amplifier. The distal end was attached to the glass hook at the bottom of the field-stimulating electrode (Radnoti model 160151). This resulted in a vertically oriented muscle suspended between the two parallel platinum electrodes. The entire preparation was then submerged in a 25-ml organ chamber (Radnoti model 158326) and allowed to equilibrate for 10 min to bring each muscle to the same temperature. The tissue bath was filled with Krebs solution (pH 7.4, concentration in mM: 122.0 NaCl, 4.7 KCl, 1.2 MgCl₂, 2.5 CaCl₂, 15.4 NaHCO₃, 1.2 KH₂PO₄, and 5.5 glucose). The solution was aerated with a 95% O₂-5% CO₂ gaseous mixture, maintained at 37°C with the help of a Cole-Parmer polystat circulator, and changed at 15-min intervals (4).

Histological examination. Two EDLs each from groups III and VI were harvested for histological examination. A piece of the muscle measuring 1.0 × 0.3 × 0.3 cm was excised and fixed to a wooden bar to prevent contraction of the muscle. The preparation was immediately immersed in 4% glutaraldehyde buffered with 0.1 M sodium cacodylate for transmission electron microscopy (TEM) examination. The remaining muscle was fixed in 10% formaldehyde and histologically examined using phosphotungstic acid hematoxylin (PTAH) and hematoxylin and eosin stain.

	RESULTS

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Systemic effects of SNAC infusion. During infusion, mean arterial pressure (MBP), HR, and RR gradually decreased in both the low-dose SNAC (100 nmol/min) and PBS-treated animals, with no significant difference between the two treatments. Average BP fell 12% in SNAC-treated rats and 11% in PBS-treated rats (Fig. 1). HR fell 8 and 5%, respectively, and RR fell 17 and 12%, respectively. Compared with the same parameters at initiation of infusion and after SNAC infusion, there was no significant difference in MBP, HR, or RR.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Dose-dependent systemic effects of NO donor S-nitroso-N-acetylcysteine (SNAC) on mean blood pressure (mmHg) of normal rat. Error bars represent SE of mean. * P < 0.05 compared with 100 nmol/min SNAC-treated or phosphate-buffered saline (PBS)-treated rats.

Influence of SNAC on contractile function in the EDL not subjected to ischemia-reperfusion (groups I and II). After 6.5 h of 100 nmol/min SNAC infusion (group I), the average maximal twitch force in the infused right EDL was 91 ± 9% (mean ± SE) of the contralateral nontreated muscle force. The average isometric tetanic contraction was 114 ± 8% of the contralateral nontreated EDL force at 40-Hz stimulation and near 100% at the remaining three stimulations. When SNAC concentration was increased to 1 µmol/min (group II), the maximal twitch force was 110 ± 6% and the isometric tetanic force remained near 100% at all four stimulation frequencies. There were no statistically significant differences between groups I and II. There was no statistically significant difference in the absolute contractile tension between the right EDL with SNAC infusion and the left EDL without SNAC infusion in either group I or group II. After fatigue induction, peak tension and FT_1/2 were 92 ± 2% and 10.7 s in group I and 88 ± 6% and 7.8 s in group II, respectively (average contralateral FT_1/2 was 9.3 s; P = NS).

Influence of SNAC on contractile function in EDL after ischemia-reperfusion (groups III and VI). The average maximal twitch force was 46 ± 3% of the contralateral nontreated EDL in the 100 nmol/min-SNAC-infused EDL (group III) and 15 ± 3% in the PBS-infused EDL (group VI). This difference was highly significant (Fig. 2). The average isometric tetanic force was 43 ± 4% in group III at 40-Hz stimulation and 59 ± 5, 80 ± 4, and 91 ± 1% at 70-, 100-, and 120-Hz stimulation, respectively. The relative values were 15 ± 4, 25 ± 7, 36 ± 6, and 42 ± 8% in group VI, respectively. When these two groups were compared, the contractile force in SNAC-treated group III muscles was found to be significantly greater than that in the PBS-treated group VI muscles at all four stimulation rates (Fig. 3). After fatigue induction, test peak tension in group III was 86.6 ± 0.96%, whereas it was only 36.3 ± 5.9% in the PBS group. This difference was highly significantly different (Table 2). The average FT_1/2 was 7.3 s in group III and only 4 s in group VI, with a significant difference.

View larger version (6K): [in this window] [in a new window]   Fig. 2.   Effects of NO donor SNAC on maximal twitch force of ischemic-reperfused extensor digitorum longus (EDL). SNAC shows a significant protective action on muscle contractile function against reperfusion injury. However, protection conferred is greater at low dose (100 nmol/min). Error bars represent SE of mean. *** P < 0.001 compared with PBS-treated muscles (group VI).

View larger version (37K): [in this window] [in a new window]   Fig. 3.   Influence of SNAC on isometric tetanic force of ischemic-reperfused rat EDL at 40, 70, 100, and 120 Hz. SNAC at low (100 nmol/min) dose shows a significant protective role in muscle isometric tetanic contractile function against reperfusion injury. When dose of SNAC is increased to 1 µmol/min, however, protection is sharply decreased and there is no significant difference between SNAC-treated and PBS-treated group. Error bars represent SE of mean. ** P < 0.01, *** P < 0.001 compared with PBS-treated muscles (group VI). + P < 0.05, ++ P < 0.02, and +++ P < 0.001 compared with low-dose SNAC group (group III).

Influence of SNAC dosage on contractile function in EDL after ischemia-reperfusion (groups III-VI). Although the maximal twitch force was significantly greater in all three SNAC-treated groups than in the PBS-treated group, the higher doses of SNAC (1 and 5 µmol/min) were less effective than the lower dose (100 nmol/min). Groups IV (1 µmol/min) and V (5 µmol/min) did not differ significantly from one another in the degree of protection conferred (Fig. 2). Measurement of the isometric tetanic contractile forces showed similar results. As the SNAC dose was increased, the contractile force was reduced to the extent that there was no statistically significant difference in the force generation at any of the four stimulation frequencies compared with the PBS-treated muscles. Moreover, the contractile force in group III was significantly greater than that in groups IV and V, which received a higher dose of SNAC (Fig. 3). After fatigue induction, peak tension in group IV was 66.8 ± 8.1% and was 60.4 ± 4.1% in group V, which was significantly higher (P < 0.01) than that in group VI but significantly lower (P < 0.05) than that in group III (Table 2). The average FT_1/2 was 5.9 and 6.4 s in groups IV and V, respectively.

Histology. Slight edema and early inflammation were seen under light microscopy. No apparent differences were discovered between groups III and VI (Fig. 4). However, TEM showed that edema and degeneration of mitochondria were less severe in the SNAC-treated EDL than in the controls (Fig. 5).

View larger version (126K): [in this window] [in a new window]   Fig. 4.   Photomicrographs of rat EDLs after 3-h ischemia and 3-h reperfusion. A: PBS-infused muscle. B: SNAC-infused muscle. There was no apparent difference between muscles, and inflammation was present in both (hematoxylin and eosin stain). ×170 Magnification.

View larger version (165K): [in this window] [in a new window]   Fig. 5.   Transmission electron micrograph of a longitudinal section of ischemia-reperfusion EDL of rat shows less severe mitochondrial swelling and degeneration in SNAC-treated EDL (100 nmol/min, ×10,400) (B) than in PBS-treated EDL (×12,000) (A). Filled arrows indicate relatively normal mitochondria, filled arrowheads indicate swelling and degenerated mitochondria, and open arrows indicate edema of sarcoplasmic reticulum.

	DISCUSSION

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Recent studies have shown that rat skeletal muscle expresses neuronal and endothelial constitutive NOSs and that NOS activity varies among several respiratory and limb muscles (15, 16, 22). Immunohistochemistry has shown that NOS activity in rat muscles correlates with type II fiber density (15). Because the EDL is a fast muscle composed mainly of type II fibers, it was hypothesized that NO may play a role in the pathophysiology of skeletal muscle injury.

The effect of ischemia-reperfusion on NO production is controversial. Although some investigators have reported that ischemia-reperfusion decreases NO_x production (15), others observed increases in NO_x in both humans (33) and animals (21). A study of rat hindlimb ischemia suggested that endothelium-derived NO plays an important role in the maintenance of vascular tone, and a reduction of NO release during reperfusion may predispose vessels to vasoconstriction (31). Although NO generation was not measured in the present study, our results suggest that ischemia-reperfusion results in a relative deficiency of NO in skeletal muscle, inasmuch as SNAC significantly protects the contractile function of reperfused skeletal muscle. Our findings are in agreement with those of others (10) who showed that constitutive NO release is impaired in rat ischemia-reperfused heart muscle because of endothelial dysfunction and can be ameliorated with L-arginine pretreatment.

We also found that SNAC exerts a complex systemic response. Although the lower dose had no significant effects, higher-dose (5 µmol/min) SNAC resulted in a reduction of blood pressure and RR of ~1/3 and an increase in HR of ~50%. However, the changes lasted only 3-4 h. Subsequently, they appeared to be diminished or reversed despite continuous SNAC infusion. The phenomenon may reflect a negative feedback regulation that protects and maintains the body in a normal physiological condition. Such tolerances and tachyphylaxis to the systemic effects of S-nitrosothiols have not been previously reported. The fact that systemic effects of SNAC at 100 nmol/min occurred mainly during the ischemic period rather than during reperfusion suggests that the changes in muscle function resulted predominantly from NO supplementation.

In the present study, a 6.5-h infusion of SNAC showed significant functional protection of both the maximal twitch and tetanic contractile forces in the EDL after 3-h ischemia and 3-h reperfusion, with no statistically significant systemic side action at <1 µmol/min. Several mechanisms may be related to the NO protective function. First, NO can reverse the vessel spasm that is known to occur during reperfusion injury (32). Our earlier study (12) showed that acetylcholine-induced NO release significantly relieved norepinephrine-induced vasoconstriction in 40- to 80-µm arteries of rat cremaster muscle. More recently, we found that SNAC infusion significantly relieved vasospasm and improved microcirculation in rat cremaster muscle after 5-h ischemia and 90-min reperfusion (unpublished observations). Therefore, the protective function by SNAC on contraction in the present study may be related to NO-mediated vasodilation, thereby improving the microcirculation and increasing nutrition input to the muscle. A feline hindlimb study (9) also suggested that basal NO production is required to maintain resting vasomotor status of vascular smooth muscle. The inhibition of such an effect, as might occur with ischemia followed by reperfusion, would be expected to increase vascular resistance and thereby impair blood flow and tissue nutrition.

Second, NO may modulate the function of blood elements that influence the microcirculation. NO reduces clotting by inhibiting platelet aggregation and adhesion (8) as well as by inhibiting the adhesion of neutrophils and monocytes (18).

Third, NO may modulate muscle contractile function directly or through effects on reactive oxygen species (ROS). Muscle contraction increases intracellular oxidant levels, and an excessive level of ROS promotes muscle fatigue (27). It is believed that the protective function of NO may relate to the scavenging of superoxide in such settings (23). Our results appear to support one of these possibilities, because fatigue peak tension and FT_1/2 were greater in the SNAC-treated EDL than in the control. Thus NO donor administration increased the resistance of skeletal muscle to fatigue after ischemia-reperfusion. That constitutive ecNOS (NOS 3) associates closely with mitochondria in skeletal muscle and influences muscle respiration is consistent with a role in scavenging ROS (16). In the present study, the mitochondrial edema seen after 3 h of reperfusion is highly suggestive of functional impairments that might adversely influence mitochondrial permeability, NO production, and reactive oxygen generation (28).

Our findings indicate that the function-sparing effect of the NO donor SNAC is critically dependent on the dosage. As an adjunct to the present study, we have examined the effects of lower (10 nmol/min) and higher (10 µmol/min) SNAC dosages in two additional small groups of rats (n = 2/group). After 3-h ischemia and 3-h reperfusion, the EDL tetanic contractile force in the animals with a 10 nmol/min SNAC infusion was <30% (23-29%) of normal at each of four stimulation rates, much lower than that observed in rats receiving a 100-nmol/min dose (group III), but not statistically different from controls (group VI). When a high dose (10 µmol/min) of SNAC was administered, the contractile forces were 30-55% of normal at the four different stimulation rates, a level less than that observed after 5-µmol/min SNAC administration. The findings suggest that there is a threshold between 10 and 100 nmol/min at which SNAC becomes dramatically more effective. As the SNAC dose was increased from 100 nmol/min to 10 µmol/min, however, SNAC progressively lost effectiveness. At the higher doses of SNAC, there was no significant difference in tetanic forces generated between the SNAC-infused and PBS-infused EDLs. This may reflect a direct inhibitory effect of SNAC on muscle under these conditions (15) or the deleterious effects of excessive NO-related activity. Indeed, S-nitrosothiols such as SNAC have been reported to exert an inhibitory effect on isolated normal diaphragm contractile function (15). The inhibitory effect of NO-related activity may be manifest in EDL after muscle function is compromised by ischemia.

The muscle function that we have documented after SNAC administration does not correlate well with the results of other investigators who reported that NOS inhibitors improved the survival of skeletal muscle (26) and skin flaps (14), suggesting that NO production was deleterious to reperfused tissue. These discrepant results may be partly due to differences in the experimental protocol, animal species, dosing, harvest time, and a multitude of related factors that might influence the contribution of NO to muscle function. For example, we initiated the SNAC infusion before ischemia in the hope of reducing the neutrophil adhesion during ischemia-reperfusion, as this is known to compromise the microcirculation (1).

Importantly, sufficient precedent and rationale exist for dual actions and paradoxical effects of NO (30). Thus the protective role of NO in ischemia-reperfusion injury of skeletal muscle in our studies cannot be generalized to all clinical ischemic conditions of the muscle or to its effects on other organs. Specifically, studies concerning NO/NOS influence on cerebral reperfusion have shown inconclusive and conflicting results (7). Infusion of L-arginine or NO donors immediately after ischemia can significantly reduce the magnitude of ischemia-reperfusion damage (34). With administration of NOS inhibitors, however, the results were inconsistent, ranging from neuroprotection, to no significant alteration, to exacerbation of damage (7). The observations in infarcted myocardial tissue have been equally contradictory (13, 25). NO donors appear to be cardioprotective in a cat model of myocardial ischemia-reperfusion (29) but deleterious in others. We conclude that judicious administration of the NO donor SNAC protects the contractile function of skeletal muscle against ischemia-reperfusion injury in the first 3 h of reperfusion.

Perspectives