Journal of Molecular and Cellular Cardiology 84 (2015) 162–169
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Original article
Effect of myofilament Ca2 + sensitivity on Ca2 + wave propagation in rat ventricular muscle Masahito Miura ⁎, Yuhto Taguchi, Tsuyoshi Nagano, Mai Sasaki, Tetsuya Handoh, Chiyohiko Shindoh Department of Clinical Physiology, Health Science, Tohoku University Graduate School of Medicine, Sendai 980-8574, Japan
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Article history: Received 8 January 2015 Received in revised form 2 April 2015 Accepted 29 April 2015 Available online 4 May 2015 Keywords: Ca2 + waves Myofilament Ca2 + sensitivity ROS Muscle stretch
a b s t r a c t Background: The propagation velocity of Ca2+ waves determines delayed afterdepolarization and affects the occurrence of triggered arrhythmias in cardiac muscle. We focused on myofilament Ca2+ sensitivity, investigating how the velocity of Ca2+ waves responds to its increased sensitivity resulting from muscle stretch or the addition of a myofilament Ca2+ sensitizer, SCH00013. We further investigated whether production of reactive oxygen species (ROS) may be involved in the change in velocity. Methods: Trabeculae were obtained from rat hearts. Force, sarcomere length, and [Ca2+]i were measured. ROS production was estimated from 2′,7′-dichlorofluorescein (DCF) fluorescence. Trabeculae were exposed to a 10 mM Ca2+ jet for the induction of Ca2+ leak from the sarcoplasmic reticulum in its exposed region. Ca2+ waves were induced by 2.5-Hz stimulus trains for 7.5 s (24 °C, 2.0 mM [Ca2+]o). Muscle stretch of 5, 10, and 15% was applied 300 ms after the last stimulus of the train. Results: Muscle stretch increased the DCF fluorescence, the amplitude of aftercontractions, and the velocity of Ca2+ waves depending on the degree of stretch. After preincubation with 3 μM diphenyleneiodonium (DPI), muscle stretch increased only the amplitude of aftercontractions but not the DCF fluorescence nor the velocity of Ca2+ waves. SCH00013 (30 μM) increased the DCF fluorescence, the amplitude of aftercontractions, and the velocity of Ca2+ waves. DPI suppressed these increases. Conclusions: Muscle stretch increases the velocity of Ca2+ waves by increasing ROS production, not by increasing myofilament Ca2+ sensitivity. In the case of SCH00013, ROS production increases myofilament Ca2+ sensitivity and the velocity of Ca2+ waves. These results suggest that ROS rather than myofilament Ca2+ sensitivity plays an important role in the determination of the velocity of Ca2+ waves, that is, arrhythmogenesis. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction It is well known that lethal arrhythmias occur in patients with acquired diseases such as myocardial infarction and heart failure [1,2], and myofilament Ca2+ sensitivity has been reported to be increased in such patients [3–7]. Also, in patients with familial hypertrophic cardiomyopathy, lethal arrhythmias occur [8], and in some cases myofilament Ca2+ sensitivity is increased because of troponin mutations [9]. In patients with this familial disease, increased myofilament Ca2+ sensitivity not only causes incomplete mechanical relaxation [10] but also underlies arrhythmia susceptibility [11]. Thus, it remains to be established whether increased myofilament Ca2 + sensitivity is involved in the occurrence of arrhythmias in patients with acquired diseases. Muscle stretch increases myofilament Ca2+ sensitivity and increases 2+ Ca binding to the myofilaments [12], thereby increasing the developed force [13–15]. Recently, it has been reported that muscle stretch ⁎ Corresponding author at: Department of Clinical Physiology, Health Science, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 9808574, Japan. Tel./fax: +81 22 717 7920. E-mail address: [email protected] (M. Miura).
http://dx.doi.org/10.1016/j.yjmcc.2015.04.027 0022-2828/© 2015 Elsevier Ltd. All rights reserved.
increases the frequency of Ca2 + sparks [16,17] due to production of reactive oxygen species (ROS) by the activation of NADPH oxidase in cardiac muscle [18]. In addition, we have reported that in rat cardiac muscle, muscle stretch increases 2′,7′-dichlorofluorescein (DCF) fluorescence, which may reflect ROS production, and increases the velocity of Ca2+ waves initiated from the region under Ca2+ overload [15]. In that study, however, it was unclear whether ROS production was involved in the increase in the propagation velocity of Ca2+ waves. Ca2 + wave propagation is deeply involved in the occurrence of arrhythmias [14,19]. Ca2 + waves are initiated not only by Ca2 + leak from the sarcoplasmic reticulum (SR) under Ca2 + overload [20] but also by Ca2 + dissociation from the myofilaments under nonuniform contraction [15,21]. These waves propagate along the cardiac muscle by the mechanism of Ca2+-induced Ca2+ release from the SR [22,23] although it has been reported in other studies that such waves may be driven intermittently by both calcium and membrane depolarization [24]. The velocity of Ca2+ waves is correlated with the amplitude of delayed afterdepolarizations (DADs) and is concerned with the occurrence of arrhythmias when it reaches a threshold for the action potential [25]. Thus, the velocity of Ca2+ waves is one of the important determinants of the occurrence of triggered arrhythmias.
M. Miura et al. / Journal of Molecular and Cellular Cardiology 84 (2015) 162–169
Therefore, in the present study we focused on myofilament Ca2 + sensitivity, investigating how the velocity of arrhythmogenic Ca2 + waves changes in response to increased myofilament Ca2+ sensitivity by muscle stretch or by the addition of SCH00013, a myofilament Ca2 + sensitizer [14,26,27]. We further investigated whether ROS production may play a role in changes in the velocity of these waves.
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the muscle for the SL of 2.1, 2.2, and 2.3 μm under an unstimulated condition was defined as 5, 10, and 15% stretch, respectively. In the present study, SL was not controlled during muscle contraction, and thus uncontrolled sarcomere shortening may have increased the peak of [Ca2+]i transients during contraction [31]. 2.4. Experimental protocol
2. Materials and methods 2+
2.1. Measurements of force, [Ca ]i, and ROS production in rat trabeculae (see Expanded materials and methods in the online data supplement) All animal procedures were performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised in 1996) and were approved by the Ethics Review Board of Tohoku University (approval reference number: 21-98). Briefly, after the rats had been adequately anesthetized, trabeculae (n = 60; length: 2.6 ± 0.3 mm; width: 281 ± 73 μm; thickness: 83 ± 3 μm in a slack condition) were dissected from the endocardial region of the rat right ventricle and mounted on an inverted microscope between a force transducer and a micromanipulator in a bath superfused with HEPES solution containing 5 mM KCl, as previously described [13–15,21,23,25]. Force was measured using a silicon strain gauge, sarcomere length (SL) using laser diffraction techniques, and [Ca2+]i using fura-2, as previously described [23]. Briefly, fura-2 pentapotassium salt was microinjected electrophoretically into a trabecula. Excitation light of 340, 360, or 380 nm was employed, and fluorescence was collected using 1) a photomultiplier tube (PMT) to assess [Ca2+]i within the entire trabecula or 2) an image intensified CCD camera at 30 frames/s to assess local [Ca2 +]i. We calculated [Ca2 +]i from the calibrated ratio of F360/F380 in the region of interest along the trabecula. To calculate the velocity of Ca2 + waves, we identified the peak of a Ca2 + transient during the Ca2 + wave at each pixel along the trabecula and plotted the time of the peak against the position of the peak. The velocity of Ca2+ waves was calculated from the slope of the line fitted to the plot when regression analysis indicated a linear relationship between the time of the peak and the position of the peak (r ≥ 0.9), as previously described [21,23]. ROS production was estimated using 2′,7′-dichlorofluorescein (DCF) fluorescence from the muscle, as previously described [15]. Briefly, a trabecula was loaded with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) [28]. DCFH-DA was cleaved to DCFH and oxidized to the fluorescent molecule DCF by a variety of ROS [29]. Average DCF fluorescence from the trabecula was measured using a PMT.
Ca2+ waves were induced by electrical stimulation at 400 ms intervals for 7.5 s (24 °C, 2.0 mM [Ca2+]o). In the present study the first Ca2+ waves were selected for analysis unless otherwise stated. To increase myofilament Ca2 + sensitivity, the muscle was stretched by 5, 10, or 15% 300 ms after the last stimulus of the electrical train. Ca2+ waves and aftercontractions during Ca2+ wave propagation were then recorded during muscle stretch. Additionally, to investigate the effect of ROS production due to muscle stretch on the Ca2 + waves and aftercontractions, the Ca2+ waves and aftercontractions were recorded after preincubation of 3 μM diphenyleneiodonium (DPI), NADPH oxidase inhibitor, for 1 h [17,32]. SCH00013 (30 μM) has been used as a Ca2+ sensitizer for myofilaments [26,27] and has been previously reported to increase the frequency of triggered arrhythmias [14]. Thus, Ca2+ waves and aftercontractions were recorded in the absence and presence of SCH00013 ([Ca2+]o = 2.0 mM). To estimate the effect of ROS production by the addition of SCH00013, the Ca2+ waves and aftercontractions were recorded again in its presence after preincubation of DPI. In addition, ROS production by the addition of SCH00013 was estimated from the changes in DCF fluorescence before (Fbefore) and after electrical stimulation (Fafter) with 2-s stimulus intervals for 30 s and 0.4-s stimulus intervals for 7.5 s ([Ca2+]o = 0.7 mM). Changes in the DCF fluorescence (%) were calculated from (Fafter − Fbefore) / Fbefore × 100. 2.5. Statistics All measurements were expressed as mean ± SEM. Statistical analysis was performed with 1-way repeated-measures ANOVA and a post-hoc test (Bonferroni) for multiple comparisons and a paired t-test for 2-group comparisons when the data were normally distributed. These analyses were performed using software for statistical analysis (Ekuseru-Toukei 2012, Social Survey Research Information Co., Ltd., Tokyo, Japan). Values of p b 0.05 were considered to be significant. The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agreed to the manuscript as written.
2.2. Regional jet exposure 3. Results A restricted region of a trabecula was exposed to a jet of solution (≈ 0.06 mL/min) directed perpendicular to a small muscle segment (≈300 μm) using a glass pipette (≈100 μm in diameter), as previously described [14,15,21]. The jet of the solution contained 10 mM Ca2+ to induce Ca2+ waves due to Ca2+ overload within the jet-exposed region. 2.3. Addition of muscle stretch SL was determined using a laser diffraction technique, as previously described [30]. Briefly, the preparations were illuminated by a He–Ne laser beam (05-LHP-925, Melles Griot, NM), and the SL was calculated from the median of the intensity distribution of the first order diffraction pattern. Trabeculae were stretched from their valvular end using a DC torque motor controlled by voltage commands from a personal computer via a 12 bit D/A converter (T-IFPC9 04120A-N, T.S.K., Japan), as previously described [13–15]. Under unstimulated conditions, the SL was first set at 2.0 μm, and then the movement distance of the valvular end was determined to increase the SL to 2.1, 2.2, or 2.3 μm. Such movement of
3.1. Muscle stretch and Ca2+ waves Muscle was stretched by 5, 10, or 15% during Ca2+ wave propagation 300 ms after the last stimulus of the electrical train, and its effect on aftercontractions and Ca2 + waves was investigated. As shown in Fig. 1Aa, after rapid electrical stimulation, regional increases in [Ca2+]i occurred within the region exposed to the high Ca2+ jet and propagated along the muscle as Ca2 + waves. The velocity of the first Ca2 + wave (white arrow) was 1.98 mm/s. In Fig. 1Ab, 15% muscle stretch enhanced the amplitude of the aftercontractions during Ca2+ wave propagation and increased the velocity of the first Ca2 + wave to 2.81 mm/s. Figs. 1B and C show summary data concerning the effect of muscle stretch on the aftercontractions and the first Ca2+ waves. As shown in Fig. 1B, muscle stretch enhanced the amplitude of aftercontractions during the first Ca2+ wave propagation, and 15% muscle stretch increased their amplitude more than that by 5% muscle stretch. Additionally, as shown in Fig. 1C, muscle stretch increased the velocity of the first Ca2 + waves, and 15% muscle stretch increased their velocity more
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Fig. 1. Effect of muscle stretch on the velocity of the first Ca2+ waves and the amplitude of their corresponding aftercontractions (ACs). A. Representative recordings of force (upper panels) and regional changes in [Ca2+]i (lower panels) during the last three electrical stimuli (ST; 400-ms stimulus interval) and Ca2+ waves without (a) and with 15% muscle stretch (b). White arrows indicate the first Ca2+ waves, and black arrowheads indicate their corresponding ACs. In panel a, a few Ca2+ waves appear within the jet-exposed region (10 mM Ca JET) after the train of electrical stimuli and propagate along the trabecula. Panel b shows that a 15% stretch increases the amplitude of ACs and the velocity of the first Ca 2 + wave ([Ca 2 + ] o = 2.0 mM, Exp. 130710). B. Summary data concerning the effect of muscle stretch on the amplitude of ACs (AC force) during the first Ca 2 + wave propagation. Blue, red, and green circles indicate the changes by 5, 10, and 15% stretch, respectively (n = 11). *p b 0.05 vs stretch(−); #p b 0.01 vs stretch(−); & p b 0.01 vs 5% stretch. C. Summary data concerning the effect of stretch on the velocity of the first Ca2+ waves. Blue, red, and green circles indicate the changes by 5, 10, and 15% stretch, respectively (n = 11). *p b 0.05 vs stretch(−); $p b 0.05 vs 5% stretch.
than that by 5% muscle stretch. These results suggest that muscle stretch, depending on its degree, increases both the amplitude of aftercontractions and the velocity of the first Ca2+ waves. It has been reported that muscle stretch increases ROS production depending on the degree of stretch in isolated single myocytes [33]. Thus, the effect of 5, 10, or 15% muscle stretch on the DCF fluorescence within trabeculae was examined. As shown in Figs. 2A and B, muscle stretch increased the DCF fluorescence depending on the degree of stretch. In addition, after preincubation with 3 μM DPI, muscle stretch did not increase the DCF fluorescence. These results suggest that muscle stretch increases ROS production depending on the degree of muscle stretch in trabeculae as well. As shown in Fig. 2A, ROS production was transiently increased just after muscle stretch. To investigate whether the velocity of Ca2 + waves was also transiently increased by stretch, the effect of 15% stretch on the first and second Ca2 + waves was measured. The peaks of aftercontractions caused by the first and second Ca2+ waves occurred at 215 ± 28 ms and 610 ± 47 ms after muscle stretch, respectively. As shown in Fig. 2C, muscle stretch increased the velocity of the first Ca2+ waves but not that of the second Ca2+ waves. When changes in the velocity of Ca2+ waves were plotted against the time of the peaks of their corresponding aftercontractions which elapsed after stretch, the changes in the velocity were larger just after muscle stretch
(Fig. 2D). These results mean that the velocity of Ca2+ waves was also transiently increased just after muscle stretch. To investigate whether ROS production was actually involved in the increases in the amplitude of aftercontractions and the velocity of the first Ca2+ waves observed in Fig. 1, muscle was stretched during Ca2+ wave propagation after preincubation with DPI. As shown in Fig. 3Aa, the high Ca2+ jet induced Ca2+ waves after the rapid electrical stimulation. The velocity of the first Ca2+ wave (white arrow) was 1.60 mm/s. In Fig. 3Ab muscle stretch enhanced the amplitude of the aftercontraction, whereas it hardly increased the velocity of the first Ca2+ wave to 1.63 mm/s. Figs. 3B and C show summary data concerning the effect of muscle stretch on the aftercontractions and velocity after preincubation with DPI. As shown in Fig. 3B, muscle stretch increased the amplitude of aftercontractions during the first Ca2+ wave propagation, and 15% muscle stretch increased their amplitude more than that by 5% muscle stretch, similar to the results shown in Fig. 1B. In contrast, muscle stretch did not increase the velocity of the first Ca2+ waves, as shown in Fig. 3C. These results suggest that muscle stretch increases the velocity of the first Ca2+ waves by increasing ROS production, whereas it increases the amplitude of aftercontractions probably because of increased myofilament Ca2+ sensitivity. They further suggest that during muscle stretch, ROS production rather than increased myofilament Ca2+ sensitivity plays an important role in the determination of the velocity of Ca2+ waves.
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Fig. 2. Effect of muscle stretch on the DCF fluorescence and the velocity of the first and second Ca2+ waves. A. The upper panel shows changes in muscle length, the middle panel shows changes in the DCF fluorescence resulting from muscle stretch, and the lower panel shows changes in the DCF fluorescence due to muscle stretch after preincubation with 3 μM DPI ([Ca2+]o = 2.0 mM, Exp. 140702). Blue, red, and green lines indicate the recordings by 5, 10, and 15% muscle stretch, respectively. B. Summary data concerning the effect of muscle stretch on the changes in the DCF fluorescence 2 s after muscle stretch (△DCF2.0) before (−) and after preincubation with 3 μM DPI (+). Blue, red, and green circles indicate the changes by 5, 10, and 15% stretch, respectively (n = 6). *p b 0.05 vs 10% stretch DPI(−); #p b 0.01 vs 15% stretch DPI(−); &p b 0.01 vs 5% stretch DPI(−). C. Summary data concerning the effect of 15% stretch on the velocity of the first and second Ca2+ waves (n = 10, [Ca2+]o = 2.0 mM). *p b 0.05 vs stretch (−); §p b 0.05 vs 1st. D. Relationship between changes in the velocity of Ca2+ waves and the time of the peaks of their corresponding aftercontractions after 15% stretch. These points are fitted to a quadratic curve (r = 0.68, n = 17, [Ca2+]o = 2.0 mM).
3.2. SCH00013, a myofilament Ca2+ sensitizer, and Ca2+ waves SCH00013 increased the developed force without changes in [Ca2+]i transients (Fig. 4), showing that SCH00013 increases myofilament Ca2+ sensitivity, as is consistent with past reports [14,26,27]. We then measured the amplitude of aftercontractions and the velocity of Ca2 + waves in the absence and presence of SCH00013 and investigated whether increased myofilament Ca2 + sensitivity affects Ca2+ waves. In the absence of SCH00013, the velocity of the first Ca2+ waves after electrical stimulation was 2.63 mm/s, as shown in Fig. 5A. After the addition of SCH00013, the amplitude of aftercontractions became higher, probably because of increased myofilament Ca2 + sensitivity, and the velocity became faster. Fig. 5B shows summary data concerning the effect of SCH00013 on the amplitude of aftercontractions and on the velocity of Ca2 + waves. SCH00013 increased both the amplitude of aftercontractions during the first Ca2 + wave propagation and the velocity of the first Ca2+ waves. To examine the effect of SCH00013 on ROS production, the DCF fluorescence was measured in the absence and presence of SCH00013. As shown in Fig. 5C, DCF fluorescence was increased after the addition of SCH00013, suggesting that SCH00013 increases ROS production. To investigate whether ROS production is involved in the increase in the velocity of Ca2+ waves in the presence of SCH00013, the effect of preincubation with DPI on the velocity of Ca2+ waves was observed. After preincubation with DPI, SCH00013 neither increased the amplitude
of aftercontractions nor the velocity of Ca2+ waves, as shown in Figs. 6A and B. In addition, SCH00013 did not increase the DCF fluorescence after preincubation with DPI, as shown in Fig. 6C. These results suggest that SCH00013 increases both the amplitude of aftercontractions and the velocity of Ca2+ waves by increasing ROS production and that ROS production plays an important role in the determination of the velocity of Ca2+ waves. It was finally examined whether SCH00013 affects myofilament Ca2+ sensitivity even after preincubation with DPI. As shown in online Fig. 1, SCH00013 did not increase myofilament Ca2 + sensitivity after preincubation with DPI. These results suggest that SCH00013 increases myofilament Ca2+ sensitivity by increasing ROS production.
4. Discussion The present study investigated how the velocity of Ca2 + waves changes in response to increased myofilament Ca2+ sensitivity by the cardiac muscle stretch and by the addition of a myofilament Ca2+ sensitizer, SCH00013. To the best of our knowledge, the present study is the first to show that muscle stretch increases the velocity of Ca2 + waves by increasing ROS production, not by increasing myofilament Ca2+ sensitivity, and that in the case of SCH00013, ROS production increases myofilament Ca2+ sensitivity and the velocity of Ca2+ waves, as discussed below.
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Fig. 3. Effect of muscle stretch on the amplitude of aftercontractions (ACs) and the velocity of the first Ca2+ waves after preincubation with 3 μM DPI. A. Representative recordings of force (upper panels) and regional changes in [Ca2+]i (lower panels) during the last three electrical stimuli (ST; 400-ms stimulus interval) and Ca2+ waves without (a) and with 15% stretch (b) after preincubation with 3 μM DPI. White arrows indicate the first Ca2+ waves, and black arrowheads indicate their corresponding ACs. In panel a, a few Ca2+ waves appear within the jet-exposed region (10 mM Ca JET) and propagate along the trabecula. In panel b, 15% stretch increases the amplitude of ACs, but hardly increases the velocity of the first Ca2+ wave ([Ca2+]o = 2.0 mM, Exp. 130221). B. Summary data concerning the effect of muscle stretch on the amplitude of ACs (AC force) during Ca2+ wave propagation. Blue, red, and green circles indicate 5, 10, and 15% stretch, respectively (n = 11). *p b 0.05 vs stretch(−); #p b 0.01 vs stretch(−); §p b 0.05 vs 5% Stretch; &p b 0.01 vs 5% Stretch. C. Summary data concerning the effect of stretch on the velocity of the first Ca2+ waves. Blue, red, and green circles indicate 5, 10, and 15% stretch, respectively (n = 11). Muscle stretch does not increase the velocity in the left panel, and in the right panel muscle stretch does not increase the changes in the velocity (p = 0.54).
4.1. ROS production and myofilament Ca2+ sensitivity Muscle stretch has been shown to produce ROS due to the activation of NADPH oxidase [16,17,33], which exists within the cardiac muscle [18]. In the present study as well, muscle stretch increased the DCF fluorescence, but did not increase it after preincubation with DPI (Fig. 2). These results suggest that muscle stretch increased ROS production probably because of the activation of NADPH oxidase although DPI also inhibits the synthesis of both oxygen- and nitrogen-derived reactive species and many other flavoproteins depending on the concentration [34]. It is unlikely, however, that this ROS production increased the myofilament Ca2 + sensitivity because muscle stretch increased the amplitude of the aftercontractions even after preincubation with DPI (Fig. 3). In contrast, it is likely that SCH00013 increased the myofilament Ca2+ sensitivity by ROS production because after preincubation with DPI, SCH00013 did not increase the DCF fluorescence (Fig. 6C), the amplitude of the aftercontractions (Fig. 6B), nor the developed force (online Fig. 1). It has been reported that SCH00013 is a Ca2+ sensitizer with a weak selective phosphodiesterase (PDE) 3 inhibitory action and class III anti-arrhythmic action [35]. In the present study, the PDE 3 inhibitory action and class III anti-arrhythmic action played only a
minor role because SCH00013 functioned without changes in [Ca2+]i transients (Fig. 4), as is consistent with a past report [26]. In our previous study, however, increased myofilament Ca2+ sensitivity by stretch increased force but accelerated the decline of [Ca2+]i transients during contractions [13] probably because of an increase in Ca2+ binding to the myofilaments [12]. In the case of SCH00013, it is still unknown why increased myofilament Ca2 + sensitivity did not change [Ca2 +]i transients, except that the increase in ROS production may be involved. Taken together, these results suggest that myofilament Ca2+ sensitivity is increased by muscle stretch with a different mechanism from that in the presence of SCH00013. 4.2. Myofilament Ca2+ sensitivity and Ca2+ waves An increase in Ca2 + buffering capacity within cardiac muscle decreases the peak of Ca2+ transient and slows the decline of Ca2+ transient [36]. It further decreases the velocity of Ca2 + waves [37] and suppresses the occurrence of arrhythmias [38,39]. It has been reported, however, that in cardiac muscle with increased myofilament Ca2+ sensitivity due to troponin T mutations, an increase in intracellular Ca2+ can easily increase Ca2+ loading within the SR, thereby causing arrhythmias, because of a decrease in the remaining Ca2+ buffering capacity
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Fig. 4. Effect of SCH00013, a myofilament Ca2+ sensitizer, on force and Ca2+ transients. A. Representative recordings of force (upper panel) and [Ca2+]i (lower panel) during electrical stimulation (ST; 2-s stimulus interval) in the absence (black lines) and presence (gray lines) of SCH00013 ([Ca2+]o = 0.7 mM, Exp. 130325). B. Summary data concerning the effect of SCH00013 on force (n = 7). #p b 0.01 vs (−). C. Summary data concerning the effect of SCH00013 on the diastolic [Ca2+]i (p = 0.75), the peak [Ca2+]i (p = 0.91), and the time constant of the [Ca2+]i decline (p = 0.55, n = 7) during electrical stimulation.
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Fig. 5. Effect of SCH00013 on the amplitude of aftercontractions (ACs), the velocity of Ca2+ waves, and the DCF fluorescence. A. Representative recordings of force (upper panels) and regional changes in [Ca2+]i (lower panels) during the last three electrical stimuli (ST; 400-ms stimulus interval) and Ca2+ waves in the absence (left panels) and presence of SCH00013 (right panels). White arrows indicate the first Ca2+ waves, and black arrowheads indicate their corresponding ACs. In the left panels, a few Ca2+ waves appear within the jet-exposed region (10 mM Ca JET) and propagate along the trabecula. In the right panels, both the amplitude of AC and the velocity of the first Ca2+ wave are increased in the presence of SCH00013 ([Ca2+]o = 2.0 mM, Exp. 130327). B. Summary data concerning the effect of SCH00013 on the amplitude of ACs (AC force) during the first Ca2+ wave propagation (left panel) and the velocity of the first Ca2+ waves (right panel). Black and blue circles indicate the changes in the absence and presence of SCH00013, respectively (n = 7). *p b 0.05 vs (−); #p b 0.01 vs (−). C. Summary data concerning the effect of SCH00013 on DCF fluorescence. Changes in the DCF fluorescence (%) was measured before and after electrical stimulation with 2-s stimulus intervals for 30 s (upper panel) and 0.4-s intervals for 7.5 s (lower panel). Black and blue circles indicate the changes in the absence and presence of SCH00013, respectively (n = 5, [Ca2+]o = 0.7 mM). #p b 0.01 vs (−).
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Fig. 6. Effect of SCH00013 on the amplitude of aftercontractions (ACs), the velocity of Ca2+ waves, and the DCF fluorescence after preincubation with 3 μM DPI. A. Representative recordings of force (upper panels) and regional changes in [Ca2+]i (lower panels) during the last three electrical stimuli (ST; 400-ms stimulus interval) and Ca2+ waves in the absence (left panels) and presence of SCH00013 (right panels) after preincubation with DPI. In the left panels, a Ca2+ wave (white arrow) appears within the jet-exposed region (10 mM Ca JET) and propagates. In the right panels, both the amplitude of AC (black arrowhead) and the velocity of the Ca2+ wave (white arrow) are hardly changed in the presence of SCH00013 ([Ca2+]o = 2.0 mM, Exp. 130128). B. Summary data concerning the effect of SCH00013 on the amplitude of ACs (AC force) during the first Ca2+ wave propagation (left panel) and the velocity of the first Ca2+ waves (right panel). Black and blue circles indicate changes in the absence (p = 0.60) and presence of SCH00013 (p = 0.31), respectively (n = 7). C. Summary data concerning the effect of SCH00013 on the DCF fluorescence after preincubation with DPI. Changes in the DCF fluorescence (%) was measured before and after electrical stimulation with 2-s stimulus intervals for 30 s (upper panel) and 0.4-s intervals for 7.5 s (lower panel). Black and blue circles indicate the changes in the absence and presence of SCH00013, respectively (n = 5, [Ca2+]o = 0.7 mM).
because the total amount of Ca2+ buffering capacity is the same [11,40]. In addition, it has been reported that blebbistatin, which has recently been used as a drug to decrease myofilament Ca2+ sensitivity, decreases the occurrence of arrhythmias [6,41]. In contrast, the results of the present study show that increased myofilament Ca2+ sensitivity by muscle stretch did not increase the velocity of Ca2+ waves after preincubation with DPI (Fig. 3), suggesting that increased myofilament Ca2+ sensitivity cannot increase the velocity of Ca2+ waves. Furthermore, we have previously reported that the presence of blebbistatin does not decrease the velocity of Ca2 + waves arising from the Ca2+-overloaded region [15]. Taken together, these results suggest that myofilament Ca2+ sensitivity does not affect the velocity of Ca2+ waves in rat cardiac muscle and that it plays only a minor role in determination of triggered arrhythmias. 4.3. ROS and Ca2+ waves We have previously reported that muscle stretch increased ROS production in trabeculae and increased the velocity of Ca2+ waves [15]. In that study, however, the muscle was stretched 10 ms after the last stimulus of the electrical train, meaning that myofilament Ca2+ sensitivity was increased by stretch during the last twitch contraction. This increase may have changed the Ca2+ dissociated from the myofilaments and thereby may have affected the velocity of the subsequent Ca2 + waves. Therefore, in the present study, the muscle was stretched 300 ms after the last stimulus to minimize the effect of the Ca2+ dissociated from the myofilaments during twitch contraction. The results in Figs. 1, 2, and 3 suggest that the degree of increase in the velocity of Ca2+ waves depended on the degree of ROS production
in response to muscle stretch. In addition, the results in Figs. 5 and 6 suggest that the velocity of Ca2+ waves was increased by ROS production in the presence of SCH00013. It has been reported that ROS increases Ca2+ release from the SR [42–44] and that Ca2+ waves propagate with the mechanism of Ca2+-induced Ca2+ release from the SR [22,23]. From these results, we hypothesize that muscle stretch and SCH00013 increase ROS production and that ROS production rather than increased myofilament Ca2+ sensitivity increases the velocity of Ca2+ waves. Because the velocity of Ca2+ waves determines the amplitude of the DADs [25], the increase in the velocity of Ca2+ waves may be related to increased arrhythmia susceptibility caused by ROS production [45] and increased frequency of triggered arrhythmias in the presence of SCH00013 [14]. In conclusion, muscle stretch increases the velocity of Ca2+ waves by increasing ROS production, not by increasing myofilament Ca2+ sensitivity. In addition, ROS production increases myofilament Ca2+ sensitivity and the velocity of Ca2+ waves in the presence of SCH00013. These results suggest that ROS rather than myofilament Ca2+ sensitivity plays an important role in the occurrence of triggered arrhythmias under conditions of regional Ca2+ overload.
4.4. Limitations Mouse models with troponin mutations would be useful to investigate the role of myofilament Ca2+ sensitivity in Ca2+ wave propagation. In the present study, however, we did not use such models because we could not create mouse models which initiate Ca2+ waves by high Ca2+ jet due to their smaller size. Therefore, in the present study we stretched
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muscle and added a Ca2 + sensitizer to increase myofilament Ca2 + sensitivity. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.yjmcc.2015.04.027. Sources of funding This work was supported by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (M. Miura, No. 23590253). Disclosures None. References [1] Packer M. Sudden unexpected death in patients with congestive heart failure: a second frontier. Circulation 1985;72:681–5. [2] Myerburg RJ, Interian A, Mitrani RM, Kessler KM, Castellanos A. Frequency of sudden cardiac death and profiles of risk. Am J Cardiol 1997;80:10F–9F. [3] van der Velden J, Papp Z, Zaremba R, Boontje NM, de Jong JW, Owen VJ, et al. Increased Ca2+-sensitivity of the contractile apparatus in end-stage human heart failure results from altered phosphorylation of contractile proteins. Cardiovasc Res 2003;57:37–47. [4] Wolff MR, Buck SH, Stoker SW, Greaser ML, Mentzer RM. Myofibrillar calcium sensitivity of isometric tension is increased in human dilated cardiomyopathies: role of altered beta-adrenergically mediated protein phosphorylation. J Clin Invest 1996; 98:167–76. [5] van der Velden J, Merkus D, Klarenbeek BR, James AT, Boontje NM, Dekkers DHW, et al. Alterations in myofilament function contribute to left ventricular dysfunction in pigs early after myocardial infarction. Circ Res 2004;95:e85–95. [6] Venkataraman R, Baldo MP, Hwang HS, Veltri T, Pinto JR, Baudenbacher FJ, et al. Myofilament calcium de-sensitization and contractile uncoupling prevent pausetriggered ventricular tachycardia in mouse hearts with chronic myocardial infarction. J Mol Cell Cardiol 2013;60:8–15. [7] Van Eyk JE, Powers F, Law W, Larue C, Hodges RS, Solaro RJ. Breakdown and release of myofilament proteins during ischemia and ischemia/reperfusion in rat hearts: identification of degradation products and effects on the pCa–force relation. Circ Res 1998;82:261–71. [8] Maron BJ, Shirani J, Poliac LC, Mathenge R, Roberts WC, Mueller FO. Sudden death in young competitive athletes. Clinical, demographic, and pathological profiles. JAMA 1996;276:199–204. [9] Knollmann BC, Potter JD. Altered regulation of cardiac muscle contraction by troponin T mutations that cause familial hypertrophic cardiomyopathy. Trends Cardiovasc Med 2001;11:206–12. [10] Davis J, Wen H, Edwards T, Metzger JM. Thin filament disinhibition by restrictive cardiomyopathy mutant R193H troponin I induces Ca2+-independent mechanical tone and acute myocyte remodeling. Circ Res 2007;100:1494–502. [11] Huke S, Knollmann BC. Increased myofilament Ca2+-sensitivity and arrhythmia susceptibility. J Mol Cell Cardiol 2010;48:824–33. [12] Kentish JC, ter Keurs HEDJ, Ricciardi L, Bucx JJJ, Noble MIM. Comparison between the sarcomere length–force relations of intact and skinned trabeculae from rat right ventricle. Influence of calcium concentrations on these relations. Circ Res 1986;58: 755–68. [13] Wakayama Y, Miura M, Sugai Y, Kagaya Y, Watanabe J, ter Keurs HEDJ, et al. Stretch and quick release of rat cardiac trabeculae accelerates Ca2+ waves and triggered propagated contractions. Am J Physiol Heart Circ Physiol 2001;281:H2133–42. [14] Miura M, Nishio T, Hattori T, Murai N, Stuyvers BD, Shindoh C, et al. Effect of nonuniform muscle contraction on sustainability and frequency of triggered arrhythmias in rat cardiac muscle. Circulation 2010;121:2711–7. [15] Miura M, Murai N, Hattori T, Nagano T, Stuyvers BD, Shindoh C. Role of reactive oxygen species and Ca2+ dissociation from the myofilaments in determination of Ca2+ wave propagation in rat cardiac muscle. J Mol Cell Cardiol 2013;56:97–105. [16] Iribe G, Ward CW, Camelliti P, Bollensdorff C, Mason F, Burton RA, et al. Axial stretch of rat single ventricular cardiomyocytes causes an acute and transient increase in Ca2+ spark rate. Circ Res 2009;104:787–95.
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Ca2+ waves during triggered propagated contractions in intact trabeculae. Am J Physiol 1998;274:H266–76. [24] Tveito A, Lines GT, Edwards AG, Maleckar MM, Michailova A, Hake J, et al. Slow calcium-depolarization-calcium waves may initiate fast local depolarization waves in ventricular tissue. Prog Biophys Mol Biol 2012;110:295–304. [25] Sugai Y, Miura M, Hirose M, Wakayama Y, Endoh H, Nishio T, et al. Contribution of Na+/Ca2+ exchange current to the formation of delayed afterdepolarizations in intact rat ventricular muscle. J Cardiovasc Pharmacol 2009;53:517–22. [26] Sugawara H, Endoh M. A novel cardiotonic agent SCH00013 acts as a Ca2+ sensitizer with no chronotropic activity in mammalian cardiac muscle. J Pharmacol Exp Ther 1998;287:214–22. [27] Tadano N, Morimoto S, Yoshimura A, Miura M, Yoshioka K, Sakato M, et al. SCH00013, a novel Ca2+ sensitizer with positive inotropic and no chronotropic action in heart failure. J Pharmacol Sci 2005;97:53–60. 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Journal of Molecular and Cellular Cardiology journal homepage: www.elsevier.com/locate/yjmcc
Original article
Effect of myofilament Ca2 + sensitivity on Ca2 + wave propagation in rat ventricular muscle Masahito Miura ⁎, Yuhto Taguchi, Tsuyoshi Nagano, Mai Sasaki, Tetsuya Handoh, Chiyohiko Shindoh Department of Clinical Physiology, Health Science, Tohoku University Graduate School of Medicine, Sendai 980-8574, Japan
a r t i c l e
i n f o
Article history: Received 8 January 2015 Received in revised form 2 April 2015 Accepted 29 April 2015 Available online 4 May 2015 Keywords: Ca2 + waves Myofilament Ca2 + sensitivity ROS Muscle stretch
a b s t r a c t Background: The propagation velocity of Ca2+ waves determines delayed afterdepolarization and affects the occurrence of triggered arrhythmias in cardiac muscle. We focused on myofilament Ca2+ sensitivity, investigating how the velocity of Ca2+ waves responds to its increased sensitivity resulting from muscle stretch or the addition of a myofilament Ca2+ sensitizer, SCH00013. We further investigated whether production of reactive oxygen species (ROS) may be involved in the change in velocity. Methods: Trabeculae were obtained from rat hearts. Force, sarcomere length, and [Ca2+]i were measured. ROS production was estimated from 2′,7′-dichlorofluorescein (DCF) fluorescence. Trabeculae were exposed to a 10 mM Ca2+ jet for the induction of Ca2+ leak from the sarcoplasmic reticulum in its exposed region. Ca2+ waves were induced by 2.5-Hz stimulus trains for 7.5 s (24 °C, 2.0 mM [Ca2+]o). Muscle stretch of 5, 10, and 15% was applied 300 ms after the last stimulus of the train. Results: Muscle stretch increased the DCF fluorescence, the amplitude of aftercontractions, and the velocity of Ca2+ waves depending on the degree of stretch. After preincubation with 3 μM diphenyleneiodonium (DPI), muscle stretch increased only the amplitude of aftercontractions but not the DCF fluorescence nor the velocity of Ca2+ waves. SCH00013 (30 μM) increased the DCF fluorescence, the amplitude of aftercontractions, and the velocity of Ca2+ waves. DPI suppressed these increases. Conclusions: Muscle stretch increases the velocity of Ca2+ waves by increasing ROS production, not by increasing myofilament Ca2+ sensitivity. In the case of SCH00013, ROS production increases myofilament Ca2+ sensitivity and the velocity of Ca2+ waves. These results suggest that ROS rather than myofilament Ca2+ sensitivity plays an important role in the determination of the velocity of Ca2+ waves, that is, arrhythmogenesis. © 2015 Elsevier Ltd. All rights reserved.
1. Introduction It is well known that lethal arrhythmias occur in patients with acquired diseases such as myocardial infarction and heart failure [1,2], and myofilament Ca2+ sensitivity has been reported to be increased in such patients [3–7]. Also, in patients with familial hypertrophic cardiomyopathy, lethal arrhythmias occur [8], and in some cases myofilament Ca2+ sensitivity is increased because of troponin mutations [9]. In patients with this familial disease, increased myofilament Ca2+ sensitivity not only causes incomplete mechanical relaxation [10] but also underlies arrhythmia susceptibility [11]. Thus, it remains to be established whether increased myofilament Ca2 + sensitivity is involved in the occurrence of arrhythmias in patients with acquired diseases. Muscle stretch increases myofilament Ca2+ sensitivity and increases 2+ Ca binding to the myofilaments [12], thereby increasing the developed force [13–15]. Recently, it has been reported that muscle stretch ⁎ Corresponding author at: Department of Clinical Physiology, Health Science, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 9808574, Japan. Tel./fax: +81 22 717 7920. E-mail address: [email protected] (M. Miura).
http://dx.doi.org/10.1016/j.yjmcc.2015.04.027 0022-2828/© 2015 Elsevier Ltd. All rights reserved.
increases the frequency of Ca2 + sparks [16,17] due to production of reactive oxygen species (ROS) by the activation of NADPH oxidase in cardiac muscle [18]. In addition, we have reported that in rat cardiac muscle, muscle stretch increases 2′,7′-dichlorofluorescein (DCF) fluorescence, which may reflect ROS production, and increases the velocity of Ca2+ waves initiated from the region under Ca2+ overload [15]. In that study, however, it was unclear whether ROS production was involved in the increase in the propagation velocity of Ca2+ waves. Ca2 + wave propagation is deeply involved in the occurrence of arrhythmias [14,19]. Ca2 + waves are initiated not only by Ca2 + leak from the sarcoplasmic reticulum (SR) under Ca2 + overload [20] but also by Ca2 + dissociation from the myofilaments under nonuniform contraction [15,21]. These waves propagate along the cardiac muscle by the mechanism of Ca2+-induced Ca2+ release from the SR [22,23] although it has been reported in other studies that such waves may be driven intermittently by both calcium and membrane depolarization [24]. The velocity of Ca2+ waves is correlated with the amplitude of delayed afterdepolarizations (DADs) and is concerned with the occurrence of arrhythmias when it reaches a threshold for the action potential [25]. Thus, the velocity of Ca2+ waves is one of the important determinants of the occurrence of triggered arrhythmias.
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Therefore, in the present study we focused on myofilament Ca2 + sensitivity, investigating how the velocity of arrhythmogenic Ca2 + waves changes in response to increased myofilament Ca2+ sensitivity by muscle stretch or by the addition of SCH00013, a myofilament Ca2 + sensitizer [14,26,27]. We further investigated whether ROS production may play a role in changes in the velocity of these waves.
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the muscle for the SL of 2.1, 2.2, and 2.3 μm under an unstimulated condition was defined as 5, 10, and 15% stretch, respectively. In the present study, SL was not controlled during muscle contraction, and thus uncontrolled sarcomere shortening may have increased the peak of [Ca2+]i transients during contraction [31]. 2.4. Experimental protocol
2. Materials and methods 2+
2.1. Measurements of force, [Ca ]i, and ROS production in rat trabeculae (see Expanded materials and methods in the online data supplement) All animal procedures were performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised in 1996) and were approved by the Ethics Review Board of Tohoku University (approval reference number: 21-98). Briefly, after the rats had been adequately anesthetized, trabeculae (n = 60; length: 2.6 ± 0.3 mm; width: 281 ± 73 μm; thickness: 83 ± 3 μm in a slack condition) were dissected from the endocardial region of the rat right ventricle and mounted on an inverted microscope between a force transducer and a micromanipulator in a bath superfused with HEPES solution containing 5 mM KCl, as previously described [13–15,21,23,25]. Force was measured using a silicon strain gauge, sarcomere length (SL) using laser diffraction techniques, and [Ca2+]i using fura-2, as previously described [23]. Briefly, fura-2 pentapotassium salt was microinjected electrophoretically into a trabecula. Excitation light of 340, 360, or 380 nm was employed, and fluorescence was collected using 1) a photomultiplier tube (PMT) to assess [Ca2+]i within the entire trabecula or 2) an image intensified CCD camera at 30 frames/s to assess local [Ca2 +]i. We calculated [Ca2 +]i from the calibrated ratio of F360/F380 in the region of interest along the trabecula. To calculate the velocity of Ca2 + waves, we identified the peak of a Ca2 + transient during the Ca2 + wave at each pixel along the trabecula and plotted the time of the peak against the position of the peak. The velocity of Ca2+ waves was calculated from the slope of the line fitted to the plot when regression analysis indicated a linear relationship between the time of the peak and the position of the peak (r ≥ 0.9), as previously described [21,23]. ROS production was estimated using 2′,7′-dichlorofluorescein (DCF) fluorescence from the muscle, as previously described [15]. Briefly, a trabecula was loaded with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) [28]. DCFH-DA was cleaved to DCFH and oxidized to the fluorescent molecule DCF by a variety of ROS [29]. Average DCF fluorescence from the trabecula was measured using a PMT.
Ca2+ waves were induced by electrical stimulation at 400 ms intervals for 7.5 s (24 °C, 2.0 mM [Ca2+]o). In the present study the first Ca2+ waves were selected for analysis unless otherwise stated. To increase myofilament Ca2 + sensitivity, the muscle was stretched by 5, 10, or 15% 300 ms after the last stimulus of the electrical train. Ca2+ waves and aftercontractions during Ca2+ wave propagation were then recorded during muscle stretch. Additionally, to investigate the effect of ROS production due to muscle stretch on the Ca2 + waves and aftercontractions, the Ca2+ waves and aftercontractions were recorded after preincubation of 3 μM diphenyleneiodonium (DPI), NADPH oxidase inhibitor, for 1 h [17,32]. SCH00013 (30 μM) has been used as a Ca2+ sensitizer for myofilaments [26,27] and has been previously reported to increase the frequency of triggered arrhythmias [14]. Thus, Ca2+ waves and aftercontractions were recorded in the absence and presence of SCH00013 ([Ca2+]o = 2.0 mM). To estimate the effect of ROS production by the addition of SCH00013, the Ca2+ waves and aftercontractions were recorded again in its presence after preincubation of DPI. In addition, ROS production by the addition of SCH00013 was estimated from the changes in DCF fluorescence before (Fbefore) and after electrical stimulation (Fafter) with 2-s stimulus intervals for 30 s and 0.4-s stimulus intervals for 7.5 s ([Ca2+]o = 0.7 mM). Changes in the DCF fluorescence (%) were calculated from (Fafter − Fbefore) / Fbefore × 100. 2.5. Statistics All measurements were expressed as mean ± SEM. Statistical analysis was performed with 1-way repeated-measures ANOVA and a post-hoc test (Bonferroni) for multiple comparisons and a paired t-test for 2-group comparisons when the data were normally distributed. These analyses were performed using software for statistical analysis (Ekuseru-Toukei 2012, Social Survey Research Information Co., Ltd., Tokyo, Japan). Values of p b 0.05 were considered to be significant. The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agreed to the manuscript as written.
2.2. Regional jet exposure 3. Results A restricted region of a trabecula was exposed to a jet of solution (≈ 0.06 mL/min) directed perpendicular to a small muscle segment (≈300 μm) using a glass pipette (≈100 μm in diameter), as previously described [14,15,21]. The jet of the solution contained 10 mM Ca2+ to induce Ca2+ waves due to Ca2+ overload within the jet-exposed region. 2.3. Addition of muscle stretch SL was determined using a laser diffraction technique, as previously described [30]. Briefly, the preparations were illuminated by a He–Ne laser beam (05-LHP-925, Melles Griot, NM), and the SL was calculated from the median of the intensity distribution of the first order diffraction pattern. Trabeculae were stretched from their valvular end using a DC torque motor controlled by voltage commands from a personal computer via a 12 bit D/A converter (T-IFPC9 04120A-N, T.S.K., Japan), as previously described [13–15]. Under unstimulated conditions, the SL was first set at 2.0 μm, and then the movement distance of the valvular end was determined to increase the SL to 2.1, 2.2, or 2.3 μm. Such movement of
3.1. Muscle stretch and Ca2+ waves Muscle was stretched by 5, 10, or 15% during Ca2+ wave propagation 300 ms after the last stimulus of the electrical train, and its effect on aftercontractions and Ca2 + waves was investigated. As shown in Fig. 1Aa, after rapid electrical stimulation, regional increases in [Ca2+]i occurred within the region exposed to the high Ca2+ jet and propagated along the muscle as Ca2 + waves. The velocity of the first Ca2 + wave (white arrow) was 1.98 mm/s. In Fig. 1Ab, 15% muscle stretch enhanced the amplitude of the aftercontractions during Ca2+ wave propagation and increased the velocity of the first Ca2 + wave to 2.81 mm/s. Figs. 1B and C show summary data concerning the effect of muscle stretch on the aftercontractions and the first Ca2+ waves. As shown in Fig. 1B, muscle stretch enhanced the amplitude of aftercontractions during the first Ca2+ wave propagation, and 15% muscle stretch increased their amplitude more than that by 5% muscle stretch. Additionally, as shown in Fig. 1C, muscle stretch increased the velocity of the first Ca2 + waves, and 15% muscle stretch increased their velocity more
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Fig. 1. Effect of muscle stretch on the velocity of the first Ca2+ waves and the amplitude of their corresponding aftercontractions (ACs). A. Representative recordings of force (upper panels) and regional changes in [Ca2+]i (lower panels) during the last three electrical stimuli (ST; 400-ms stimulus interval) and Ca2+ waves without (a) and with 15% muscle stretch (b). White arrows indicate the first Ca2+ waves, and black arrowheads indicate their corresponding ACs. In panel a, a few Ca2+ waves appear within the jet-exposed region (10 mM Ca JET) after the train of electrical stimuli and propagate along the trabecula. Panel b shows that a 15% stretch increases the amplitude of ACs and the velocity of the first Ca 2 + wave ([Ca 2 + ] o = 2.0 mM, Exp. 130710). B. Summary data concerning the effect of muscle stretch on the amplitude of ACs (AC force) during the first Ca 2 + wave propagation. Blue, red, and green circles indicate the changes by 5, 10, and 15% stretch, respectively (n = 11). *p b 0.05 vs stretch(−); #p b 0.01 vs stretch(−); & p b 0.01 vs 5% stretch. C. Summary data concerning the effect of stretch on the velocity of the first Ca2+ waves. Blue, red, and green circles indicate the changes by 5, 10, and 15% stretch, respectively (n = 11). *p b 0.05 vs stretch(−); $p b 0.05 vs 5% stretch.
than that by 5% muscle stretch. These results suggest that muscle stretch, depending on its degree, increases both the amplitude of aftercontractions and the velocity of the first Ca2+ waves. It has been reported that muscle stretch increases ROS production depending on the degree of stretch in isolated single myocytes [33]. Thus, the effect of 5, 10, or 15% muscle stretch on the DCF fluorescence within trabeculae was examined. As shown in Figs. 2A and B, muscle stretch increased the DCF fluorescence depending on the degree of stretch. In addition, after preincubation with 3 μM DPI, muscle stretch did not increase the DCF fluorescence. These results suggest that muscle stretch increases ROS production depending on the degree of muscle stretch in trabeculae as well. As shown in Fig. 2A, ROS production was transiently increased just after muscle stretch. To investigate whether the velocity of Ca2 + waves was also transiently increased by stretch, the effect of 15% stretch on the first and second Ca2 + waves was measured. The peaks of aftercontractions caused by the first and second Ca2+ waves occurred at 215 ± 28 ms and 610 ± 47 ms after muscle stretch, respectively. As shown in Fig. 2C, muscle stretch increased the velocity of the first Ca2+ waves but not that of the second Ca2+ waves. When changes in the velocity of Ca2+ waves were plotted against the time of the peaks of their corresponding aftercontractions which elapsed after stretch, the changes in the velocity were larger just after muscle stretch
(Fig. 2D). These results mean that the velocity of Ca2+ waves was also transiently increased just after muscle stretch. To investigate whether ROS production was actually involved in the increases in the amplitude of aftercontractions and the velocity of the first Ca2+ waves observed in Fig. 1, muscle was stretched during Ca2+ wave propagation after preincubation with DPI. As shown in Fig. 3Aa, the high Ca2+ jet induced Ca2+ waves after the rapid electrical stimulation. The velocity of the first Ca2+ wave (white arrow) was 1.60 mm/s. In Fig. 3Ab muscle stretch enhanced the amplitude of the aftercontraction, whereas it hardly increased the velocity of the first Ca2+ wave to 1.63 mm/s. Figs. 3B and C show summary data concerning the effect of muscle stretch on the aftercontractions and velocity after preincubation with DPI. As shown in Fig. 3B, muscle stretch increased the amplitude of aftercontractions during the first Ca2+ wave propagation, and 15% muscle stretch increased their amplitude more than that by 5% muscle stretch, similar to the results shown in Fig. 1B. In contrast, muscle stretch did not increase the velocity of the first Ca2+ waves, as shown in Fig. 3C. These results suggest that muscle stretch increases the velocity of the first Ca2+ waves by increasing ROS production, whereas it increases the amplitude of aftercontractions probably because of increased myofilament Ca2+ sensitivity. They further suggest that during muscle stretch, ROS production rather than increased myofilament Ca2+ sensitivity plays an important role in the determination of the velocity of Ca2+ waves.
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Fig. 2. Effect of muscle stretch on the DCF fluorescence and the velocity of the first and second Ca2+ waves. A. The upper panel shows changes in muscle length, the middle panel shows changes in the DCF fluorescence resulting from muscle stretch, and the lower panel shows changes in the DCF fluorescence due to muscle stretch after preincubation with 3 μM DPI ([Ca2+]o = 2.0 mM, Exp. 140702). Blue, red, and green lines indicate the recordings by 5, 10, and 15% muscle stretch, respectively. B. Summary data concerning the effect of muscle stretch on the changes in the DCF fluorescence 2 s after muscle stretch (△DCF2.0) before (−) and after preincubation with 3 μM DPI (+). Blue, red, and green circles indicate the changes by 5, 10, and 15% stretch, respectively (n = 6). *p b 0.05 vs 10% stretch DPI(−); #p b 0.01 vs 15% stretch DPI(−); &p b 0.01 vs 5% stretch DPI(−). C. Summary data concerning the effect of 15% stretch on the velocity of the first and second Ca2+ waves (n = 10, [Ca2+]o = 2.0 mM). *p b 0.05 vs stretch (−); §p b 0.05 vs 1st. D. Relationship between changes in the velocity of Ca2+ waves and the time of the peaks of their corresponding aftercontractions after 15% stretch. These points are fitted to a quadratic curve (r = 0.68, n = 17, [Ca2+]o = 2.0 mM).
3.2. SCH00013, a myofilament Ca2+ sensitizer, and Ca2+ waves SCH00013 increased the developed force without changes in [Ca2+]i transients (Fig. 4), showing that SCH00013 increases myofilament Ca2+ sensitivity, as is consistent with past reports [14,26,27]. We then measured the amplitude of aftercontractions and the velocity of Ca2 + waves in the absence and presence of SCH00013 and investigated whether increased myofilament Ca2 + sensitivity affects Ca2+ waves. In the absence of SCH00013, the velocity of the first Ca2+ waves after electrical stimulation was 2.63 mm/s, as shown in Fig. 5A. After the addition of SCH00013, the amplitude of aftercontractions became higher, probably because of increased myofilament Ca2 + sensitivity, and the velocity became faster. Fig. 5B shows summary data concerning the effect of SCH00013 on the amplitude of aftercontractions and on the velocity of Ca2 + waves. SCH00013 increased both the amplitude of aftercontractions during the first Ca2 + wave propagation and the velocity of the first Ca2+ waves. To examine the effect of SCH00013 on ROS production, the DCF fluorescence was measured in the absence and presence of SCH00013. As shown in Fig. 5C, DCF fluorescence was increased after the addition of SCH00013, suggesting that SCH00013 increases ROS production. To investigate whether ROS production is involved in the increase in the velocity of Ca2+ waves in the presence of SCH00013, the effect of preincubation with DPI on the velocity of Ca2+ waves was observed. After preincubation with DPI, SCH00013 neither increased the amplitude
of aftercontractions nor the velocity of Ca2+ waves, as shown in Figs. 6A and B. In addition, SCH00013 did not increase the DCF fluorescence after preincubation with DPI, as shown in Fig. 6C. These results suggest that SCH00013 increases both the amplitude of aftercontractions and the velocity of Ca2+ waves by increasing ROS production and that ROS production plays an important role in the determination of the velocity of Ca2+ waves. It was finally examined whether SCH00013 affects myofilament Ca2+ sensitivity even after preincubation with DPI. As shown in online Fig. 1, SCH00013 did not increase myofilament Ca2 + sensitivity after preincubation with DPI. These results suggest that SCH00013 increases myofilament Ca2+ sensitivity by increasing ROS production.
4. Discussion The present study investigated how the velocity of Ca2 + waves changes in response to increased myofilament Ca2+ sensitivity by the cardiac muscle stretch and by the addition of a myofilament Ca2+ sensitizer, SCH00013. To the best of our knowledge, the present study is the first to show that muscle stretch increases the velocity of Ca2 + waves by increasing ROS production, not by increasing myofilament Ca2+ sensitivity, and that in the case of SCH00013, ROS production increases myofilament Ca2+ sensitivity and the velocity of Ca2+ waves, as discussed below.
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Fig. 3. Effect of muscle stretch on the amplitude of aftercontractions (ACs) and the velocity of the first Ca2+ waves after preincubation with 3 μM DPI. A. Representative recordings of force (upper panels) and regional changes in [Ca2+]i (lower panels) during the last three electrical stimuli (ST; 400-ms stimulus interval) and Ca2+ waves without (a) and with 15% stretch (b) after preincubation with 3 μM DPI. White arrows indicate the first Ca2+ waves, and black arrowheads indicate their corresponding ACs. In panel a, a few Ca2+ waves appear within the jet-exposed region (10 mM Ca JET) and propagate along the trabecula. In panel b, 15% stretch increases the amplitude of ACs, but hardly increases the velocity of the first Ca2+ wave ([Ca2+]o = 2.0 mM, Exp. 130221). B. Summary data concerning the effect of muscle stretch on the amplitude of ACs (AC force) during Ca2+ wave propagation. Blue, red, and green circles indicate 5, 10, and 15% stretch, respectively (n = 11). *p b 0.05 vs stretch(−); #p b 0.01 vs stretch(−); §p b 0.05 vs 5% Stretch; &p b 0.01 vs 5% Stretch. C. Summary data concerning the effect of stretch on the velocity of the first Ca2+ waves. Blue, red, and green circles indicate 5, 10, and 15% stretch, respectively (n = 11). Muscle stretch does not increase the velocity in the left panel, and in the right panel muscle stretch does not increase the changes in the velocity (p = 0.54).
4.1. ROS production and myofilament Ca2+ sensitivity Muscle stretch has been shown to produce ROS due to the activation of NADPH oxidase [16,17,33], which exists within the cardiac muscle [18]. In the present study as well, muscle stretch increased the DCF fluorescence, but did not increase it after preincubation with DPI (Fig. 2). These results suggest that muscle stretch increased ROS production probably because of the activation of NADPH oxidase although DPI also inhibits the synthesis of both oxygen- and nitrogen-derived reactive species and many other flavoproteins depending on the concentration [34]. It is unlikely, however, that this ROS production increased the myofilament Ca2 + sensitivity because muscle stretch increased the amplitude of the aftercontractions even after preincubation with DPI (Fig. 3). In contrast, it is likely that SCH00013 increased the myofilament Ca2+ sensitivity by ROS production because after preincubation with DPI, SCH00013 did not increase the DCF fluorescence (Fig. 6C), the amplitude of the aftercontractions (Fig. 6B), nor the developed force (online Fig. 1). It has been reported that SCH00013 is a Ca2+ sensitizer with a weak selective phosphodiesterase (PDE) 3 inhibitory action and class III anti-arrhythmic action [35]. In the present study, the PDE 3 inhibitory action and class III anti-arrhythmic action played only a
minor role because SCH00013 functioned without changes in [Ca2+]i transients (Fig. 4), as is consistent with a past report [26]. In our previous study, however, increased myofilament Ca2+ sensitivity by stretch increased force but accelerated the decline of [Ca2+]i transients during contractions [13] probably because of an increase in Ca2+ binding to the myofilaments [12]. In the case of SCH00013, it is still unknown why increased myofilament Ca2 + sensitivity did not change [Ca2 +]i transients, except that the increase in ROS production may be involved. Taken together, these results suggest that myofilament Ca2+ sensitivity is increased by muscle stretch with a different mechanism from that in the presence of SCH00013. 4.2. Myofilament Ca2+ sensitivity and Ca2+ waves An increase in Ca2 + buffering capacity within cardiac muscle decreases the peak of Ca2+ transient and slows the decline of Ca2+ transient [36]. It further decreases the velocity of Ca2 + waves [37] and suppresses the occurrence of arrhythmias [38,39]. It has been reported, however, that in cardiac muscle with increased myofilament Ca2+ sensitivity due to troponin T mutations, an increase in intracellular Ca2+ can easily increase Ca2+ loading within the SR, thereby causing arrhythmias, because of a decrease in the remaining Ca2+ buffering capacity
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Fig. 4. Effect of SCH00013, a myofilament Ca2+ sensitizer, on force and Ca2+ transients. A. Representative recordings of force (upper panel) and [Ca2+]i (lower panel) during electrical stimulation (ST; 2-s stimulus interval) in the absence (black lines) and presence (gray lines) of SCH00013 ([Ca2+]o = 0.7 mM, Exp. 130325). B. Summary data concerning the effect of SCH00013 on force (n = 7). #p b 0.01 vs (−). C. Summary data concerning the effect of SCH00013 on the diastolic [Ca2+]i (p = 0.75), the peak [Ca2+]i (p = 0.91), and the time constant of the [Ca2+]i decline (p = 0.55, n = 7) during electrical stimulation.
A
B
C
Fig. 5. Effect of SCH00013 on the amplitude of aftercontractions (ACs), the velocity of Ca2+ waves, and the DCF fluorescence. A. Representative recordings of force (upper panels) and regional changes in [Ca2+]i (lower panels) during the last three electrical stimuli (ST; 400-ms stimulus interval) and Ca2+ waves in the absence (left panels) and presence of SCH00013 (right panels). White arrows indicate the first Ca2+ waves, and black arrowheads indicate their corresponding ACs. In the left panels, a few Ca2+ waves appear within the jet-exposed region (10 mM Ca JET) and propagate along the trabecula. In the right panels, both the amplitude of AC and the velocity of the first Ca2+ wave are increased in the presence of SCH00013 ([Ca2+]o = 2.0 mM, Exp. 130327). B. Summary data concerning the effect of SCH00013 on the amplitude of ACs (AC force) during the first Ca2+ wave propagation (left panel) and the velocity of the first Ca2+ waves (right panel). Black and blue circles indicate the changes in the absence and presence of SCH00013, respectively (n = 7). *p b 0.05 vs (−); #p b 0.01 vs (−). C. Summary data concerning the effect of SCH00013 on DCF fluorescence. Changes in the DCF fluorescence (%) was measured before and after electrical stimulation with 2-s stimulus intervals for 30 s (upper panel) and 0.4-s intervals for 7.5 s (lower panel). Black and blue circles indicate the changes in the absence and presence of SCH00013, respectively (n = 5, [Ca2+]o = 0.7 mM). #p b 0.01 vs (−).
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Fig. 6. Effect of SCH00013 on the amplitude of aftercontractions (ACs), the velocity of Ca2+ waves, and the DCF fluorescence after preincubation with 3 μM DPI. A. Representative recordings of force (upper panels) and regional changes in [Ca2+]i (lower panels) during the last three electrical stimuli (ST; 400-ms stimulus interval) and Ca2+ waves in the absence (left panels) and presence of SCH00013 (right panels) after preincubation with DPI. In the left panels, a Ca2+ wave (white arrow) appears within the jet-exposed region (10 mM Ca JET) and propagates. In the right panels, both the amplitude of AC (black arrowhead) and the velocity of the Ca2+ wave (white arrow) are hardly changed in the presence of SCH00013 ([Ca2+]o = 2.0 mM, Exp. 130128). B. Summary data concerning the effect of SCH00013 on the amplitude of ACs (AC force) during the first Ca2+ wave propagation (left panel) and the velocity of the first Ca2+ waves (right panel). Black and blue circles indicate changes in the absence (p = 0.60) and presence of SCH00013 (p = 0.31), respectively (n = 7). C. Summary data concerning the effect of SCH00013 on the DCF fluorescence after preincubation with DPI. Changes in the DCF fluorescence (%) was measured before and after electrical stimulation with 2-s stimulus intervals for 30 s (upper panel) and 0.4-s intervals for 7.5 s (lower panel). Black and blue circles indicate the changes in the absence and presence of SCH00013, respectively (n = 5, [Ca2+]o = 0.7 mM).
because the total amount of Ca2+ buffering capacity is the same [11,40]. In addition, it has been reported that blebbistatin, which has recently been used as a drug to decrease myofilament Ca2+ sensitivity, decreases the occurrence of arrhythmias [6,41]. In contrast, the results of the present study show that increased myofilament Ca2+ sensitivity by muscle stretch did not increase the velocity of Ca2+ waves after preincubation with DPI (Fig. 3), suggesting that increased myofilament Ca2+ sensitivity cannot increase the velocity of Ca2+ waves. Furthermore, we have previously reported that the presence of blebbistatin does not decrease the velocity of Ca2 + waves arising from the Ca2+-overloaded region [15]. Taken together, these results suggest that myofilament Ca2+ sensitivity does not affect the velocity of Ca2+ waves in rat cardiac muscle and that it plays only a minor role in determination of triggered arrhythmias. 4.3. ROS and Ca2+ waves We have previously reported that muscle stretch increased ROS production in trabeculae and increased the velocity of Ca2+ waves [15]. In that study, however, the muscle was stretched 10 ms after the last stimulus of the electrical train, meaning that myofilament Ca2+ sensitivity was increased by stretch during the last twitch contraction. This increase may have changed the Ca2+ dissociated from the myofilaments and thereby may have affected the velocity of the subsequent Ca2 + waves. Therefore, in the present study, the muscle was stretched 300 ms after the last stimulus to minimize the effect of the Ca2+ dissociated from the myofilaments during twitch contraction. The results in Figs. 1, 2, and 3 suggest that the degree of increase in the velocity of Ca2+ waves depended on the degree of ROS production
in response to muscle stretch. In addition, the results in Figs. 5 and 6 suggest that the velocity of Ca2+ waves was increased by ROS production in the presence of SCH00013. It has been reported that ROS increases Ca2+ release from the SR [42–44] and that Ca2+ waves propagate with the mechanism of Ca2+-induced Ca2+ release from the SR [22,23]. From these results, we hypothesize that muscle stretch and SCH00013 increase ROS production and that ROS production rather than increased myofilament Ca2+ sensitivity increases the velocity of Ca2+ waves. Because the velocity of Ca2+ waves determines the amplitude of the DADs [25], the increase in the velocity of Ca2+ waves may be related to increased arrhythmia susceptibility caused by ROS production [45] and increased frequency of triggered arrhythmias in the presence of SCH00013 [14]. In conclusion, muscle stretch increases the velocity of Ca2+ waves by increasing ROS production, not by increasing myofilament Ca2+ sensitivity. In addition, ROS production increases myofilament Ca2+ sensitivity and the velocity of Ca2+ waves in the presence of SCH00013. These results suggest that ROS rather than myofilament Ca2+ sensitivity plays an important role in the occurrence of triggered arrhythmias under conditions of regional Ca2+ overload.
4.4. Limitations Mouse models with troponin mutations would be useful to investigate the role of myofilament Ca2+ sensitivity in Ca2+ wave propagation. In the present study, however, we did not use such models because we could not create mouse models which initiate Ca2+ waves by high Ca2+ jet due to their smaller size. Therefore, in the present study we stretched
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muscle and added a Ca2 + sensitizer to increase myofilament Ca2 + sensitivity. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.yjmcc.2015.04.027. Sources of funding This work was supported by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (M. Miura, No. 23590253). Disclosures None. References [1] Packer M. Sudden unexpected death in patients with congestive heart failure: a second frontier. Circulation 1985;72:681–5. [2] Myerburg RJ, Interian A, Mitrani RM, Kessler KM, Castellanos A. Frequency of sudden cardiac death and profiles of risk. Am J Cardiol 1997;80:10F–9F. [3] van der Velden J, Papp Z, Zaremba R, Boontje NM, de Jong JW, Owen VJ, et al. Increased Ca2+-sensitivity of the contractile apparatus in end-stage human heart failure results from altered phosphorylation of contractile proteins. Cardiovasc Res 2003;57:37–47. [4] Wolff MR, Buck SH, Stoker SW, Greaser ML, Mentzer RM. Myofibrillar calcium sensitivity of isometric tension is increased in human dilated cardiomyopathies: role of altered beta-adrenergically mediated protein phosphorylation. J Clin Invest 1996; 98:167–76. [5] van der Velden J, Merkus D, Klarenbeek BR, James AT, Boontje NM, Dekkers DHW, et al. Alterations in myofilament function contribute to left ventricular dysfunction in pigs early after myocardial infarction. Circ Res 2004;95:e85–95. [6] Venkataraman R, Baldo MP, Hwang HS, Veltri T, Pinto JR, Baudenbacher FJ, et al. Myofilament calcium de-sensitization and contractile uncoupling prevent pausetriggered ventricular tachycardia in mouse hearts with chronic myocardial infarction. J Mol Cell Cardiol 2013;60:8–15. [7] Van Eyk JE, Powers F, Law W, Larue C, Hodges RS, Solaro RJ. Breakdown and release of myofilament proteins during ischemia and ischemia/reperfusion in rat hearts: identification of degradation products and effects on the pCa–force relation. Circ Res 1998;82:261–71. [8] Maron BJ, Shirani J, Poliac LC, Mathenge R, Roberts WC, Mueller FO. Sudden death in young competitive athletes. Clinical, demographic, and pathological profiles. JAMA 1996;276:199–204. [9] Knollmann BC, Potter JD. Altered regulation of cardiac muscle contraction by troponin T mutations that cause familial hypertrophic cardiomyopathy. Trends Cardiovasc Med 2001;11:206–12. [10] Davis J, Wen H, Edwards T, Metzger JM. Thin filament disinhibition by restrictive cardiomyopathy mutant R193H troponin I induces Ca2+-independent mechanical tone and acute myocyte remodeling. Circ Res 2007;100:1494–502. [11] Huke S, Knollmann BC. Increased myofilament Ca2+-sensitivity and arrhythmia susceptibility. J Mol Cell Cardiol 2010;48:824–33. [12] Kentish JC, ter Keurs HEDJ, Ricciardi L, Bucx JJJ, Noble MIM. Comparison between the sarcomere length–force relations of intact and skinned trabeculae from rat right ventricle. Influence of calcium concentrations on these relations. Circ Res 1986;58: 755–68. [13] Wakayama Y, Miura M, Sugai Y, Kagaya Y, Watanabe J, ter Keurs HEDJ, et al. Stretch and quick release of rat cardiac trabeculae accelerates Ca2+ waves and triggered propagated contractions. Am J Physiol Heart Circ Physiol 2001;281:H2133–42. [14] Miura M, Nishio T, Hattori T, Murai N, Stuyvers BD, Shindoh C, et al. Effect of nonuniform muscle contraction on sustainability and frequency of triggered arrhythmias in rat cardiac muscle. Circulation 2010;121:2711–7. [15] Miura M, Murai N, Hattori T, Nagano T, Stuyvers BD, Shindoh C. Role of reactive oxygen species and Ca2+ dissociation from the myofilaments in determination of Ca2+ wave propagation in rat cardiac muscle. J Mol Cell Cardiol 2013;56:97–105. [16] Iribe G, Ward CW, Camelliti P, Bollensdorff C, Mason F, Burton RA, et al. Axial stretch of rat single ventricular cardiomyocytes causes an acute and transient increase in Ca2+ spark rate. Circ Res 2009;104:787–95.
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