Regional increase in ROS within stretched region exacerbates arrhythmias in rat trabeculae with nonuniform contraction

Abstract

In diseased hearts, impaired muscle within the hearts is passively stretched by contractions of the more viable neighboring muscle during the contraction phase. We investigated whether in the myocardium with nonuniform contraction such passive stretch regionally generates ROS within the stretched region and exacerbates arrhythmias. In trabeculae from rat hearts, force, intracellular Ca²⁺, and membrane potential were measured. To assess regional ROS generation, the slope of the change in the 2′,7′-dichlorofluorescein fluorescence (DCF_slope) was calculated at the each pixel position along the long axis of trabeculae using DCF fluorescence images. Ca²⁺ waves and arrhythmias were induced by electrical stimulation. A H₂O₂ (1 mmol/L) jet regionally increased the DCF_slope within the jet-exposed region. A blebbistatin (10 μmol/L) jet caused passive stretch of the muscle within the jet-exposed region during the contraction phase and increased the DCF_slope within the stretched region, the velocity of Ca²⁺ waves, and the number of beats after electrical stimulation (0.2 μmol/L isoproterenol), while 3 μmol/L diphenyleneiodonium (DPI), NADPH oxidase inhibitor, decreased them. A jet of a solution containing 0.2 mmol/L H₂O₂ in addition to 10 µmol/L blebbistatin also increased them. A H₂O₂ jet within the region where Ca²⁺ waves propagated increased their velocity. In the myocardium with nonuniform contraction, passive stretch of the muscle by contractions of the neighboring muscle regionally increases ROS within the stretched region, and the regional ROS exacerbates arrhythmias by activating the propagation of Ca²⁺ waves.

Introduction

In patients with heart failure and myocardial infarction, reactive oxygen species (ROS) is increased [16, 17], probably due to an increase in NADPH oxidase activity [13, 22] or a decrease in hydrogen peroxide scavenging enzyme catalase activity [2]. This increase in ROS is involved in the exacerbation of heart failure [37] as well as in the occurrence of arrhythmias [7, 19, 46] by increasing Ca²⁺ release from the sarcoplasmic reticulum (SR) [43, 47]. Actually, in patients with diseased hearts, the occurrence of lethal arrhythmias is an important determinant of their prognosis [29, 30].

In a diseased heart, impaired muscle is widely distributed throughout the heart, causing nonuniform muscle contraction [34, 45]. In such myocardium with nonuniform contraction, impaired muscle with weaker contractile strength is stretched by contractions of the more viable neighboring muscle during the contraction phase. Conversely, during the relaxation phase, the impaired muscle is passively shortened and dissociates Ca²⁺ from the myofilaments within the region due to a decrease in myofilament Ca²⁺ sensitivity [20], thereby inducing Ca²⁺ waves [27, 40] and arrhythmias [25]. Additionally, it has been reported that stretch of cardiac muscle increases ROS generation in isolated single myocytes [32, 33] and trabeculae [27, 28] and further increases the frequency of Ca²⁺ sparks [18, 31, 33] and the velocity of Ca²⁺ waves [28]. It has not yet been established, however, whether in the myocardium with nonuniform contraction stretch of the impaired muscle by contractions of the neighboring muscle also increases ROS generation within the stretched region. Furthermore, it has not yet been established whether such an increase in regional ROS affects the propagation velocity of Ca²⁺ waves and the occurrence of arrhythmia.

Therefore, in the present study, we focused on regional changes in ROS generation in the myocardium with nonuniform contraction, investigating whether ROS is regionally increased within its stretched region and affects the propagation velocity of Ca²⁺ waves and the occurrence of arrhythmias. Our results indicate that in the myocardium with nonuniform contraction, passive stretch of the muscle by contractions of the neighboring muscle regionally increases ROS generation within the stretched region and exacerbates arrhythmias by increasing the velocity of Ca²⁺ waves.

Materials and methods (see expanded materials and methods in the Online Data Supplement)

Measurements of force, sarcomere length, membrane potential, [Ca²⁺]_i, and ROS

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 1996). All experimental protocols were approved by the Ethics Review Board of Tohoku University (approval reference number 2014-004, 2015-023). After rats had been adequately anesthetized, trabeculae were obtained from their right ventricles. Force, sarcomere length, membrane potential, and [Ca²⁺]_i were measured as previously described [23,24,25,26,27,28, 40]. To estimate regional changes in ROS, trabeculae were loaded with 2′,7′-dichlorofluorescein (DCF) as previously described [27, 28]. As shown in Fig. 1a, regional change in the DCF fluorescence (DCF_slope) was calculated at each pixel along the long axis of trabeculae using the DCF fluorescence images before and after exposure to a H₂O₂ jet or a blebbistatin jet, and the profile of DCF_slope along the trabeculae was then obtained. To create a nonuniform contraction model, trabeculae were regionally exposed to a jet of a solution containing 10 μmol/L blebbistatin, as previously described [25, 27, 40]. When a blebbistatin jet was used, measurements were performed a few minutes after the stoppage of the blebbistatin jet because blebbistatin has fluorescent properties [10, 11].

Fig. 1

a Analysis of 2′,7′-dichlorofluorescein (DCF) fluorescence images. DCF fluorescence images were recorded before (DCF_pre) and after (DCF_post) exposure to a H₂O₂ jet without stimulation or a blebbistatin jet with/without electrical stimulation (4 Hz for 30 s) (a). A region of interest (ROI; 50 × 720 pixels) was set along the long axis of a trabecula (T), and the profile of DCF fluorescence along the trabecula was calculated by vertically averaging the values of pixels within the ROI across the trabecula (b). To obtain the slope of the changes in the DCF fluorescence (DCF_slope) along the trabecula, the difference in the profile of DCF fluorescence between the DCF_pre and DCF_post was calculated pixel by pixel at the identical position along the trabecula and was divided by ΔTime. b Effect of a H₂O₂ jet on DCF fluorescence within trabeculae. Representative recordings of the profile of the DCF_slope along a trabecula with (light blue) and without (black) exposure to a 1 mmol/L H₂O₂ jet (a). The trabecula was exposed to the jet in the region of X. Y indicates the region 0.5 mm apart from X (Exp. 170529). Summary data concerning the effect of a H₂O₂ jet on the DCF_slope (n = 6) (b). Exposure to a H₂O₂ jet increased the DCF_slope within X compared to that within Y (right panel). #p < 0.01 vs Y

Experimental protocol with trabeculae

Ca²⁺ waves were induced by electrical stimulation (400-ms stimulus intervals for 7.5 s), and arrhythmias were induced by electrical stimulation (250-ms stimulus intervals for 15 s) in the presence of 0.2 μmol/L isoproterenol. All measurements were performed at 24 °C.

Statistics

All measurements were expressed as mean ± SEM. Statistical analysis was performed with a paired t test for two-group comparisons and one-way repeated-measures ANOVA with Tukey-Kramer for multiple comparisons when the data were normally distributed. Otherwise, the Wilcoxon signed-ranks test was used for two-group comparisons, unless otherwise mentioned. These analyses were performed using software for statistical analysis (Ekuseru-Toukei 2012, Social Survey Research Information Co., Ltd., Tokyo, Japan). Values of p < 0.05 were considered to be significant.

Results

Effect of a H₂O₂ jet on ROS generation

To confirm whether the DCF_slope calculated in the present study actually reflects regional changes in ROS generation, trabeculae were regionally exposed to a 1 mmol/L H₂O₂ jet. As shown in Fig. 1b, regional exposure to a H₂O₂ jet for 30 s increased the DCF_slope within the jet-exposed region (X) compared with that within the region 0.5 mm apart from the jet-exposed region (Y), whereas the DCF_slope showed no regional changes within trabeculae without exposure to a H₂O₂ jet. These results suggest that the DCF_slope within trabeculae reflects regional changes in ROS.

Effect of a blebbistatin jet on ROS generation

Regional exposure of trabeculae to a jet of a solution that reduces muscle contraction causes regional stretch within the jet-exposed region by contractions of the neighboring muscle during the contraction phase, as previously reported [27, 40]. As shown in Fig. 2a, the sarcomere was stretched within the region exposed to a 10 μmol/L blebbistatin jet (stretched region: X), whereas it was shortened within the region apart from the jet-exposed region (contracting region: Y) during the contraction phase, representing nonuniform contraction. Regional changes in the DCF fluorescence along the long axis of trabeculae were then recorded when trabeculae contracted nonuniformly in response to regional exposure to the 10 μmol/L blebbistatin jet. As shown in Fig. 2b, c (a), electrical stimulation for 30 s increased the DCF_slope within the region exposed to the blebbistatin jet (X) compared with that within the region 0.5 mm apart from the jet-exposed region (Y). This regional increase in the DCF_slope was not detected without electrical stimulation, as shown in Fig. 2c (b). Besides, this regional increase was not detected after superfusion with 3 μmol/L diphenyleneiodonium (DPI), NADPH oxidase inhibitor, for 1 h (Fig. 2c (b)). These results suggest that when cardiac muscle contracts nonuniformly, ROS is regionally increased within the stretched region, at least in part, due to the activation of NADPH oxidase.

Fig. 2

Regional effect of a blebbistatin jet on the DCF_slope. a The upper panel shows force, and the lower panel shows changes in sarcomere length (SL). The trabecula was exposed to a 10 μmol/L blebbistatin jet in the region of X. Y indicates the region apart from X. The sarcomere within X (red line) was stretched, while the sarcomere within Y (purple line) was shortened by electrical stimulation (ST; 2-s stimulus intervals, 0.7 mmol/L [Ca²⁺]_o; Exp. 151214). b Representative recordings of the profile of DCF_slope along a trabecula exposed to a 10 μmol/L blebbistatin jet in the absence (black line) and presence (red line) of 3 μmol/L DPI. X indicates the region exposed to a blebbistatin jet, and Y indicates the region 0.5 mm apart from X (Exp. 150521). c Summary data concerning the effect of a blebbistatin jet on the DCF_slope with 4 Hz electrical stimulation (n = 5) (a). Exposure to a blebbistatin jet increased the DCF_slope within X compared to that within Y with electrical stimulation. *p < 0.01 vs Y. Summary data concerning the effect of a blebbistatin jet on the DCF_slope without electrical stimulation (left panel) and that after superfusion with 3 μmol/L diphenyleneiodonium (DPI, n = 5, right panel) (b)

Roles of ROS within the stretched region in Ca²⁺ waves and arrhythmias

In trabeculae with nonuniform contraction, Ca²⁺ waves are initiated from the border zone between a contracting region and a stretched region due to Ca²⁺ dissociation from the myofilaments and propagate along trabeculae by Ca²⁺-induced Ca²⁺ release (CICR) from the SR, as previously reported [25, 27, 40]. To investigate whether ROS generation within the stretched region affects Ca²⁺ wave propagation and arrhythmias, we examined the effect of DPI on the propagation features of Ca²⁺ waves and the occurrence of arrhythmias in trabeculae exposed to a 10 μmol/L blebbistatin jet. As shown in Fig. 3a, electrical stimulation induced Ca²⁺ waves arising around the region exposed to a blebbistatin jet. Within the jet-exposed region, the peak [Ca²⁺]_i of the Ca²⁺ waves ([Ca²⁺]_CW) was higher than that within the region 0.4 mm apart from the jet-exposed region (Fig. 3b (a)). Besides, superfusion with DPI decreased the [Ca²⁺]_CW within the jet-exposed region and the velocity of Ca²⁺ waves (Fig. 3a, b (b)), suggesting that ROS generation within the stretched region enhances Ca²⁺ release from the SR induced by the Ca²⁺ dissociated from the myofilaments and increases the velocity of Ca²⁺ waves even outside the jet-exposed region. Concerning the occurrence of arrhythmias, we have previously reported that in the presence of isoproterenol, electrical stimulation induces arrhythmias due to acceleration of Ca²⁺ waves in the myocardium with nonuniform contraction [25, 26, 36]. Also in the present study, electrical stimulation induced arrhythmias in the presence of 0.2 μmol/L isoproterenol, as shown in Fig. 3c. Superfusion with DPI decreased the number of beats induced by electrical stimulation (Fig. 3c, d), suggesting that ROS generation within the stretched region is involved with the occurrence of arrhythmias.

Fig. 3

Effect of DPI on Ca²⁺ waves and arrhythmias in the myocardium exposed to a blebbistatin jet. 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 intervals for 7.5 s) and Ca²⁺ waves in the absence (left panels) and presence (right panels) of 3 μmol/L DPI. White arrows indicate the first Ca²⁺ waves. Yellow dotted lines a and b indicate the jet-exposed region and the region 0.4 mm apart from the jet-exposed region where the peaks of [Ca²⁺]_i of Ca²⁺ waves ([Ca²⁺]_CW) were calculated, respectively. In the left panel, Ca²⁺ waves appeared around the region exposed to a 10 μmol/L blebbistatin jet and propagated along the trabecula. In the right panel, the velocity of the Ca²⁺ wave was decreased in the presence of DPI (2.0 mmol/L [Ca²⁺]_o; Exp. 140528). b Summary data concerning the [Ca²⁺]_CW within the region indicated by lines a and b in the left panel of a. #p < 0.01 vs line a (a). Summary data concerning the effect of DPI on the [Ca²⁺]_CW within the region indicated by lines a in the panels of a (left panel) and the velocity of Ca²⁺ waves (right panel; n = 7). *p < 0.05 vs (–) (b). c Representative recordings of membrane potential (upper panels) and force (lower panels) after the last three electrical stimuli (ST; 250-ms stimulus intervals for 15 s) in the absence (left panels) and presence (right panels) of 3 μmol/L DPI in a trabecula exposed to a blebbistatin jet (2.0 mmol/L [Ca²⁺]_o, 0.2 μmol/L isoproterenol; Exp. 150129). d Summary data concerning the effect of DPI on the number of beats induced by electrical stimulation (n = 5; 2.0 ± 0.5 mmol/L [Ca²⁺]_o). *p < 0.05 vs (–)

In order to further examine whether ROS within the stretched region increased the velocity of Ca²⁺ waves outside the jet-exposed region and induced arrhythmias, we added H₂O₂ to a solution used for a blebbistatin jet. Electrical stimulation induced a Ca²⁺ wave arising around the region exposed to a 10 μmol/L blebbistatin jet (Fig. 4a). Addition of 0.2 mmol/L H₂O₂ to the blebbistatin jet increased the [Ca²⁺]_CW within the jet-exposed region and the velocity of the Ca²⁺ wave (Fig. 4a, b). Furthermore, addition of H₂O₂ increased the number of beats induced by electrical stimulation (Fig. 4c, d). Taken together, these results suggest that a regional increase in ROS within the stretched region enhances Ca²⁺ release from the SR within the region and that this enhanced Ca²⁺ release works as an enhanced initiator of CICR for propagation of Ca²⁺ waves and induces arrhythmias.

Fig. 4

Effect of a jet of a solution containing H₂O₂ in addition to blebbistatin on Ca²⁺ waves and arrhythmias. 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 intervals for 7.5 s) and a Ca²⁺ wave. The left panels show changes in a trabecula exposed to a 10 μmol/L blebbistatin jet, and the right panels show changes in the trabecula exposed to a jet of a solution containing 0.2 mmol/L H₂O₂ in addition to 10 μmol/L blebbistatin. White arrows indicate the first Ca²⁺ waves. In the left panel, a Ca²⁺ wave appeared around the jet-exposed region and propagated along the trabecula. In the right panel, the velocity of the Ca²⁺ wave was increased to 4.7 mm/s. Yellow dotted line a in both panels indicates the regions where the [Ca²⁺]_CW were calculated (2.0 mmol/L [Ca²⁺]_o; Exp. 151015). b Summary data concerning the effect of addition of H₂O₂ to the blebbistatin jet on the [Ca²⁺]_CW within the region indicated lines a in the panels of a (left panel) and the velocity of Ca²⁺ waves (right panel; n = 6). *p < 0.05 vs blebbistatin jet. c Representative recordings of membrane potential (upper panels) and force (lower panels) after the last three electrical stimuli (ST; 250-ms stimulus intervals for 15 s) before (left panels) and after (right panels) addition of 0.2 mmol/L H₂O₂ to the blebbistatin jet. In the right panels, addition of H₂O₂ to the blebbistatin jet increased the number of beats induced by electrical stimulation (2.0 mmol/L [Ca²⁺]_o, 0.2 μmol/L isoproterenol; Exp. 151026). d Summary data concerning the effect of addition of H₂O₂ to the blebbistatin jet on the number of beats induced by electrical stimulation (n = 6; 1.9 ± 0.1 mmol/L [Ca²⁺]_o). *p < 0.05 vs bleb jet

It is possible, however, that addition of H₂O₂ to the blebbistatin jet may have affected the regional contractile strength [12], thereby increasing the velocity of Ca²⁺ waves and the number of beats after electrical stimulation. We thus examined the effect of H₂O₂ on the developed force. The bath superfusate containing both 0.2 mmol/L H₂O₂ and 10 μM blebbistatin decreased the developed force to the level similar to that in the superfusate containing only 10 μmol/L blebbistatin (data not shown), meaning that the addition of H₂O₂ to a blebbistatin jet does not affect the contractile features within the stretched region.

Roles of ROS in Ca²⁺ wave propagation

Finally, to examine whether ROS affected the CICR mechanism, trabeculae were exposed to a 0.2 mmol/L H₂O₂ jet during propagation of Ca²⁺ waves. To minimize the effect of H₂O₂ on the contractile strength, the bath was superfused with a solution containing 10 μmol/L blebbistatin. This bath superfusion with blebbistatin decreased the force developed by electrical stimulation to 8.7 ± 1.4% of its initial value. To induce Ca²⁺ waves due to Ca²⁺ leak from the SR, trabeculae were exposed to a 10 mmol/L Ca²⁺ jet. As shown in Fig. 5a, electrical stimulation induced spontaneous increases in [Ca²⁺]_i (white arrowheads) within the jet-exposed region just before electrical stimulation and induced Ca²⁺ waves arising from the jet-exposed region after electrical stimulation. When a H₂O₂ jet was directed to the region where Ca²⁺ waves propagated, it increased the velocity of Ca²⁺ waves (Fig. 5a). Figure 5b shows the summary data. A H₂O₂ jet increased the velocity of Ca²⁺ waves, suggesting that ROS accelerates Ca²⁺ waves probably activating the CICR mechanism.

Fig. 5

Effect of a H₂O₂ jet on Ca²⁺ waves induced by a 10 mmol/L Ca²⁺ jet. 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 intervals for 7.5 s) in the absence (left panels) and presence (right panels) of a 0.2 mmol/L H₂O₂ jet. Muscle contractions were minimized by the bath superfusion with 10 μmol/L blebbistatin, and Ca²⁺ waves were induced by a 10 mmol/L Ca²⁺ jet. White arrows indicate the first Ca²⁺ waves, and white arrowheads indicate spontaneous increases in [Ca²⁺]_i just before electrical stimulation. In the left panel, Ca²⁺ waves appeared within the region exposed to the 10 mmol/L Ca²⁺ jet and propagated along the trabecula. In the right panel, exposure to the H₂O₂ jet increased the velocity of the first Ca²⁺ wave (2.0 mmol/L [Ca²⁺]_o; Exp. 140910). b Summary data concerning the effect of the 0.2 mmol/L H₂O₂ jet on the velocity of Ca²⁺ waves (n = 6). *p < 0.05 vs (–)

Discussion

The present study characterized the effect of regional muscle stretch on ROS generation, Ca²⁺ waves, and arrhythmias using the cardiac muscle model representing nonuniform contraction. To the best of our knowledge, it shows for the first time that in the myocardium with nonuniform contraction, passive stretch of the muscle by contractions of the neighboring muscle regionally generates ROS within the stretched region and that such regional ROS generation exacerbates arrhythmias by activating the propagation of Ca²⁺ waves, as discussed below.

Regional ROS generation within the stretched region

It has been reported that in cardiac muscle, stretch of the muscle increases ROS [27, 28, 32, 33], the frequency of Ca²⁺ sparks [18, 31, 33], and the velocity of Ca²⁺ waves [28]. In the present study, the DCF_slope was increased within the region stretched by contractions of the neighboring muscle during the contraction phase in trabeculae exposed to a blebbistatin jet (Fig. 2c (a)). We assume that this regional increase in the DCF_slope reflects a regional increase in ROS generation for the following reasons. First, the blebbistatin jet regionally increased the DCF_slope in the manner similar to the H₂O₂ jet (Fig. 1b). Second, the DCF_slope was not increased within the blebbistatin jet-exposed region after superfusion with DPI (Fig. 2c (b)), suggesting that the DCF_slope was increased due to the activation of NADPH oxidase although DPI inhibits the synthesis of both oxygen- and nitrogen-derived reactive species and many other flavoproteins depending on the concentration [1]. Third, the DCF_slope was measured a few minutes after the stoppage of a blebbistatin jet because blebbistatin has fluorescent properties by itself. Fourth, the DCF_slope was not increased within the blebbistatin jet-exposed region without electrical stimulation (Fig. 2c (b)). Thus, the results in the present study suggest that in the myocardium with nonuniform contraction, passive muscle stretch during the contraction phase regionally increases ROS within the stretched region, at least in part, due to the activation of NADPH oxidase.

Roles of ROS in Ca²⁺ waves and arrhythmias

ROS increases Ca²⁺ release from the SR [39, 43, 47] by oxidizing ryanodine receptors (RyRs) [6] or activating calcium/calmodulin-dependent protein kinase II (CaMKII) [8, 9]. It further increases the velocity of Ca²⁺ waves [18] and exacerbates arrhythmias [7, 19]. Likewise, H₂O₂ causes triggered arrhythmias [42] by directly activating RyRs [39] or by impairing Na⁺ current inactivation [35] through activation of CaMKII [39] or protein kinase C [41]. In addition, H₂O₂ changes force and Ca²⁺ transients [12] through the modulation of the Ca²⁺ current [15], HERG [3], and the sodium-calcium exchange current [14, 21].

As for the initiation mechanism of Ca²⁺ waves, two mechanisms have been proposed [27]. One is Ca²⁺ leak from the SR due to Ca²⁺ overload [23, 28], and the other is Ca²⁺ dissociation from the myofilaments in the myocardium with nonuniform contraction [25, 40]. In the latter mechanism, regional differences in contractile strength causes stretching of muscle by contractions of the more viable neighboring muscle. During the relaxation phase, Ca²⁺ is dissociated from the myofilaments due to the passive shortening and initiates Ca²⁺ waves from the border zone between the contracting and stretched region [40]. As for the propagation mechanism, CICR has been believed to underlie both the Ca²⁺ waves. In the present study, the blebbistatin jet caused nonuniform contraction (Fig. 2a) and induced Ca²⁺ waves from the jet-exposed region (Figs. 3a and 4a), suggesting that Ca²⁺ waves in Figs. 3a and 4a were initiated by Ca²⁺ dissociation from the myofilaments, while Ca²⁺ waves using a high Ca²⁺ jet in Fig. 5 were initiated by SR Ca²⁺ leak.

Concerning an increase in the propagation velocity of Ca²⁺ waves outside the jet-exposed region in Figs. 3 and 4, we assume that an increase in [Ca²⁺]_i due to ROS generation within the stretched region works as an enhanced initiator of CICR for propagation of Ca²⁺ waves for the following reasons. First, the [Ca²⁺]_CW within the stretched region was higher than that outside the blebbistatin jet-exposed region (Fig. 3b (a)). Second, superfusion with DPI decreased the [Ca²⁺]_CW within the jet-exposed region and the velocity of Ca²⁺ waves (Fig. 3b (b)), while addition of H₂O₂ to a blebbistatin jet increased them (Fig. 4b). Third, we have previously reported that the velocity of Ca²⁺ waves increases depending on the [Ca²⁺]_CW in trabeculae [23]. Fourth, we have also reported that the velocity of Ca²⁺ waves increases depending on the Ca²⁺ dissociated from the myofilaments within the jet-exposed region when trabeculae are shortened [24].

In the present study, superfusion with DPI decreased the number of beats after electrical stimulation (Fig. 3), and addition of H₂O₂ to a blebbistatin jet increased it (Fig. 4). Besides, as shown in Fig. 5, a H₂O₂ jet directed to the region where Ca²⁺ waves propagated increased the velocity of Ca²⁺ waves. Furthermore, we have previously reported that an increase in the velocity of Ca²⁺ waves enhances the amplitude of delayed after depolarizations and cause arrhythmias [36]. Taken together, these results suggest that in the myocardium with nonuniform contraction, an increase in ROS within the stretched region increases the velocity of Ca²⁺ waves by activating CICR [5, 43, 47] and thereby induces arrhythmias.

Clinical implications

In patients with heart failure and myocardial infarction, lethal arrhythmias frequently occur [29, 30]. Within such diseased hearts, impaired muscle is widely distributed, and thus, the hearts exhibit nonuniform contraction due to the regional difference in contractile strength. Results of the present study suggest that in patients with diseased hearts, stretch of the impaired muscle by contractions of the more viable neighboring muscle increases ROS, especially within the stretched region and that such an increase in ROS causes arrhythmias by activating Ca²⁺ waves, which is induced by the Ca²⁺ dissociated from the myofilaments due to the difference in contractile strength.

Study limitations

In diseased hearts, abnormal Ca²⁺ handling frequently occurs, especially within the impaired muscle, causing ROS generation [44] and arrhythmias [4, 38]. In the present study, however, the region showing stretch by exposure to a blebbistatin jet was not impaired but was just paralyzed.

Conclusion

In the myocardium with nonuniform contraction, passive stretch of the muscle by contractions of the neighboring muscle regionally increases ROS generation within the stretched region, and the regional ROS exacerbates arrhythmias by activating the propagation of Ca²⁺ waves.