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ROS-Mediated Decline in Maximum Ca 2+ -Activated Force in Rat Skeletal Muscle Fibers following In Vitro and In Vivo Stimulation

Travis L. Dutka

1 Department of Zoology, La Trobe University, Melbourne, Australia,

Esther Verburg

2 Institute for Experimental Medical Research, University of Oslo and Oslo University Hospital, Oslo, Norway,

Noni Larkins

1 Department of Zoology, La Trobe University, Melbourne, Australia,

Kristin H. Hortemo

2 Institute for Experimental Medical Research, University of Oslo and Oslo University Hospital, Oslo, Norway,

Per K. Lunde

2 Institute for Experimental Medical Research, University of Oslo and Oslo University Hospital, Oslo, Norway,

Ole M. Sejersted

2 Institute for Experimental Medical Research, University of Oslo and Oslo University Hospital, Oslo, Norway,

Graham D. Lamb

1 Department of Zoology, La Trobe University, Melbourne, Australia,

Conceived and designed the experiments: TLD EV NL OMS GDL. Performed the experiments: TLD EV NL KHH PKL. Analyzed the data: TLD EV NL KHH PKL GDL. Contributed reagents/materials/analysis tools: OMS GDL. Wrote the paper: TLD NL EV GDL.


We hypothesised that normal skeletal muscle stimulated intensely either in vitro or in situ would exhibit reactive oxygen species (ROS)-mediated contractile apparatus changes common to many pathophysiological conditions. Isolated soleus (SOL) and extensor digitorum longus (EDL) muscles of the rat were bubbled with 95% O2 and stimulated in vitro at 31°C to give isometric tetani (50 Hz for 0.5 s every 2 s) until maximum force declined to ≤30%. Skinned superficial slow-twitch fibers from the SOL muscles displayed a large reduction (∼41%) in maximum Ca 2+ -activated specific force (Fmax), with Ca 2+ -sensitivity unchanged. Fibers from EDL muscles were less affected. The decrease in Fmax in SOL fibers was evidently due to oxidation effects on cysteine residues because it was reversed if the reducing agent DTT was applied prior to activating the fiber. The GSH∶GSSG ratio was ∼3-fold lower in the cytoplasm of superficial fibers from stimulated muscle compared to control, confirming increased oxidant levels. The presence of Tempol and L-NAME during in vitro stimulation prevented reduction in Fmax. Skinned fibers from SOL muscles stimulated in vivo at 37°C with intact blood supply also displayed reduction in Fmax, though to a much smaller extent (∼12%). Thus, fibers from muscles stimulated even with putatively adequate O2 supply display a reversible oxidation-induced decrease in Fmax without change in Ca 2+ -sensitivity, consistent with action of peroxynitrite (or possibly superoxide) on cysteine residues of the contractile apparatus. Significantly, the changes closely resemble the contractile deficits observed in a range of pathophysiological conditions. These findings highlight how readily muscle experiences ROS-related deficits, and also point to potential difficulties when defining muscle performance and fatigue.


Reactive oxygen and nitrogen species (ROS and RNS) are thought to have a major role in the skeletal muscle weakness observed in a host of pathophysiological conditions such as sepsis [1], [2], rheumatoid arthritis and other inflammatory conditions [3], [4], and heart failure and stroke [5], [6], [7], [8]. In many of these conditions the muscle weakness is associated with a reduction in maximum specific force (Fmax) independent of muscle atrophy, and occurs without any change in Ca 2+ -sensitivity [9]. However, it is not known which specific oxidant(s) causes the dysfunction, which protein sites are involved, whether the dysfunction is acutely reversible, and whether different fiber types are affected to the same degree. It is also unclear whether or how readily normal skeletal muscle displays the same specific dysfunction in conditions where ROS and RNS levels are elevated.

Application of exogenous ROS and RNS have been shown to have various effects on maximum force production and/or Ca 2+ sensitivity of the myofilaments [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], as well as on the activity of the sarcoplasmic reticulum (SR) Ca 2+ pumps (SERCA) [20], and the SR Ca 2+ release channels (ryanodine receptors) [21], [22] (for review see [23], [24], [25]). ROS and RNS are generated in muscle with both hypoxia and hyperoxia [26], [27], as well as with elevated muscle temperature in vitro [28], [29]. Interestingly, it has been found that a 30 min period of anoxia reduced Fmax in rat soleus (SOL) muscle bundles by ∼32% without change in Ca 2+ -sensitivity, in association with increased nitrotyrosine levels, and also that the effects could be prevented with the nitric oxide synthase inhibitor L-NMMA [30]. It was concluded that the contractile changes likely resulted from the effects of increased levels of peroxynitrite. Furthermore, when mouse EDL muscles bubbled in vitro with 95% O2 were maintained at 37°C for 30 min, Fmax also decreased substantially (∼50%) without change in Ca 2+ -sensitivity, with effects prevented by the superoxide dismutase mimetic Tempol [28]. The decrease in Fmax was suggested to be due to increased superoxide, but might also be due to effects of peroxynitrite, which is readily generated when superoxide and nitric oxide levels are elevated [31], [32], [33], [34]. In apparent accord, application of peroxynitrite causes a reduction in Fmax with little or no change in Ca 2+ -sensitivity in both limb muscles and diaphragm [18], [35], interestingly with greater effect in slow-twitch SOL muscle fibers than in fast-twitch EDL fibers [18]. Application of superoxide also causes a decrease in Fmax in diaphragm fibers, though its effects on Ca 2+ -sensitivity were not ascertained [11]. In contrast, related ROS and RNS, including H2O2, hydroxyl, NO and GSNO, have quite different actions on limb muscle fibers, changing Ca 2+ -sensitivity more readily than Fmax or having little if any effect (as in case of H2O2) [12], [13], [17], [19].

Importantly, ROS and RNS are known to be generated in muscle with activity [23], [24], [36]. Various studies of muscle function have utilized in vitro stimulation of whole muscles or muscle bundles with 95% O2 at temperatures ≥30°C. The high O2 levels are used to avoid hypoxia in deep regions of the muscle [37], but the combination of stimulation and hyperoxia seem assured to lead to elevated levels of ROS and RNS, raising the question of whether resulting ROS-related effects influence the muscle properties found. An important related question and comparison is what effects, if any, occur in muscles stimulated in situ with their blood supply intact.

This study investigates whether the increased levels of ROS/RNS generated by stimulating an in vitro whole muscle preparation in typical oxygenation conditions, causes dysfunction of the contractile proteins in superficial, well-oxygenated fibers, and compares results found in an in situ muscle preparation with more physiological oxygenation and perfusion. We hypothesised that normal healthy muscle can be made to exhibit the hallmarks of ROS-mediated contractile apparatus changes characteristic of many pathophysiological conditions if the muscles experience abnormal O2 supply and perfusion. We examined maximum force production and Ca 2+ -sensitivity in skinned fibers from muscle stimulated in vitro and in situ, comparing each to matching fibers from the non-stimulated contralateral muscles, and further tested the effects of particular treatments to counter or reverse possible oxidation effects. By examining contractile properties of skinned fibers under standardized conditions mimicking the resting cytoplasmic environment, it was possible to determine whether there were any changes in fiber characteristics without confounding effects arising from the alterations in cytoplasmic conditions that occur with repeated stimulation.


Ethics Statement

All animal in vitro experiments were performed at La Trobe University (Melbourne Campus, Australia) with approval of the La Trobe University Animal Ethics Committee (animal ethics number AEC06-09-L). All animal in vivo experiments were conducted at the University of Oslo (Norway) in accordance with current regulations and approved by the Norwegian Animal Research Authority, approval ID 2310.

In vitro whole muscle preparation and experimental protocol

Seventeen male Long-Evans hooded rats and one male Wistar rat, 3–8 mo old, were kept at the La Trobe University Central Animal House in cages in a climate-controlled room with 12/12 h light/dark cycle and food and water given ad libidum. The rats were killed by overdose of isoflurane and their soleus ‘SOL’ or extensor digitorum longus ‘EDL’ muscles dissected out and attached between a glass hook and an insulated force-transducer in an in vitro bath filled with Krebs-Ringer solution at 31°C. The solution was bubbled with 95% O2 and 5% CO2, with final pH of ∼7.4. Force responses were sampled at 1000 Hz on a personal computer (Powerlab hardware and Chart 5 software, ADInstruments Australia). Following 10 min of equilibration, the muscle was electrically stimulated with field stimulation to contract isometrically. Muscle length and stimulation voltage were varied to give maximum twitch force and voltage set at >1.5 times this level, and then the force frequency behaviour examined with ∼5 tetani evoked at various frequencies (10–100 Hz). After a further 5 min rest the stimulation protocol was started. In one set of experiments one of each contralateral pair of SOL muscles was subjected to a prolonged train of tetani, each elicited with 1 ms pulses at 50 Hz for 0.5 s every 2 s, until the peak tetanic force dropped to less than 30% of its initial level (on average muscles were stimulated for ∼5 min). In most instances, following two minutes rest the muscle was subjected to: a) one further 0.5 s 50 Hz stimuli in order to test whether there was any rapid recovery of the tetanic force, b) another such stimulus at higher voltage to verify that the stimulating voltage was still supramaximal, and c) finally tested with a single prolonged 50 Hz stimulus until the force plateau was reached in order to assess maximum tetanic force. Immediately afterwards the muscle was removed from the in vitro bath and pinned at resting length in a silica-gel lined Petri dish filled with paraffin oil. The dish was then put on ice in order to maintain the muscle temperature at ∼6–8°C. Contralateral muscles were placed directly into paraffin oil in a Petri dish and put on ice, and served as the rested control muscle. In another set of experiments, both SOL muscles from a given rat were stimulated in vitro as above, but one was left to recover in oxygenated Krebs-Ringer solution for 45 min following stimulation before being put in oil in the Petri dish and on ice.

In vivo whole muscle preparation and experimental protocol

Five male Wistar rats, 3–4 months old (supplied by Taconic, Skensvedt Denmark) were kept for at least 1 wk at the animal facility at Oslo University Hospital Ullevaal in cages in a climate-controlled room with 12/12 h light/dark cycle and food and water given ad libidum. Each rat was anesthetised in a chamber filled with 1∶3 O2/N2O with 4% isoflurane (Abbott no. 506949, Chicago, Ill. USA) and then the rat was intubated and attached to a respirator (Zoovent, Triumph Technical Services LTD, London, UK) and kept under anesthesia with 1∶3 O2/N2O gas mixture with 2–3% isoflurane for the remainder of the experiment. Blood pressure was measured with a microtip pressure catheter (SPR-407, Millar Instruments Inc, Houston TX, USA) inserted via the right arteria carotis communis and led retrograde to the aortic arch, and used to monitor anesthesia depth and general condition of the animal. The animal was kept on a heated table (37°C) during the experiment.

The right SOL muscle was carefully dissected free from the surrounding tissues, leaving the muscles blood supply intact. The calcaneus was cut and tendons to the other calf muscles separated and cut, leaving just the SOL tendon. This tendon was attached to the lever of a servo-controlled force transducer (model 305B Aurora Scientific, Canada). The tibia was clamped immobile. The muscle was kept moist and at 37°C by dripping warmed 0.9% NaCl solution onto the muscle. The sciatic nerve was cut to prevent retrograde transmission of the stimulation current. The SOL muscle was activated to contract isometrically by direct stimulation with platinum electrodes with 1 ms pulses at 8 V (pulsar 6 bp, FHC Brunswick, ME, USA), at the proximal and distal end of the muscle. Optimum length and voltage was set using twitch force, followed by a few test tetani at various frequencies (10–100 Hz). After 5 min rest, the stimulation protocol was started, consisting of the same repeated 50 Hz tetani protocol as the in vitro experiments (0.5 s every 2 s, 1 ms pulses), with the only difference being that the protocol was continued for 10 min in every case. Force, aortic pressure, muscle surface temperature and stimulation pulses were sampled at 2000 Hz (National Instruments hardware and Labview software). Immediately after the stimulation protocol, the muscle was removed and pinned under paraffin oil in a Petri dish, which was put on ice. The contralateral muscle was dissected out and also pinned in the Petri dish, and served as resting control. At the end of the experiment the rat was sacrificed by decapitation.

Single skinned fiber preparation

Skinned fibers were prepared from SOL and EDL muscles as described previously [12], [38]. Using the muscles kept under paraffin oil on ice, single fibers were dissected from the superficial region of the muscle ( 2+ (giving 1 mM free), 126 K + , 36 Na + , 8 ATP and 10 creatine phosphate (CP), pH 7.10, pCa ( = −log10[Ca 2+ ])>9. Maximal Ca 2+ -activating solution contained (in mM) 50 CaEGTA, 9, Hepes, 8.1 total Mg 2+ (giving 1 mM free), 126 K + , 36 Na + , 8 total ATP, 10 CP, pH 7.10 and pCa 4.7. Solutions with free [Ca 2+ ] heavily buffered at intermediate levels (pCa 6.7 to 4.7) were obtained by mixing appropriate volumes of the relaxing solution and maximal activating solutions. The strontium (Sr) activating solution at pSr 5.3 was made by mixing relaxing buffer with maximal Sr activating solution containing (mM): 40 SrEGTA, 10 EGTA, 90 Hepes, 8.5 Mg 2+ (giving 1 mM free), 126 K + , 36 Na + , 8 ATP, 10 CP, pH 7.10, pSr 3.7. Where required, 10 mM dithiodithreitol (DTT) was added to relaxing solution from a 1 M stock prepared in distilled water. Tempol (4-Hydroxy-2, 2, 6, 6-tetramethylpiperidine), a superoxide dismutase mimetic, and L-NAME (N-Nitro-L-arginine methyl ester hydrochloride), a nitric oxide (NO • ) scavenger, were directly dissolved in the Krebs-Ringer solution at a concentration of 1 and 3 mM respectively.

ROS-Mediated Decline in Maximum Ca 2+ -Activated Force in Rat Skeletal Muscle Fibers following In Vitro and In Vivo Stimulation Travis L. Dutka 1 Department of Zoology, La Trobe ]]>