Role of intracellular calcium in fatigue in single skeletal muscle fibers isolated from the rat

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1 Pathophysiology 6 (2000) Role of intracellular calcium in fatigue in single skeletal muscle fibers isolated from the rat Hiroyuki Hirano a, Eiji Takahashi b, *, Katsuhiko Doi b, Yoshihiro Watanabe a a Department of Orthopedics, Yamagata Uni ersity School of Medicine, Yamagata , Japan b Department of Physiology, Yamagata Uni ersity School of Medicine, Yamagata , Japan Received 16 September 1998; received in revised form 4 March 1999; accepted 19 April 1999 Abstract The aim of the present study was to demonstrate the role of intracellular calcium ([Ca 2+ ] i ) in the performance of fatigued muscle fibers isolated from the skeletal muscle of the rat. We measured developed tension of a single myocyte during short tetanic electrical stimulation of various intensities along with [Ca 2+ ] i dynamics by fura-2. The performance of individual muscle fiber was assessed by developed tension during 100 Hz tetanic stimulation ( 100 Hz force ). We regarded the muscle fiber fatigued when, after repeated tetanic stimulations, the developed tension declined to 50% of the initial level. When fatigue was induced by maximal stimulation (100 Hz tetani), the 100 Hz force measured immediately following completion of fatigue was considerably decreased (48% of control). This change in the muscle performance was associated with significant increase in the resting [Ca 2+ ] i (280% of control) and decrease in Ca 2+ transient (54% of control). The 50% relaxation time after cessation of tetanic stimulation (RT 50 ) was also prolonged. In contrast, when fatigue was induced by low frequency electrical stimulation (30 Hz tetani), neither the 100 Hz force, RT 50, nor Ca 2+ transient in fatigue were significantly different from the controls, while the resting [Ca 2+ ] i increased only slightly. These findings suggest a tight relationship between [Ca 2+ ] i and the performance of fatigued single isolated skeletal muscles. Also, the results show that performance of the fatigued muscle fiber may in part depend on the protocol used to produce muscle fatigue Elsevier Science Ireland Ltd. All rights reserved. Keywords: Calcium transient; Fatigue; Fura-2; Intracellular calcium; Single skeletal muscle 1. Introduction In skeletal muscles, fatigue is defined as a reduction in performance (i.e. force production and speed) as a result of prolonged activity [1]. The cause of muscle fatigue is obviously diverse and involves factors such as the central command, activation of motor neurons, neuro-muscular transmission, action potential generation at the sarcolemma, the T-tubular system, Ca 2+ release and reuptake by the sarcoplasmic reticulum (SR), and activation and restoration of the contractile mechanism [1,2]. Although the relative contribution of these factors vary considerably according to the fiber type and characteristics of muscle activation, recent studies have indicated that the central factors (central * Corresponding author. Tel.: ; fax: address: eiji@med.id.yamagata-u.ac.jp (E. Takahashi) nervous system or neuromuscular junction) play a minor role [3 5] and that alterations of cellular processes regulating muscle contraction are mainly responsible for muscle fatigue [1,2]. One of the major intracellular mechanisms responsible for muscle fatigue is the change in metabolism including ATP depletion and accumulation of metabolites such as lactate, protons, and inorganic phosphates [6 8]. Another important factor is the transduction of excitatory signal from sarcolemma to contractile proteins, particularly signal transduction via dynamic changes in intracellular calcium concentration ([Ca 2+ ] i ) [9 11]. To elucidate the relative importance of these intracellular factors in the development of fatigue, it is important to exclude the neural factors and to precisely regulate the external milieu of individual cell. These are best fulfilled by using a single muscle fiber isolated from skeletal muscles. Hence, in the present study, we measured the dynamic changes in [Ca 2+ ] i that are associ /00/$ - see front matter 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S (99)

2 212 H. Hirano et al. / Pathophysiology 6 (2000) ated with muscle contraction (Ca 2+ transient) in a single intact muscle fiber isolated from a mammalian skeletal muscle, the flexor digitorum brevis of the rat. While any prolonged muscle activation may cause fatigue, fatigue produced by prolonged submaximal contraction of muscle may differ from that produced by short strenuous exercise, in terms of intracellular energy state and signal transduction processes, thereby affecting performance of the fatigued muscle. In the present study, we produced fatigue in the single fast-twitch fiber using repetitive short tetanic stimulation of various intensities. To clarify the role of [Ca 2+ ] i in the power output (performance) of fatigued muscle fibers, we compared the developed tensions during 100 Hz tetanic electrical stimulation of a single muscle fiber before and after induction of fatigue achieved by different stimulus intensities, along with measurements of Ca 2+ transients by fura Methods 2.1. Isolation of single skeletal muscle fibers We isolated single intact muscle fibers from the flexor digitorum brevis muscle of the 6 8 weeks old male Sprague Dawley rats. The muscle has been well characterized and is known to be composed mainly ( 90%) of the fast-twitch muscle fibers [12]. Following anesthesia by i.p. sodium pentobarbital (Nembutal, 5 mg/100 g b.w.), the flexor digitorum brevis was isolated as a whole muscle. Then, single fibers were isolated from the muscle in a buffer solution (in mm, NaCl 133.5, KCl 4.0, CaCl 2 1.8, MgCl 2 0.5, NaH 2 PO 4 1.2, Hepes 10.0, glucose 5.5, 2% bovine fetal serum albumin, ph 7.33 at 30 C, bubbled with 100% O 2 ). The muscle is a pennate muscle where the fibers are supported by three thick tendons. We collected fibers in between two of these tendons as a large bundle. A large bundle was cut along the fibers into half, and this procedure was repeated until the whole muscle was divided into many small bundles each consisting of five muscle fibers. We took special care to collect the large bundles from the same portion of the muscle. The cut ends of the small bundle were fixed using adhesive to the hook and cantilever of the tension measuring device described below. Finally, we obtained single isolated muscle fiber by compressing and stretching outward the four fibers while leaving only one fiber at the center intact. During the isolation procedures, field electrical stimulation was occasionally applied to the fiber to evaluate viability Measurement of de eloped force The isolated muscle fiber was placed in the measuring cuvette (volume 5 ml) and perfused with the buffer solution (24 ml/min) at room temperature. The fiber was fixed to the cantilever where a semiconductor strain gauge (KSP-2-12-E4, Kyowa, Tokyo, Japan) was placed. The position of the cantilever could be adjusted with a micro manipulator. Mechanical strain of the cantilever associated with muscle contraction was detected with a sensitivity of V/mgf (mgf is a unit representing force. 1 mgf= N). The developed force was continuously recorded on a chart recorder (PH-635, Denki Keisoku, Tokyo, Japan) and also stored in a computer (PC9801VM, NEC, Tokyo, Japan) following analog-to-digital conversion at 200 Hz. The resonance frequency of the measuring system was approximately 1 khz Stimulation protocols Bipolar electrical stimulation of the fiber was conducted using a pair of platinum electrodes placed parallel to the fiber. The intensity of the electrical stimulation was set at approximately 120% of the threshold (10 14 V). Duration of tetanic pulse train was 350 msec and the frequency of individual electrical pulses was varied from 25 to 1000 Hz using an electronic stimulator (SEN-3301, Nihon Kohden, Tokyo, Japan). Muscle fatigue was defined in such a way that, following repeated short tetanic stimulation of the muscle fiber (at 3 s intervals), the developed tension of a single muscle fiber declined to 50% of the initial tension [11,13,14]. This stimulation protocol did not produce irreversible muscle injury [Ca 2+ ] i measurements During the course of fiber isolation, the small bundle of muscle fibers was incubated with the calcium indicator fluorescent dye fura-2 (30 M as acetoxymethyl ester, Dojindo, Kumamoto, Japan) for min at 30 C. We measured simultaneously with high time resolution the dynamic changes in fura-2 fluorescence and force. Details of the measuring system and the calibration procedure were reported previously [15]. Synchronizing with a command pulse just prior to the onset of the tetanic stimulation, 1000 fluorescence data points (emission wavelength 510 nm) were continuously sampled at 2 msec interval while the dye was excited at 340 nm (F 340 ). Then, the procedure was repeated while the dye was excited at 380 nm (F 380 ). Note that the fluorescence signals were collected from different, but successive contractions. We assessed [Ca 2+ ] i using the following equation. [Ca 2+ ] i =K D (R R min )/(R max R) where R=F 340 /F 380. R min and R max were the fluores-

3 cence ratios where the fibers were perfused with the buffer solution containing 20 mm EGTA and 2 mm CaCl 2, respectively, in the presence of the calcium ionophore, ionomycin (10 M) [9]. R max and R min determined by this method were (n= 9) and (n=6), respectively. We used a K D value of 150 nm according to the literature [16]. is the ratio of F 380 in Ca 2+ free solution and F 380 under conditions where fura-2 was saturated with Ca 2+. In our experiments, the value of was 1.36 that is substantially smaller than the value (3.34) reported by Westerblad and Allen [9]. Background autofluorescence and photo bleaching of the dye were negligible. H. Hirano et al. / Pathophysiology 6 (2000) Protocol First, to evaluate the performance of the individual fiber, we measured the tetanic tension and the associated [Ca 2+ ] i transient at 100 Hz stimulation and this response was defined as the 100 Hz force response. Then, we started repetitive tetanic stimulation at a predetermined pulse frequency at 3 s interval until fatigue was produced, i.e. developed tension decreased to 50% of the tetanic force response at the particular frequency of stimulation. Immediately following the completion of the fatigue protocol, we measured the 100 Hz force and the fura-2 fluorescence responses to evaluate the performance of the fatigued muscle fiber Statistical analysis All the data are represented as mean S.D. Significance of the difference between two data sets was judged by Student s t-test using StatView statistical package (Abacuns Concepts, Berkeley, CA). P values less than 0.05 were considered significant. Fig. 1. The relationship between magnitude of tetanic stimulation and tension developed in isolated perfused single muscle fibers. tude of tetanic frequency, whereas no significant difference in the endurance time was found at pulse frequencies between 25 and 30 Hz. Fig. 3 compares the 100 Hz force response before (open circles) and after (closed circles) induction of fatigue. When fatigue was induced by using relatively low stimulation frequency ( 30 Hz), no significant reduction of the 100 Hz force was observed in fatigued fibers. This was in marked contrast to the clear reduction of the 100 Hz force response in the muscle fiber in which fatigue was induced using relatively higher stimulation frequency ( 30 Hz). The 50% relaxation time (time required for the developed tension falling to 50% of the 100 Hz force response after cessation of the stimulation, RT 50 ) was significantly (P 0.05) increased from ms (n=6) to ms (n=6) when fatigue was induced by tetanic stimulation at 100 Hz. In contrast, RT 50 was unchanged ( ms, n=5) when the fatigue was induced by stimulation at 30 Hz (Fig. 4). 3. Results The isometric tetanic force was maximal for fiber length at mm (n=31). At this fiber length, the magnitude of the developed tension was a function of pulse frequency of the tetanic stimulation, the maximum tension being achieved at Hz (Fig. 1). Accordingly, we assumed that tension developed at 100 Hz tetanic stimulation ( 100 Hz force ) represents the largest level of force which the individual muscle fiber could develop under given conditions. The 100 Hz force of the control muscle fiber was mgf (n=31). Fig. 2 illustrates the relationship between pulse frequency of the tetanic stimulation and time to fatigue (endurance time). At pulse frequency 30 Hz, the endurance time was a reciprocal function of the magni- Fig. 2. The relationship between pulse frequency during tetanic stimulation and time required for development of fatigue (50% reduction in tetanic force at a particular frequency of stimulation).

4 214 H. Hirano et al. / Pathophysiology 6 (2000) Fig. 3. The relationship between the ability of the muscle fibers to develop force when tetanically stimulated at 100 Hz before and after development of fatigue with different pulse frequency. Open and closed circles represent the 100 Hz force in control and fatigued fibers. Fig. 5 illustrates changes in the Ca 2+ transient (i.e. tetanic [Ca 2+ ] i resting [Ca 2+ ] i ) of a single muscle fiber when fatigue was induced by 100 Hz tetani. There was a significant increase in the basal [Ca 2+ ] i (280% of the control, Table 1) and a decrease in the amplitude (54% of the control, Table 1). Changes in Ca 2+ transient associated with fatigue caused by 30 Hz tetani are shown in Fig. 6. Although fatigue was certainly produced (i.e. developed tension halved), no significant changes were observed in the 100 Hz force responses or the [Ca 2+ ] i transients (Table 1). The base line [Ca 2+ ] i slightly increased after development of fatigue (135% of the control, Table 1). Fig. 5. Representative data of the changes in [Ca 2+ ] i and the 100 Hz force response before and after induction of fatigue. Fatigue was induced by repeated trains of 100 Hz tetanic stimuli. 4. Discussion In the present study, using a well characterized single muscle fiber isolated from the rat skeletal muscle, we have demonstrated that the performance of the muscle fiber as assessed by the 100 Hz force response test was not significantly affected by fatigue if fatigue was produced by relatively low frequency ( 30 Hz) tetani, while the 100 Hz force response of the fatigued muscle was significantly reduced when the fibers were fatigued at higher frequencies ( 30 Hz). The amplitude of the tetanic [Ca 2+ ] i of the fatigued muscle fiber showed a similar pattern; no change and 50% reduction following the low frequency and high frequency fatigue protocols, respectively. Table 1 Changes in [Ca 2+ ] i before and after fatigue induced by repeated short tetani a Tetanic stimulation frequency (Hz) Resting [Ca 2+ ] i (nm) Before After Ca 2+ transient (nm) Before After 30 (n=5) * (n=6) * * Fig. 4. Comparisons of time required for the developed tension to fall to 50% of the 100 Hz force response after cessation of tetanic stimulation (RT 50 ). a Values are mean S.D. * Indicates significant (P 0.05) difference as compared to controls before fatigue. Ca 2+ transient is the difference between resting and tetanic [Ca 2+ ] i.

5 H. Hirano et al. / Pathophysiology 6 (2000) Fig. 6. Representative data demonstrating changes in [Ca 2+ ] i and the 100 Hz force response before and after induction of fatigue. Fatigue was induced by repeated trains of 30 Hz tetanic stimuli. Development of muscle fatigue depends on a variety of factors such as the central command, excitation transmission to muscles, cellular energy production and stores, intracellular ionic balance, washout of metabolites, and intracellular signal transduction by intracellular free calcium. Use of a perfused single muscle fiber in the present study gave us an opportunity to accurately define changes in intracellular signal transduction, Ca 2+ transient, associated with fatigue. Alterations of coupling between [Ca 2+ ] i and force development in fatigue can be as a result of several factors [1,2]. Firstly, muscle fatigue may arise from diminished maximum calcium activated tension of a fiber caused by increased concentrations of inorganic phosphates and protons [6,14,17 19]. Approximately 20 30% of the tension reduction in intermittent tetanic fatigue may be attributed to this mechanism [1]. Unfortunately, we were unable to measure changes in the resting tension of a single muscle fiber throughout the entire course of the fatigue protocol, as a result of random drift of the tension signal. Therefore, although [Ca 2+ ] i was almost maximally increased during the 100 Hz stimulation (Table 1), we could not precisely determine whether the maximum calcium activated tension was in fact diminished with the two distinct fatigue procedures. Second, a decrease in the sensitivity of myofilaments to Ca 2+ can also account for the reduction of tension development in fatigue. This may occur as a result of accumulation of metabolites in the intracellular space, particularly proton and inorganic phos- phates [11,17,19]. We demonstrated that the relatively mild ( 30 Hz) fatigue protocol hardly affected the Ca 2+ transients induced at 100 Hz stimulation (Table 1). Hence, these alterations in the relationship between [Ca 2+ ] i and force (i.e. myofilament Ca 2+ sensitivity) may account for both the tension decline and unchanged 100 Hz force response observed in muscle fibers in which fatigue was induced by 30 Hz tetani. In addition to alterations of [Ca 2+ ] i -force relationship, fatigue may produce significant changes in Ca 2+ transients. A reduction of Ca 2+ release from the intracellular Ca 2+ store (sarcoplasmic reticulum, SR) in fatigue has been suggested to occur [9,10,19]. Furthermore, dysfunction of Ca 2+ uptake by SR Ca 2+ pump may also occur in fatigue, because the rate of muscle relaxation is slow and the resting [Ca 2+ ] i remains elevated in fatigue [1,20], although the precise role of decreased SR Ca 2+ pumping in fatigue has not been determined [20,21]. Hence, these factors together may cause decreased Ca 2+ transients and reduced force development. In the present study, when fatigue was induced by the high frequency stimulation (100 Hz), we demonstrated a significant increase in the resting [Ca 2+ ] i from 35 to 98 nm, whereas the peak [Ca 2+ ] i achieved during tetanic stimulation at 100 Hz was comparable to the control value (229 vs 202 nm before and after the fatigue procedure, respectively, Table 1). These findings, along with the significant prolongation of RT 50 (Fig. 4), strongly suggest the importance of Ca 2+ reuptake by SR Ca 2+ pump in the formation of Ca 2+ transients. These changes in the resting [Ca 2+ ] i elicited by fatigue thus appear to account for approximately 50% reductions of both Ca 2+ transient and the performance of the fatigued muscle fiber. On the other hand, the fatigue protocol using low frequency stimulation (30 Hz) did not affect the performance of the muscle fiber even though fatigue was certainly induced (Fig. 3). This result was consistent with the observed Ca 2+ transient in fatigue; unchanged Ca 2+ transient (amplitude) with slight elevation of the resting [Ca 2+ ] i (Table 1). Also, these changes in Ca 2+ transients are consistent with the finding that significant prolongation of RT 50 was not demonstrated in this low frequency fatigue protocol (Fig. 4). We conclude from these findings that [Ca 2+ ] i is one of the major determinant of the performance of fatigued single muscle fibers in vitro. We found that both 100 Hz force response and the associated Ca 2+ transient were not affected by fatigue induced by relatively low frequency tetani (30 Hz). This apparently paradoxical result may be explained if repeated applications of short tetani shifted the relationship between stimulation frequency and developed tension (Fig. 1) to the right, presumably as a result of increased intracellular proton and/or inorganic phosphates concentrations. In the case where fatigue was

6 216 H. Hirano et al. / Pathophysiology 6 (2000) induced by 30 Hz tetani, only a slight rightward shift of the relationship, or a decrease of Ca 2+ sensitivity of myofilaments, would result in considerable decrease in the developed tension (i.e. fatigue) even in the absence of a significant reduction of Ca 2+ transient, because the relationship is the steepest for pulse frequencies less than 50 Hz (Fig. 1). In contrast, this rightward shift of the frequency tension relationship would not affect the tension generation at 100 Hz because the developed tension is independent of the stimulation frequency around 100 Hz, thus resulting in unchanged 100 Hz force response in fatigued muscle. The present results, therefore, suggest that the performance (maximum power output) of the fatigued muscle fiber is determined at least in part by the protocol used to induce muscle fatigue. References [1] H. Westerblad, J.A. Lee, J. Lännergren, D.G. Allen, Cellular mechanisms of fatigue in skeletal muscle, Am. J. Physiol. 261 (1991) C195 C209. [2] R.H. Fitts, Cellular mechanisms of muscle fatigue, Physiol. Rev. 74 (1994) [3] N.K. Vøllestad, O.M. Sejersted, R. Bahr, J.J. Woods, B. Bigland-Ritchie, Motor drive and metabolic responses during repeated submaximal contractions in humans, J. Appl. Physiol. 64 (1988) [4] J. Duchateau, K. Hainaut, Electrical and mechanical failures during sustained and intermittent contractions in humans, J. Appl. Physiol. 58 (1985) [5] B. Bigland-Ritchie, R. Johansson, O.C.J. Lippold, J. Woods, The absence of neuromuscular transmission failure in sustained maximal voluntary contractions, J. Physiol. (Lond.) 330 (1982) [6] R. Cooke, K. Franks, G.B. Luciani, E. Pate, The inhibition of rabbit skeletal muscle contraction by hydrogen ions and phosphate, J. Physiol. (Lond.) 395 (1988) [7] R.G. Miller, M.D. Boska, R.S. Moussavi, P.J. Carson, M.W. Weiner, 31 P nuclear magnetic resonance studies of high energy phosphates and ph in human muscle fatigue, J. Clin. Invest. 81 (1988) [8] J.R. Wilson, K.K. McCully, D.M. Mancini, B. Boden, B. Chance, Relationship of muscular fatigue to ph and diprotonated P i in humans, a 31 P-NMR study, J. Appl. Physiol. 64 (1988) [9] H. Westerblad, D.G. Allen, Changes of myoplasmic calcium concentration during fatigue in single mouse muscle fibers, J. Gen. Physiol. 98 (1991) [10] H. Westerblad, S. Duty, D.G. Allen, Intracellular calcium concentration during low-frequency fatigue in isolated single fibers of mouse muscle, J. Appl. Physiol. 75 (1993) [11] J.A. Lee, H. Westerblad, D.G. Allen, Changes in tetanic and resting [Ca 2+ ] i during fatigue and recovery of single muscle from Xenopus lae is, J. Physiol. (Lond.) 433 (1991) [12] R.B. Armstrong, R.O. Phelps, Muscle fiber type composition of the rat hindlimb, Am. J. Anat. 171 (1984) [13] D.G. Allen, J.A. Lee, H. Westerblad, Intracellular calcium and tension during fatigue in isolated single muscle fibres from Xenopus Lae is, J. Physiol. (Lond.) 415 (1989) [14] J. Lännergren, H. Westerblad, Force decline due to fatigue and intracellular acidification in isolated fibres from mouse skeletal muscle, J. Physiol. (Lond.) 434 (1991) [15] I. Shibuya, K. Matsuyama, K. Tanaka, K. Doi, A microfluorometric method for simultaneous measurement of changes in cytosolic free calcium concentration and ph in single cardiac myocytes, Jpn. J. Physiol. 41 (1991) [16] M. Konishi, A. Olson, S. Hollingworth, S.M. Baylor, Myoplasmic binding of fura-2 investigated by steady-state fluorescence and absorbance measurements, Biophys. J. 54 (1988) [17] R.E. Godt, T.M. Nosek, Changes of intracellular milieu with fatigue or hypoxia depress contraction of skinned rabbit skeletal and cardiac muscle, J. Physiol. (Lond.) 412 (1989) [18] T.M. Nosek, J.H. Leal-Cardoso, M. McLaughlin, R.E. Godt, Inhibitory influence of phosphate and arsenate on contraction of skinned skeletal and cardiac muscle, Am. J. Physiol. 259 (1990) C933 C939. [19] M.W. Fryer, V.J. Owen, G.D. Lamb, D.G. Stephenson, Effects of creatin phosphate and P i on Ca 2+ movements and tension development in rat skinned skeletal muscle fibres, J. Physiol. (Lond.) (1995) [20] H. Westerblad, D.G. Allen, The contribution of [Ca 2+ ] i to the slowing of relaxation in fatigued single fibres from mouse skeletal muscle, J. Physiol. (Lond.) 468 (1993) [21] H. Westerblad, D.G. Allen, The role of sarcoplasmic reticulum in relaxation of mouse muscle; effects of 2,5-di(tert-butyl)-1,4- benzohydroquinone, J. Physiol. (Lond.) 474 (1994)