Preconditioning improves function and recovery of single muscle fibers during severe hypoxia and reoxygenation

Suzanne Kohin, Creed M. Stary, Richard A. Howlett, and Michael C. Hogan

Department of Medicine, University of California, San Diego, La Jolla, California 92093


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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Reperfusion following prolonged ischemia induces cellular damage in whole skeletal muscle models. Ischemic preconditioning attenuates the deleterious effects. We tested whether individual skeletal muscle fibers would be similarly affected by severe hypoxia and reoxygenation (H/R) in the absence of extracellular factors and whether cellular damage could be alleviated by hypoxic preconditioning. Force and free cytosolic Ca2+ ([Ca2+]c) were monitored in Xenopus single muscle fibers (n = 24) contracting tetanically at 0.2 Hz during 5 min of severe hypoxia and 5 min of reoxygenation. Twelve cells were preconditioned by a shorter bout of H/R 1 h before the experimental trial. In preconditioned cells, force relative to initial maximal values (P/Po) and relative peak [Ca2+]c fell (P < 0.05) during 5 min of hypoxia and recovered during reoxygenation. In contrast, P/Po and relative peak [Ca2+]c fell more during hypoxia (P < 0.05) and recovered less during reoxygenation (P < 0.05) in control cells. The ratio of force to [Ca2+]c was significantly higher in the preconditioned cells during severe hypoxia, suggesting that changes in [Ca2+]c were not solely responsible for the loss in force. We conclude that 1) isolated skeletal muscle fibers contracting in the absence of extracellular factors are susceptible to H/R injury associated with changes in Ca2+ handling; and 2) hypoxic preconditioning improves contractility, Ca2+ handling, and cell recovery during subsequent hypoxic insult.

skeletal muscle; postischemic injury; cytosolic calcium; ischemia; reperfusion


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PROLONGED ISCHEMIA AND REPERFUSION result in tissue damage, vascular dysfunction, and necrosis in many organs. Mechanisms by which damage is incurred are widely studied, yet still not fully understood. In skeletal muscle, the deleterious effects appear to be closely associated with proinflammatory processes, perhaps mediated by activation of complement, circulating neutrophils, reactive oxygen species (ROS) generation, and/or Ca2+ overload from extracellular sources (7, 8, 16). Ischemic preconditioning, by which the tissue is exposed to brief periods of vascular occlusion and reperfusion before a more prolonged ischemic event, confers some protection from ischemia and reperfusion injury (11). Acute ischemic preconditioning reduces infarct size (10, 12), alleviates no-reflow in capillaries (6, 24), and improves contractile force generation (3, 9) following ischemia and reperfusion in skeletal muscle models.

While circulating factors are known to contribute to postischemic cellular injury in intact animals and whole muscle models, intracellular processes are also implicated. Of the proposed mechanisms not dependent on circulating factors, increased intracellular Ca2+ during ischemia and reperfusion appears to be a primary cause of postischemic injury in muscle cells (1, 27). Ca2+ homeostasis may initially be disrupted through ROS-induced injuries and subsequent disturbances in Ca2+ handling (4, 15, 20). Elevations in intracellular Ca2+ activate proteases and phospholipases that cause irreversible cellular and mitochondrial damage (4, 14, 20). In addition, high intracellular Ca2+ can uncouple oxidative phosphorylation, exacerbating the low energy supply (4, 23).

The goal of the present study was to examine the effects of severe hypoxia (PO2 = 3-5 Torr) and reoxygenation (H/R), the in vitro analog to ischemia and reperfusion, on muscle function and Ca2+ handling in working single isolated muscle fibers. The single isolated cell model is ideal to test whether extracellular components are required to induce cellular damage to skeletal muscle during H/R. A second goal was to determine whether preconditioning confers protection from damage during a subsequent H/R event in this model. Force generation and free cytosolic Ca2+ concentration ([Ca2+]c) were monitored in preconditioned and nonpreconditioned working single muscle cells during severe H/R. The results indicate that acute severe H/R causes considerable loss in muscle cell performance in the absence of extracellular factors. Preconditioning attenuated losses in muscle function and improved cell recovery.


    METHODS
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INTRODUCTION
METHODS
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Single fiber preparation. Adult female Xenopus laevis were doubly pithed and decapitated. Lumbrical muscles II-IV were removed, and single living muscle fibers free of adherent microvessels were microdissected from the muscle. Dissections and experiments were performed in Ringer solution (112 mM NaCl, 1.87 mM KCl, 0.82 mM CaCl2, 2.38 mM NaHCO3, 0.07 mM NaH2PO4, 0.1 mM EGTA, and 5 mM glucose) at 20°C and pH 7.0. After dissection, the cells were pressure-injected with the Ca2+ indicator fura 2 (F-1200; Molecular Probes). Platinum clips were attached to the tendons, and the fibers were mounted in a glass chamber and placed on the stage of an inverted microscope configured for epi-illumination. Experiments were begun after a 1-h equilibration period to allow fura 2 to diffuse evenly throughout each cell from the site of injection.

Tetanic contractions were induced by direct stimulation (70 impulses/s of 1-ms duration at 9 V, with a train duration of 200 ms) with platinum conducting electrodes on either side of the fiber using a Grass S48 stimulator (Quincy, MA).

Experimental protocols. High-PO2 (159 Torr) and low-PO2 (3-5 Torr) solutions were generated by bubbling Ringer solution with mixed gases of 21% O2-5% CO2-74% N2 and 5% CO2-95% N2, respectively. In several experiments, superfusate PO2 was monitored with a Clark-style electrode (model 733; Diamond General, Ann Arbor, MI) to confirm that the desired PO2 was achieved.

Control cells (C cells, n = 12) were stimulated with tetanic contractions at a frequency of 0.2 Hz for 13 min. During the 13-min stimulation period, the cells were continually superfused, first for 3 min with the high-PO2 solution and then for 5 min with the low-PO2 solution and were finally reoxygenated for 5 min with the high-PO2 solution. Preconditioned cells (PC cells, n = 12) were subjected to two periods of tetanic contractions at 0.2 Hz separated by a 1-h rest period. During the first 10-min stimulation period, PC cells were superfused initially for 3 min with the high-PO2 solution and then for 2 min with the low-PO2 solution and were finally reoxygenated for 5 min with the high-PO2 solution. After a 1-h recovery period, PC cells were then subjected to the same stimulation protocol as the C cells (3 min of high PO2, 5 min of low PO2, 5 min of reoxygenation).

Force and [Ca2+]c measurements. Force development was measured with a force transducer system (model 400A; Aurora Scientific, Aurora, ON, Canada). A MP100WSW analog-to-digital converter (Biopac Systems, Santa Barbara, CA) was used to transform the analog force signal, and the digital data were collected and analyzed with AcqKnowledgeIII 3.2.6 software (Biopac Systems). Individual peak generated forces (P) are reported relative to the initial maximal force measured during the steady-state contractile period (Po).

[Ca2+]c was obtained using fluorescence spectroscopy. Fibers were illuminated with two rapidly alternating (20 Hz) excitation wavelengths of 340 and 380 nm, and the resulting fluorescence emissions at 510 nm were divided (340/380) to obtain the Ca2+-dependent signal (2). Fluorescence was measured with an illumination and detection system (DeltaScan model; Photon Technology International) integrated with a Nikon inverted microscope with a 40× Fluor objective. Relative peak [Ca2+]c measurements are reported as the average of five 340/380 excitation ratio maxima at the measurement time point compared with the highest five excitation ratio maxima measured during the initial 3-min steady-state contractile period. Relative baseline Ca2+ measurements (340/380 baseline) were obtained in a similar fashion and compared with the lowest baseline Ca2+ level within each run.

Generated force and [Ca2+]c were monitored simultaneously throughout all trials. Data are reported for five time points throughout each experiment: at the end of the 3-min steady-state period, 2 min into hypoxia, at the point where generated force was minimal during hypoxia; at the point where generated force was maximal during reoxygenation; and at the end of the trial.

Statistics. Statistical analyses were carried out with SYSTAT software (Evanston, IL). For all dependent variables, two-way analysis of variance was performed. Dunnett's test was used to determine significant changes from steady-state values as necessary. For significant treatment effects, one-way analysis of variance was used to compare the two groups. In all analyses, significance was determined at the P < 0.05 level. Results are reported as means ± SE.


    RESULTS
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During the 3-min high-PO2 superfusion period, the muscle cells achieved a near steady state of force development. Generated force subsequently fell in both groups of cells during 5 min of severe hypoxia (PO2 = 3-5 Torr; Fig. 1). Force relative to initial maximal values (P/Po) fell significantly less in PC than in C cells (to 0.64 ± 0.07 vs. 0.30 ± 0.05, respectively; P < 0.001). During reoxygenation, P/Po recovered in PC cells (0.90 ± 0.05) but remained depressed in C cells (0.68 ± 0.08; P = 0.024). To determine that the fall in force throughout the stimulation period was not due to fatigue but, rather, to the H/R protocol, we performed an experiment in which we examined force in single fibers during prolonged tetanic stimulation at 0.2 Hz (n = 3). No significant fatigue was observed after 21 min (P/Po was maintained at 0.92 ± 0.02).


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Fig. 1.   Mean (±SE) force production relative to initial maximal force (P/Po) vs. time during 5 min of severe hypoxia and 5 min of reoxygenation for 12 preconditioned and 12 control cells. Force fell during severe hypoxia in both groups of cells. The fall in force was less during hypoxia, and the recovery was greater during reoxygenation, in the preconditioned cells than in the control cells. Horizontal error bars reflect differences in the time to reach minimum force during severe hypoxia and maximum force during reoxygenation. *P < 0.05 compared with steady-state value. dagger P < 0.05 compared with control cells.

Changes in relative peak [Ca2+]c followed a pattern similar to the changes in relative force: relative peak [Ca2+]c fell during severe hypoxia and partially recovered during reoxygenation (Fig. 2). The fall in relative peak [Ca2+]c was significantly less in PC cells than in C cells (0.80 ± 0.06 vs. 0.59 ± 0.05, respectively; P = 0.014). Relative peak [Ca2+]c recovered to a significantly higher level during reoxygenation in PC cells (0.88 ± 0.02) than in C cells (0.77 ± 0.03; P = 0.01). Despite similarities in the force and peak [Ca2+]c records within each group, the ratio of relative force to peak [Ca2+]c was significantly greater (P = 0.001) during severe hypoxia in PC cells than in C cells (Fig. 3); however, it recovered during reoxygenation in both groups. Relative baseline [Ca2+]c increased during hypoxia and remained elevated during reoxygenation, but the responses were not different between PC and C cells (Fig. 2).


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Fig. 2.   Mean (±SE) relative free cytosolic Ca2+ concentration ([Ca2+]c) vs. time during 5 min of severe hypoxia and 5 min of reoxygenation. Upper curves show relative peak [Ca2+]c, and lower curves show relative baseline [Ca2+]c. There was a fall in relative peak [Ca2+]c during severe hypoxia in both groups of cells that persisted during reoxygenation in the control cells. Relative baseline [Ca2+]c rose during severe hypoxia and remained elevated during reoxygenation in both groups. Horizontal error bars reflect differences in the time to reach minimum force during severe hypoxia and maximum force during reoxygenation.*P < 0.05 compared with steady-state value. dagger P < 0.05 compared with control cells.



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Fig. 3.   Ratio of generated force to peak [Ca2+]c during severe hypoxia and reoxygenation. Both groups of cells showed a decrease in the force-to-peak [Ca2+]c ratio during severe hypoxia; however, the ratio was significantly lower in the control cells than in the preconditioned cells. Horizontal error bars reflect differences in the time to reach minimum force during severe hypoxia and maximum force during reoxygenation. *P < 0.05 compared with steady-state value. dagger P < 0.05 compared with control cells.


    DISCUSSION
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INTRODUCTION
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DISCUSSION
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These results clearly show that working single muscle fibers are susceptible to severe H/R injury. Five minutes of near anoxic conditions caused a marked decline in force generation despite the absence of vascular endothelium and circulating factors. The decline in force was associated with an even greater decrease in relative peak [Ca2+]c, suggestive of a loss in myofibrillar Ca2+ sensitivity. Preconditioning with a brief bout of hypoxia improved force production and attenuated changes in Ca2+ handling during the subsequent H/R event in this model.

Ischemia and reperfusion injury in skeletal muscle. It is well documented that prolonged ischemia and reperfusion lead to skeletal muscle injury (3, 9, 12, 13, 23, 24). Several extracellular and intracellular factors have been implicated in muscle damage. In an isolated skeletal muscle fiber model, processes likely to contribute to injury during H/R include elevations in [Ca2+]c and/or the generation of ROS from subcellular sources (16). [Ca2+]c rises as a consequence of increases in H+ during ischemia, which is exchanged across the membrane for Na+ on the Na+/H+ exchanger. Na+ subsequently leaves the cell in exchange for Ca2+ on the Na+/Ca2+ exchanger (14, 27). In addition, during contractions, a significant amount of Ca2+ entry is mediated by voltage-gated Na+ channels (1). In isolated Xenopus muscle fibers, Stary and Hogan (18) observed elevations in baseline [Ca2+]c during fatiguing stimulation under hypoxic conditions (extracellular PO2 = 22 Torr) relative to higher extracellular PO2 conditions. Excessive intracellular Ca2+ activates proteases and phospholipases that can cause irreversible cellular damage (14, 20).

In the present study, we have demonstrated increases in relative baseline [Ca2+]c during severe H/R; however, there was no significant difference in the average change between the PC and C cells. After further analysis, we found that some C cells were more susceptible to changes in baseline [Ca2+]c than others. Of the 12 nonpreconditioned fibers, 7 did not survive the H/R event as indicated by the inability to generate measurable force 1 h after the experimental trial. The five fibers that survived severe H/R displayed almost no change in relative baseline [Ca2+]c, whereas the fibers that did not survive H/R had significantly greater increases in relative baseline [Ca2+]c. Relative baseline [Ca2+]c rose 21 ± 15% (n = 5) in the surviving cells during severe hypoxia vs. a rise of 104 ± 26% (n = 7, P = 0.032) above the pre-hypoxic levels in those cells that did not survive H/R, suggesting that the rise in intracellular Ca2+ could have contributed to cellular injury in some cells. These results parallel those of Smith et al. (17), who demonstrated in an in situ canine gracilis muscle model that myocyte viability following ischemia and reperfusion was associated with the ability to extrude excessive Ca2+.

The other possible contributor to cellular injury in this model is an increase in ROS during H/R. ROS can be released from circulating neutrophils, but sources of ROS also exist subcellularly. Vanden Hoek et al. (21) recently demonstrated that ROS of mitochondrial origin are released during hypoxic preconditioning in cultured cardiomyocytes and may be important in signaling a protective cascade (see below). A larger burst of ROS, however, is released during reoxygenation from an alternate subcellular source and is correlated to cardiomyocyte death (22). Deleterious effects of increased oxidants include damage to mitochondria and increased cell permeability. Blocking the ROS burst upon reoxygenation conferred protection to the cells (22). The potential sources of ROS in isolated muscle fibers during reoxygenation are the same as in cardiomyocytes, e.g., NADH, NADPH, cytochrome P-450, or other endogenous oxidases, the mitochondrial electron transport chain, and possibly nitric oxide synthase (21), and it is reasonable to assume that ROS may have contributed to muscle cell injury in the present experiment. The fact that nonpreconditioned muscle fiber function was compromised relative to the preconditioned fibers even before reoxygenation suggests that a combination of factors may have contributed to muscle cell injury in this model.

Ischemic injury to skeletal muscle may also vary depending on muscle fiber type. Xenopus lumbrical muscles contain muscle fibers of three types (25): a high-mitochondrial density fiber that has slow-twitch characteristics and is very fatigue resistant (type 3), a low-mitochondrial density fiber that has fast-twitch characteristics and fatigues very easily (type 1), and an intermediate type (type 2). One study reported that mammalian skeletal muscle composed predominately of fast-twitch fibers had greater areas of necrosis than slow-twitch muscle after 5 h of hindlimb ischemia and reperfusion (13). However, another study demonstrated no difference in sensitivity of resting type I and type II fibers to ischemia and reperfusion injury (19). In fast-twitch fibers, it is expected that there be a greater depletion of ATP during prolonged ischemia, and perhaps a loss of precursors for oxidative phosphorylation during reperfusion precludes adequate restoration of ATP (23). In addition, Gissel and Clausen (1) reported that Ca2+ uptake in the rat extensor digitorum longus (EDL; composed predominantly of fast-twitch fibers) is higher than in the soleus muscle (predominantly slow-twitch fibers), leading to a greater degree of cellular damage and leakiness in the EDL. In the present investigation, muscles of all three fiber types were studied to determine the general response of the muscle fibers to severe H/R. Whether the cell injury incurred was fiber type dependent in this model remains to be determined.

Protective mechanisms of preconditioning. Ischemic preconditioning may serve to protect skeletal muscle by promoting energy sparing or reducing anaerobic ATP production during the subsequent ischemic event. Pang et al. (12) demonstrated that preconditioned porcine latissimus dorsi muscle flaps had less lactate accumulation and higher levels of high-energy phosphates after prolonged ischemia than did nonpreconditioned controls. Lee et al. (9) also showed preservation of ATP and creatine phosphate levels by ischemic preconditioning followed by prolonged ischemia and reperfusion relative to nonpreconditioned controls. However, Gürke et al. (3) found that ischemic preconditioning improved postischemic muscle force generation, contractility, and endurance in a rat hindlimb model without preservation of high-energy phosphates relative to nonpreconditioned controls. Similarly, in our experiments, force generation during 5 min of severe hypoxia and 5 min of reoxygenation was greater in PC than in C cells. It is possible that energy sparing may have occurred through a tightening of excitation-contraction coupling, a reduction in futile ion pumping, and/or through a relatively lower reliance on anaerobic energy production without compromising force generation.

A number of studies focusing on the molecular mechanisms of ischemic preconditioning in cardiomyocytes have recently helped to describe signaling pathways important in the protective effects (for recent review, see Ref. 26). Initiation of the protective response appears to be dependent on activation of protein kinase C and the subsequent activation of ATP-dependent K+ (KATP) channels. Activation of sarcolemmal KATP channels may lead to hyperpolarization and energy sparing (24), whereas activation of mitochondrial KATP channels may be an important early step in protective responses involving heat shock proteins and antioxidants (3, 5, 22, 24). The molecular mechanisms of ischemic preconditioning are currently under intense investigation in cardiac models, and it is likely that molecular mechanisms of preconditioning in skeletal muscle are similar. The results of the present investigation provide support for the contribution of intracellular factors in the protective response.

In conclusion, research in isolated single muscle fibers permits examination of some of the cellular mechanisms contributing to severe hypoxia and reoxygenation injury. Here we have shown for the first time that isolated muscle fibers are susceptible to severe H/R injury despite the absence of vascular endothelium and circulating factors and that the effects are attenuated by hypoxic preconditioning. The results demonstrate that a loss in function is associated with a decrease in the force-to-peak Ca2+ ratio and is perhaps also mediated by increases in [Ca2+]c. Further work with this model is needed to help establish the intracellular mechanisms underlying H/R injury in skeletal muscle.


    ACKNOWLEDGEMENTS

This research was supported by a National Research Service Award postdoctoral fellowship to S. Kohin, a National Sciences and Engineering Research Council (Canada) postdoctoral fellowship to R. A. Howlett, and National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-40155 to M. C. Hogan.


    FOOTNOTES

Address for reprint requests and other correspondence: S. Kohin, Dept. of Medicine, 0623A, Univ. of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0623 (E-mail:skohin{at}ucsd.edu).

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.

Received 16 November 2000; accepted in final form 6 February 2001.


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RESULTS
DISCUSSION
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