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Contractile response of skeletal muscle to low peroxide concentrations: myofibrillar calcium sensitivity as a likely target for redox-modulation ¹

FRANCISCO H. ANDRADE^*,2, MICHAEL B. REID and HÅKAN WESTERBLAD^*

^* Department of Physiology and Pharmacology, Karolinska Institutet, S-171 77 Stockholm, Sweden;
Department of Neurology, Case Western Reserve University and The Research Institute of University Hospitals of Cleveland, Cleveland, Ohio 44106, USA;
Pulmonary and Critical Care Medicine Section, Baylor College of Medicine, Houston, Texas 77030, USA

²Correspondence: Wearn 650, University Hospitals of Cleveland, 11100 Euclid Avenue, Cleveland, OH 44106-5068. E-mail: fha{at}po.cwru.edu

SPECIFIC AIMS

Starting with the premise that reactive oxygen species (ROS) may function as biological signals, we wanted to test whether the contractile function of skeletal muscle is influenced by peroxide concentrations that approximate the physiological levels. Our initial hypothesis was that myofibrillar Ca²⁺ sensitivity, and not mean tetanic free cytosolic Ca²⁺ concentration ([Ca²⁺]_i), would be altered by low hydrogen peroxide (H₂O₂) and t-butyl hydroperoxide (t-BOOH) concentrations.

PRINCIPAL FINDINGS

1. Low peroxide concentrations decrease mean tetanic [Ca²⁺]_i and increase submaximal force
H₂O₂ (10^–10 to 10^–5 M) decreased mean tetanic [Ca²⁺]_i and increased force (30–60 Hz tetani) by about 10%. The effects on tetanic [Ca²⁺]_i and force were largely concentration-independent; the exception was 10^–5 M H₂O₂, where force increased more markedly, 38% over the control level. t-BOOH decreased mean tetanic [Ca²⁺]_i by 10% across the same concentration range. The resulting increase in force due to t-BOOH was more variable: 10% at the lowest concentration, 37% at 10^–6 M, and 78% at 10^–5 M (Fig. 1 ).

View larger version (17K): [in this window] [in a new window]   Figure 1. . Tetanic [Ca2+]i and force are very sensitive to peroxides. A) original [Ca2+]i (top) and force (bottom) records from a single muscle fiber during 40 Hz tetani. The horizontal bars between the tracings represent the 350-ms stimulation periods; dotted lines mark the control values for [Ca2+]i and force. Responses before (control) and during exposure to 100 pM H2O2 are shown. H2O2 decreased tetanic [Ca2+]i and increased force. B) changes in force (30–60 Hz tetani) and mean tetanic [Ca2+]i in single skeletal muscle fibers during incubation with different concentrations of H2O2 and t-BOOH. From 10–10 to 10–6 M, H2O2 () and t-BOOH () produce the same decrease in [Ca2+]i. Force increases by about 10% during exposure to 10–10 or 10–8 M H2O2 () or t-BOOH (•). At higher concentrations, t-BOOH induces greater increases in force than H2O2.

Catalase blocked the response to H₂O₂. Because catalase is H₂O₂-specific, it did not prevent the response to t-BOOH. The decrease in tetanic [Ca²⁺]_i during exposure to H₂O₂ or t-BOOH was constant during the 30 min incubation period. The response of force was biphasic: an initial increase that peaks within 5 min of exposure, followed by a monotonic decline towards the control level. Removal of the peroxide does not result in an immediate restoration of function. Instead, [Ca²⁺]_i increases above the control level and remains slightly elevated during the 30 min washout period. Meanwhile, force decreases within the first 10 min of washout and slowly recovers up to the control level.

2. Myofibrillar Ca²⁺ sensitivity increases during exposure to low H₂O₂ or t-BOOH concentrations
The opposing changes in force (increased) and [Ca²⁺]_i (decreased) that result from exposure to H₂O₂ or t-BOOH (1 µM) suggest that the myofibrillar Ca²⁺ sensitivity of the single fibers increases and the force-[Ca²⁺]_i relationship shifts to the left. While the [Ca²⁺]_i decrease is constant during incubation with either peroxide, force shows a biphasic response. Therefore, the moment-to-moment change in Ca₅₀ was calculated from the measured force and [Ca²⁺]_i transients. Figure 2A presents the estimated Ca₅₀ during incubation in H₂O₂ or t-BOOH (1 µM) at the time of the lowest tetanic [Ca²⁺]_i (•), and after 30 min washout ). Ca₅₀ remained to the left of the control force-[Ca²⁺]_i relationship during the incubation in H₂O₂ or t-BOOH, and shifted to the right during washout. The peroxides induced an immediate stepwise decrease in the Ca₅₀. Conversely, washout resulted in a drastic increase in Ca₅₀ to 10% over the initial value that slowly declined towards the control level (Fig. 2B ).

View larger version (16K): [in this window] [in a new window]   Figure 2. . Peroxides shift the force-[Ca2+]i relation. A) average force-[Ca2+]i relationship obtained under control conditions (solid line). The mean Ca50 estimated at the time of the lowest tetanic [Ca2+]i during peroxide exposure (•) is shifted to the left of the control line. After 30 min of washout, Ca50 shifts to the right of the initial control value (). B) mean Ca50 at 2, 5, 10, 20, and 30 min of exposure to 1 µM H2O2 or t-BOOH (•), and 5, 10, 20, and 30 min of washout (). The dotted line denotes the control level (100%).

3. The peroxides alter cross-bridge kinetics
The effect of H₂O₂ and t-BOOH on maximal force was tested with 100 Hz tetani in the presence of caffeine, which results in [Ca²⁺]_i levels high enough to produce maximum cross-bridge activation. The combination of caffeine with either peroxide resulted in higher tetanic forces than those obtained during incubation with caffeine alone. In addition, both peroxides increased rate-of-force redevelopment after a shortening step (slack tests), and slightly decreased maximum shortening velocity. These data indicate that the peroxides influence several aspects of cross-bridge function.

4. Cellular Ca²⁺ handling is relatively insensitive to H₂O₂ and t-BOOH
Resting [Ca²⁺]_i increased less than 15% after 30 min in 1 µM H₂O₂ and t-BOOH. This was not different from the change in resting [Ca²⁺]_i measured in single fibers not exposed to peroxide. By contrast, H₂O₂ and t-BOOH at 10 µM for 30 min increased mean resting [Ca²⁺]_i by 78% and 74%, respectively. Moreover, 10 µM of either peroxide slowed the decline of [Ca²⁺]_i to resting levels after stimulation: Analyses of [Ca²⁺]_i after contractions indicated that the rate of Ca²⁺ re-uptake by the sarcoplasmic reticulum (SR) was reduced by more than 70%. Lower concentrations of peroxides had no effect on SR Ca²⁺ uptake.

5. Mild thiol alkylation blocks the response to the peroxides
The role of free thiol groups in mediating the effects of H₂O₂ and t-BOOH was explored by using the alkylating agent N-ethylmaleimide (NEM). When used at 250 µM, NEM almost completely inhibited force production, while tetanic [Ca²⁺]_i was still very close to normal. This experiment suggests that thiol groups in the contractile filaments are either more accessible that those in the SR or are more important for normal function. In contrast, a short pre-exposure to 25 µM NEM did not alter force or [Ca²⁺]_i during submaximal contractions, and it completely blocked the response to 1 µM H₂O₂.

CONCLUSIONS

ROS are generated by cellular enzymatic and nonenzymatic oxidation-reduction reactions. Physiologically production of ROS is now recognized, and the list of processes that are regulated by them is growing. ROS can also influence normal contractile function. This study demonstrates that the contractile filaments are sensitive to peroxide levels that approximate physiological concentrations, while Ca²⁺ handling is much less so (Fig. 3 ). Our data suggest that the contractile function of mammalian skeletal muscle may be regulated by small shifts in the intracellular ROS concentration.

View larger version (44K): [in this window] [in a new window]   Figure 3. . Diagram of the potential role of ROS in the contractile process. Physiological intra- and extracellular ROS sources are in equilibrium with cellular antioxidant systems, which results in a steady-state intracellular ROS concentration. The function of the contractile filaments is more sensitive to changes in ROS concentration (thick arrows) than to SR Ca2+ handling (thin arrow). The redox-mediated changes in myofibrillar function may result from the activation of protein kinases or other redox-sensitive signaling pathways upstream of the contractile filaments.

Both force and [Ca²⁺]_i were changed by peroxide concentrations that include the physiological range of 10^–9–10^–7 M. The decrease in [Ca²⁺]_i was not anticipated because of reports that SR Ca²⁺ release is facilitated and that re-uptake is inhibited by oxidants. Both changes would combine to increase [Ca²⁺]_i. Moreover, the peroxide-induced decrease in [Ca²⁺]_i did not show concentration-dependence, while the increase in force was larger at the highest concentration used (10 µM). This remarkable divergence between [Ca²⁺]_i and force at low peroxide concentrations has not been reported previously and indicates that ROS effects on contractile function can be independent of changes in SR Ca²⁺ handling.

Resting skeletal muscles generate ROS. Therefore, the control of these compounds within physiological limits may be an important regulatory mechanism for various cellular functions. Our data are consistent with myofibrillar Ca²⁺ sensitivity being particularly susceptible to the influence of ROS. This is probably not due to increased Ca²⁺ affinity of Ca²⁺-binding proteins that share the E-F hand motif (regulatory myosin light chains, troponin C, calmodulin): oxidants may actually decrease Ca²⁺ binding to these proteins. On the one hand, because NEM-accessible thiols in the contractile filaments are necessary for normal force production, it seems less likely that direct oxidation of thiol sub-populations in myosin by H₂O₂ or t-BOOH is the mechanism that mediates the changes in [Ca²⁺]_i and force. This suggests that NEM blocks thiol-containing elements upstream of the contractile machinery. That is, it may prevent the activation of a redox-sensitive signaling pathway that culminates with the modification of key contractile elements. Thus, we speculate that H₂O₂ and t-BOOH would enter the fiber and activate a kinase, which would phosphorylate protein(s) involved in the contractile process (Fig. 3) . That myofibrillar Ca²⁺ sensitivity returns to the control level after washout of the peroxides suggests that the response is self-limiting.

We have demonstrated that myofibrillar function is more redox-sensitive than SR Ca²⁺ handling. However, the mechanisms mediating the ROS-induced functional responses remain unresolved. First, it is not clear if H₂O₂ and t-BOOH mediate their effects by direct interaction with redox-sensitive sites in the contractile filaments themselves or, instead, indirectly by activating redox-sensitive signaling pathways. Our results suggest that redox-sensitive thiol groups, upstream of those in the myosin head, participate in the response to low peroxide concentrations. Second, the identity of this putative "sensor" for changes in intracellular ROS levels is yet to be determined. Third, the relative importance of ROS sources in skeletal muscle has not been elucidated. Clearly, the steady-state intracellular ROS concentration results from the interplay of intra- and extracellular ROS sources and antioxidant systems. How these factors interact under various physiological conditions to shift the intracellular oxidant load is still unknown. Finally, the divergence between our results and those obtained at higher oxidant concentrations may reflect the differential sensitivity of the various redox-sensitive targets. We propose that increases in intracellular ROS concentration beyond homeostatic boundaries, such as in fatigue, may be sufficient to alter SR function and explain previous descriptions of increased Ca²⁺ release and impaired re-uptake.

Current knowledge of ROS biology in skeletal muscle is mostly in the context of deviations from homeostasis: fatigue, sepsis, and aging. However, ROS are normally present in the skeletal muscle milieu and may influence contractile function via mechanisms that are overshadowed during fatigue and disease. This report shows that myofibrillar function, and not SR Ca²⁺ handling, is sensitive to peroxide concentrations that approximate the physiological range.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0507fje To cite this article, use (December 8, 2000) FASEB J. 10.1096/fj.00-0507fje

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