INTRODUCTION

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RECENT WORK HAS SHOWN that the development of endotoxemia is associated with profound reductions in skeletal muscle force-generating capacity (15, 16, 18). In fact, in one in vivo animal study of endotoxemia, diaphragm force generation decreased so severely that respiratory failure and death occurred as a direct result of diaphragmatic dysfunction. Subsequent studies have extended this work, showing that oxygen-derived free radicals are involved in the genesis of this form of muscle dysfunction (15, 16, 18, 20).

One issue not completely resolved by previous experiments is the identification of the specific cellular sites altered in endotoxemia and responsible for derangements of muscle function. In particular, no study has examined the contractile apparatus of skeletal muscle to determine if there are distinct alterations in the force-generating capacity of the contractile proteins in endotoxemia.

The purpose of the present study, therefore, was to determine whether endotoxin administration alters the intrinsic properties of the contractile apparatus of limb and respiratory skeletal muscle. Studies were performed on two groups of rats, one which was given endotoxin and one which served as a saline-treated control. To examine the contractile proteins alone, independent of the influence of any other component of the contractile machinery, we studied single, Triton X-100-skinned muscle fibers from the diaphragm, soleus, and extensor digitorum longus (EDL) muscles of these animals. Triton X-100 exposure results in the removal of all cellular membranes, thereby leaving only the contractile proteins functional. Single fiber force vs. pCa relationships were determined for each muscle (2, 6), and each fiber was typed by examining myosin isoforms electrophoretically (19). Comparison was made between responses in control and endotoxin-treated groups as a function of muscle and fiber type.

	METHODS

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Solutions. The composition of all solutions used in this study was calculated using a computer program (Borland International, Scotts Valley, CA) that takes into account stability constants and stock solutions to produce final solutions of the correct ionic strength and pCa (7). The skinning solution used was composed of (in mM) 50.0 potassium methane sulfonate, 15.0 phosphocreatine, 10.0 EGTA, 1.0 Mg²⁺, 2.0 MgATP, 20.0 imidazole, with ionic strength 150.0, pCa > 8.5, and pH 7.0 at 22°C (7). When needed for storage of muscles, a cytidine-5-triphosphate (CTP) relaxing solution consisting of 110.0 mM potassium methane sulfonate, 5.0 mM EGTA, 1.0 mM Mg²⁺, 2.0 mM MgCTP, 20.0 mM imidazole, with ionic strength 150.0, pCa > 8.5, and pH 7.0 was employed. To prevent phosphorylation of the myosin light chains in these stored muscles, CTP was used in place of ATP because CTP is not a substrate for myosin light-chain kinase. All solutions contained protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride, 0.1 mM leupeptin, 10 µM aprotinin, and 1.0 mM benzamidine) to prevent muscle breakdown during experimental manipulation.

Experimental procedures. Studies were performed using single fibers from the diaphragm, soleus, and EDL muscles of 12 adult male, Zivic Miller rats weighing 300-500 g. Before experimentation, these animals were housed and cared for in accordance with American Association for Accreditation of Laboratory Animals Care guidelines in the Case Western Reserve University Animal Resource Center. Rats were divided into two groups: 1) control and 2) endotoxemic. Endotoxemic animals were injected intraperitoneally with 8 mg/kg lipopolysaccharide (from Escherichia coli serotype 055:B5); controls were given 1 ml saline intraperitoneally. After incubation for 18 h, all animals were anesthetized and killed by decapitation. Muscles (i.e., the diaphragm, soleus, and EDL) were dissected, rinsed in skinning solution, and transferred to a 50% CTP relaxing-50% glycerol solution (containing protease inhibitors) for storage at 20°C. Previous studies have indicated that fibers isolated from muscles and stored in this fashion are usable for single fiber assessment for at least 3 mo. In the present study, all single-fiber assessments were completed within 1 wk of dissection.

On the day that fiber characteristics were assessed, muscles were removed from the relaxing glycerol solution, placed in a petri dish containing fresh skinning solution (including protease inhibitors), and allowed to warm to room temperature. Small bundles of ~10 fibers were then separated from the whole muscle by gently pulling on one end of the muscle with a pair of fine-tipped forceps while the other end of the muscle was held stationary with a second pair of forceps. Fiber bundles were immersed for 30 min in 0.1% Triton X-100, an ionic detergent that permeabilizes and disrupts all membranes, leaving only the contractile proteins intact.

After incubation bundles were removed from Triton X-100 and placed back in skinning solution, and individual fibers were teased away from bundles. The ends of fibers were carefully wound around two platinum posts, which protruded into the skinning solution. One of these posts was attached to a microforce transducer (Harvard Apparatus, South Natick, MS) mounted on a movable gear assembly, whereas the other was fixed to a stationary metal support. Sarcomere length was set to 2.6 µm using a helium-neon laser. All procedures involving the manipulation of single muscle fibers were conducted with the aid of a dissecting microscope.

Force vs. pCa curves were then constructed for each fiber by immersing fibers in solutions of increasing calcium concentrations and recording tension on a strip recorder (6). Once peak tension was achieved in a given solution, fibers were rapidly submerged in the next solution by means of a spring-loaded, Plexiglas tray. Initially, fibers were submerged in a relaxing solution containing no added calcium (pCa 8.5). Each fiber was then sequentially exposed to 13 different calcium solutions: pCa 6.0, 5.90, 5.80, 5.75, 5.70, 5.65, 5.60, 5.55, 5.50, 5.40, 5.30, 5.20, and 5.0. These solutions correspond to a range of calcium concentrations of 10⁶ to 10⁵ M. After exposure to pCa 5.0, fibers were transferred back to relaxing solution. The diameter of fibers was measured using a micrometer mounted in the eyepiece of the dissecting microscope. Fibers were stored in SDS sample buffer at 70°C and later typed using gel electrophoresis as described below.

A total of 144 single muscle fibers were examined in this study (6 diaphragm fibers, 3 soleus fibers, and 3 EDL fibers from each of the 12 rats studied). For each muscle fiber tested, one force vs. pCa curve was constructed.

Fiber typing. The myosin heavy chain (MHC) isoform of each single fiber studied was determined using the SDS gel electrophoresis procedure of Talmedge and Roy (19). After boiling in SDS sample buffer for 4 min, MHCs were separated using an 8% running gel and a 6% stacking gel, both of which contained glycerol. Gels were run at 70 volts for 20 h in a cold room. After separation, MHC isoforms were visualized by Coomassie blue staining. Myosin isoforms were identified by comparison to the MHC separation pattern of extracted myosin samples from normal rat diaphragm (these samples contained all MHC isoforms) and by comparison to previously published data (19).

Calculations. Absolute force was normalized to cross-sectional area; for this calculation, fibers were assumed to be cylindrical and fiber diameter was used to calculate cross-sectional area. The maximum calcium-activated force (F_max) of fibers was expressed in kilo Pascals (kPa). Using computer software (SigmaPlot, Statistical Package for the Social Sciences, SPSS; San Rafael, CA), force data were fit to the Hill equation
where Ca₅₀ is the Ca²⁺ concentration required to produce half-maximal activation and n is the Hill coefficient. The Ca₅₀ was used as an index of calcium sensitivity and the Hill coefficient was used as a measure of myofilament cooperativity.

Except where otherwise noted, all data are expressed as means ± SE. Statistical calculations were performed using SigmaStat (SPSS). P < 0.05 was taken to indicate statistical significance.

	RESULTS

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Diaphragm fiber force generation. The endotoxin regimen employed in the present study had a marked effect on the force production of Triton X-100-skinned muscle fibers. Figure 1 displays the absolute force vs. pCa relationship of single diaphragm fibers taken from control and endotoxemic animals. Fibers from endotoxemic animals had reductions in force generation over the entire range of pCa tested compared with control fibers, without an apparent left- or rightward shift in the calcium sensitivity. On average, maximum calcium- activated force of diaphragm fibers from endotoxemic animals (80.25 ± 2.30 kPa, n = 36) was 29% lower than for fibers from saline-treated controls (112.34 ± 2.64 kPa, n = 36; P < 0.001 for this comparison).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Mean data presenting averaged absolute (Abs) force vs. pCa relationship for diaphragmatic fibers from control () and endotoxemic () animals. Error bars represent SE.

Despite the observed difference in absolute force generation, endotoxin administration did not alter the calcium sensitivity of single diaphragm fibers. Figure 2 illustrates that when force vs. pCa was normalized to the F_max of each fiber, no significant effect of endotoxin on calcium sensitivity was observed. The Ca₅₀ for control and endotoxemic fibers was 1.61 ± 0.05 × 10⁶ M and 1.69 ± 0.05 × 10⁶ M, respectively, and the Hill coefficients were 3.944 ± 0.130 and 3.708 ± 0.131 for the same groups.

View larger version (10K): [in this window] [in a new window]   Fig. 2.   Mean data presenting force expressed as percentage maximum calcium-activated force (Fmax) vs. pCa for diaphragmatic fibers from control and endotoxemic experiments. Symbols same as Fig. 1.

Leg fiber force generation. The pattern of contractile alterations observed in diaphragm skinned fibers in response to endotoxin was replicated for fibers isolated from the soleus and EDL. The mean F_max of soleus fibers was reduced 36% from 111.55 ± 3.66 to 72.47 ± 2.97 kPa by the administration of endotoxin (Fig. 3A; P < 0.001 for this comparison, n = 18 for each group), whereas the F_max of EDL single fibers was lowered 25% from a control value of 104.05 ± 4.33 to 78.32 ± 2.43 kPa (P < 0.001, n = 18 for each group; results shown in Fig. 3B). As in the diaphragm, no significant alterations in calcium sensitivity (Ca₅₀) or the Hill coefficient were detected.

View larger version (10K): [in this window] [in a new window]   Fig. 3.   Mean data presenting averaged absolute force vs. pCa relationship for soleus fibers (A) and extensor digitorum longus (EDL) fibers (B) from control and endotoxemic animals. , Control soleus fibers; , endotoxemic soleus fibers; , control EDL fibers; , endotoxemic EDL fibers.

MHC isoforms. When examined as a function of myosin isoform, control diaphragm force generation was similar across all fiber types (Fig. 4A). Specifically, control group F_max was 103.2 ± 9.9 kPa for type IIa fibers, 116.9 ± 4.6 kPa for type IIx fibers, 104.0 ± 4.3 kPa for type IIb fibers, and 105.1 ± 0.4 kPa for slow fibers. The contractile response to endotoxin administration was similar in all fiber types and, as a result, F_max was uniformly lower compared with control. These values were 71.1 ± 8.3 kPa for type IIa fibers, 78.4 ± 3.2 kPa for type IIx fibers, 82.9 ± 5.4 kPa for type IIb fibers, and 94.3 ± 5.3 kPa for slow fibers. The Ca₅₀ and the Hill coefficient were also similar across all diaphragm fiber types in both control and endotoxemic groups. Specifically, Ca₅₀ was 1.56 ± 0.12 × 10⁶ M, 1.56 ± 0.06 × 10⁶ M, 1.67 ± 0.08 × 10⁶ M, and 1.68 ± 0.25 × 10⁶ M for control IIa, IIx, IIb, and slow fibers and 1.62 ± 0.05 × 10⁶ M, 1.66 ± 0.07 × 10⁶ M, 1.75 ± 0.11 × 10⁶ M, and 1.75 ± 0.16 × 10⁶ M for endotoxemic IIa, IIx, IIb, and slow fibers. The Hill coefficients were 4.640 ± 0.406, 3.862 ± 0.180, 3.599 ± 0.177, 4.100 ± 0.351, and 3.778 ± 0.336, 3.549 ± 0.214, 4.049 ± 0.251, 3.533 ± 0.245 for type IIa, IIx, IIb, and slow muscle fibers from control and endotoxemic diaphragms, respectively.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   A: absolute mean maximum fiber force generation as function of fiber type for diaphragm fibers taken from control (hatched bars) and endotoxemic (solid bars) animals. B: absolute mean maximum fiber force generation as function of fiber type for EDL fibers taken from control (hatched bars) and endotoxemic (solid bars) animals. Error bars represent SE.

Figure 4B displays EDL single fiber F_max plotted by MHC isoform. This muscle is composed entirely of fast fibers, and therefore no slow myosin isoform fibers were found. As observed in the diaphragm, endotoxin administration elicited similar F_max reductions in all EDL fiber types. As for the diaphragm, calcium sensitivity and the Hill coefficient were similar across all EDL fiber types (i.e., for IIa, IIx, and IIb) from control and endotoxin-treated animals. All soleus fibers were typed and found to contain a slow myosin isoform (data not shown).

	DISCUSSION

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A number of previous studies have reported reductions in both respiratory and limb muscle force-generating capacity in response to sepsis (8) and endotoxin administration (1, 15, 16, 18, 20). For example, van Surell et al. (20) found that injection of E. coli lipopolysaccharide was followed by significant reductions in the pressures generated by the in situ diaphragm in response to both low (1-20 Hz) and high (50-100 Hz) frequencies of electrical stimulation (20). In another study, Shindoh et al. (15) administered endotoxin to hamsters and observed a pronounced reduction in the capacity of excised diaphragm muscle strips to generate force, again observing reductions in response to both low and high frequencies of electrical stimulation. In addition, alterations in muscle force-generating capabilities in endotoxemia have been reported for limb muscles by several groups (5, 14). Moreover, the magnitude of skeletal muscle force reductions elicited by endotoxin appears to be dose dependent, and there appears to be a strong correlation between reductions in limb muscle and diaphragmatic force-generating capacity (16).

In the present study, we used an endotoxin regimen similar to that used in several previous studies by our laboratory. In this previous work, we observed significant decreases in in vitro intact muscle fiber force production in both diaphragm and leg muscles of endotoxemic rats (16). We now demonstrate that this same endotoxin regimen elicits a marked shift in the force vs. pCa relationship of Triton-skinned single muscle fibers taken from the diaphragm, EDL, and soleus, reducing the maximum force-generating capacity of the contractile elements of these fibers. The reduction in force generation observed in this study can only be attributed to the contractile proteins themselves, since incubation of fibers in Triton results in the elimination of all other functional components of the contractile process.

The administration of endotoxin results in a myriad of changes in cellular metabolism (8) and affects sarcolemmal membrane function (9), and it is probable that this stress also influences the function of a number of other cellular organelles (e.g., the sarcoplasmic reticulum) involved in muscle force-generating capacity. It would be expected that endotoxin-induced alterations in the function of these other organelles could further influence muscle force-generating capacity by either altering the time course and concentration of the calcium presented to the contractile elements during a contraction or by producing metabolic by-products (phosphate and hydrogen ions) that influence myofilament calcium sensitivity (12). It is also possible that endotoxin administration could activate the production of systemic cytokines and that these substances could target skeletal muscle fibers and lead to damage that results in decreased force production (4). Nevertheless, the present data indicate that very large reductions in muscle force-generating capacity would result from endotoxin-induced alterations in muscle fiber contractile protein characteristics even if this stress produced absolutely no additional effect on muscle function. For example, we observed a 30% average reduction in maximum diaphragm skinned fiber force-generating capacity in the present study, whereas we found only a slightly larger (45%) reduction in diaphragm fiber bundle force-generating capacity in response to a slightly different regimen of endotoxin administration in one previous report (16) and a virtually identical reduction in fiber bundle force generation (29%) in another report (17). These data argue that the shifts in contractile protein function observed in the present study in response to endotoxin administration may well account for most of the force reduction previously reported for intact muscles in this pathophysiological condition.

The changes we report here regarding force-generating capacity of skinned fibers from endotoxemic animals represent the first time, to our knowledge, that this particular pathophysiological process has been shown to produce a physiological alteration in the intrinsic force-generating capacity of the contractile proteins. The observed alterations in fiber force-generating capacity were quite stable in vitro, i.e., multiple sequential determinations of force vs. pCa relationships in a given fiber yielded the same maximum force generation in a fiber, and the duration of incubation in relaxing solution prior to the first determination had no effect on the force vs. pCa relationship. It is likely that endotoxin administration alters F_max by inducing one or more chemical modifications of the contractile apparatus that impair myosin-actin interactions. The fact that Ca₅₀ and the Hill coefficient were not affected would make it unlikely that troponin, tropomyosin, or myosin light-chain alterations were responsible for the observed shift in the force vs. pCa relationship. In theory, direct biochemical modification of the myosin head or actomyosin ATPase would result in a reduction in F_max without an alteration in Ca₅₀ or the Hill coefficient and would be possible candidates to explain the changes observed in this study (13).

Although the present study does not identify the mechanism by which the contractile proteins are modified in endotoxemia, it is worth noting that muscles taken from animals injected with endotoxin have been shown recently to generate substantially more reactive oxygen species (i.e., free radicals) during contraction than muscles taken from controls (11). In addition, several reports have shown that decrements in whole muscle force-generating capacity in endotoxemia can be blunted by the administration of scavengers of free radicals (15, 16, 18). This implies that one or more free radical species (superoxide, nitric oxide, peroxynitrite, hydrogen peroxide, hydroxyl radicals) are responsible for inducing endotoxin-related alterations in muscle function. As a result, we would speculate that the changes in contractile protein function observed in the present study may have been free-radical induced. In keeping with this possibility, incubation of Triton- skinned muscle fibers with either peroxynitrite, nitric oxide, hydroxyl radicals, or superoxide has been shown recently to produce functional alterations very similar to those observed in the present study (2, 3, 13, 16a).

This study represents the first time to our knowledge that the force-generating capacity of the contractile proteins themselves has been shown to be altered in any pathophysiological condition. The specific biochemical alteration or alterations responsible for this decrease in muscle fiber force generation remains to be elucidated. Nevertheless, this finding is of importance because it may represent an underlying mechanism by which skeletal muscle function is diminished in other situations.