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Possible contribution of the sarcoplasmic reticulum Ca2+ pump function to electrical and mechanical alternans
Journal of Electrocardiology Vol. 36 No. 2 2003
Possible Contribution of the Sarcoplasmic Reticulum Ca2ⴙ Pump Function to Electrical and Mechanical Alternans
Mikihiko Kameyama, MD, Yoshiyuki Hirayama, MD, PhD, Hirokazu Saitoh, MD, PhD, Mitsunori Maruyama, MD, Hirotsugu Atarashi, MD, PhD, and Teruo Takano, MD, PhD
Abstract: We investigated the role of the sarcoplasmic reticulum’s (SR) Ca2⫹ pump function of the in the mechanism of alternans. We recorded the surface ECG, monophasic action potential (MAP) and left ventricular pressure (LVP) in the canine beating heart. Alternans was induced with an abrupt shortening of the cycle length from 1000 to 350 ms. After the control studies, we administered propranolol or isoproterenol. In the presence of propranolol, we administered milrinone or 4,4⬘-diisothiocyanostilbene-2,2⬘-disulfonic acid (DIDS). In the presence of isoproterenol, we administered thapsigargin. Isoproterenol and milrinone attenuated both the electrical and mechanical alternans. Thapsigargin, a specific SR Ca2⫹ pump inhibitor, and propranolol magnified both types of alternans. DIDS, a Ca2⫹-activated Cl⫺ current (ICl(Ca)) inhibitor, attenuated the MAP alternans without an affect on the LVP alternans. Thus, the delayed intracellular Ca2⫹ cycling caused by the impaired SR Ca2⫹ pump function might produce electrical and mechanical alternans. -adrenergic stimulation eliminated these alternans. The ICl(Ca) contributed to the appearance of the electrical alternans. Key words: Alternans, arrhythmia, calcium, Ca2⫹ pump, sarcoplasmic reticulum (SR), Cl⫺ channel.
Electrical alternans can precipitate serious ventricular arrhythmias (1), and sensitive ECG processing techniques have been established to detect T wave alternans at the microvolt-level (2). Despite
the clinical recognition of the importance of T wave alternans, the mechanism of alternans is not yet fully understood. We proposed that a delayed intracellular Ca2⫹ cycling mediated by the sarcoplasmic reticulum (SR) plays an important role in the concomitant occurrence of electrical and mechanical alternans (3), but the precise mechanism of how the intracellular Ca2⫹ cycling is delayed has not been examined. We reported that a high stimulation frequency combined with a lowering of the blood temperature produced an increase in the magnitude of the alternans (3). It is reported that a reduced temperature decreases the Ca2⫹ ATPase activity of SR (4)
From the First Department of Internal Medicine, Nippon Medical School, Tokyo, Japan. Presented in part at the 66th Annual Scientific Meeting of the Japanese Circulation Society, Sapporo, Japan, April 24, 2002. Reprint requests: Yoshiyuki Hirayama, MD, PhD, First Department of Internal Medicine, Nippon Medical School, 1-1-5 Sendagi Bunkyo-ku, Tokyo 113-8603, Japan; e-mail: [email protected]. © 2003 Elsevier Inc. All rights reserved. 0022-0736/03/3602-0007$30.00/0 doi:10.1054/jelc.2003.50021
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126 Journal of Electrocardiology Vol. 36 No. 2 April 2003 and may inhibit the SR Ca2⫹ pump function. In the myocardium, the Ca2⫹ pump constitutes nearly 40% of the protein component of the SR (5). The Ca2⫹ pump of the SR reduces the cytosolic Ca2⫹ level and plays a central role in Ca2⫹ recycling (6). We hypothesized that the impaired Ca2⫹ pump function in the SR causes a delayed intracellular Ca2⫹ cycling, and thus produces alternans. Several authors have investigated the mechanism of alternans by using excised hearts or the Langendorf heart model (7). But these models have several potential limitations. For example, sympathetic stimulation is absent in isolated heart preparations. -adrenergic stimulation activates the ryanodine channels (8) and the Ca2⫹ pump function of the SR (5), and seems to accelerate the intracellular Ca2⫹ cycling. Our second hypothesis was that -adrenergic stimulation suppresses electrical and mechanical alternans. The transient outward currents play an important role in the action potential repolarization (9). The Ca2⫹ sensitive component of this current has been identified as the Ca2⫹-activated Cl⫺ current (ICl(Ca)) (10). The amplitude of the ICl(Ca) changes depending on the intracellular Ca2⫹ concentration ([Ca2⫹]i) (10), and there has been speculation by several authors on the importance of the ICl(Ca) in electrical alternans (3,11). However, this hypothesis remains unproven. To confirm these hypotheses and extend our understanding, we conducted the following experiments on a beating canine heart.
Materials and Methods In Vivo Canine Beating Heart We anesthetized 18 mongrel dogs of both sexes (weight, 20 to 30 Kg) with sodium pentobarbital 30 mg/kg, i.v., and made a midsternal incision in each dog. Supplemental pentobarbital (1–2 mg/kg i.v.) was given every 1 to 2 hours to maintain general anesthesia. The dogs were ventilated with a respirator (model SN-480, Shimano Inc., Tokyo, Japan). Each dog’s heart was suspended in a pericardial cradle and a complete atrioventricular block was induced with an injection of 40% formaldehyde through the right ventricular free wall into the bundle of His region. The arterial pH and PCO2 were measured periodically and maintained at normal levels by adjusting the ventilatory volume or frequency or by administering intravenous sodium bicarbonate. Electrical stimuli with a 2 ms pulse
duration and twice the diastolic threshold were delivered through bipolar platinum wire electrodes positioned in both the right atrial appendage and left ventricular apex to produce simultaneous atrioventricular pacing to avoid the effects of the atrial contraction on the alternans. Simultaneous Recording of ECG, Monophasic Action Potential, and Left Ventricular Pressure We used a suction electrode to record the monophasic action potential (MAP) by the technique described previously (3). In this experiment, we kept the negative pressure of the suction electrode between 25 and 30 mm Hg to minimize the myocardial cell damage from the combined effects of the pressure and resultant ischemia. The MAP configurations were checked to satisfy the MAP quality criteria described by Franz (12) before and after the recordings. The Left ventricular pressure (LVP) was also measured with a tip-manometer angiocatheter. We simultaneously recorded one MAP, ECG limb lead II and the LVP with a multichannel recorder (MP100 system; BIOPAC Systems, Inc, Gioleta, CA). These signals were digitized at 5 ms intervals with an on-line analog-to-digital converter, then displayed and stored on the hard drive of a computer. Monophasic action potential durations at 30% and 100% repolarization levels (MAPD30, MAPD100, respectively) were measured (12) with the investigator blinded to the pharmacological conditions under which the analysis recordings were obtained. The time constant of the isovolumic LV relaxation () was calculated from a semilogarhythmic plot of pressure against time using the first 40 ms after the peak negative dP/dt (13). Induction of Alternans We measured the surface temperature of the heart directly with a thermistor probe and lowered it to 32°C with ice packs on both the cervical and femoral regions (3). We paced the heart at a basic cycle length of 1000 ms for 30 seconds and abruptly changed the cycle length to 350 ms. The coupling interval of the first stimulus at a cycle length of 350 ms to the last stimulus at a cycle length of 1000 ms was fixed at 350 ms. The magnitude of the MAP or LVP alternans was defined as the difference between the 30th and 31st MAPD30 or LVP after changing the cycle length, respectively.
Role of the Ca2⫹ Pump Function in Alternans •
Serum Catecholamine Levels To evaluate the effect of the -adrenergic signaling pathway on the alternans, venous blood samples from the coronary sinus were obtained to measure the catecholamine levels during basic stimulation at 1 Hz and after rapid stimulation at a cycle length of 350 ms for 1 minute. Study Protocols After the control studies, we administered propranolol (with a loading dose of 0.1 mg/kg followed by a maintenance infusion of 0.02 mg/kg/h) in 12 experiments and isoproterenol (0.01 g/kg/min) in 6 experiments to investigate the effects of the -adrenergic stimulation on the alternans of the MAP and LVP. In the presence of propranolol, we additionally administered milrinone (with a loading dose of 5 g/kg/min for 10 minutes followed by a maintenance infusion of 0.75 g/kg/min) in 6 experiments to investigate the contribution of the cAMP-dependent phosphorylation pathway to the magnitude of the alternans. We hypothesized that an impaired Ca2⫹ pump function in the SR causes a delayed intracellular Ca2⫹ cycling and thus produces alternans. To confirm our hypothesis, we additionally administered the specific inhibitor of the SR Ca2⫹ pump, thapsigargin (10 g/kg), in the presence of isoproterenol in 6 experiments. We previously reported that electrical alternans was eliminated by verapamil and that the L-type Ca2⫹ current (ICa) contributed to the formation of the MAP alternans (3). Ca2⫹ entry through the L-type Ca2⫹ channel induces Ca2⫹ release from the SR, which activates several ion channels including the ICl(Ca). We hypothesized that the ICl(Ca) is another major current which forms the MAP alternans. To test this hypothesis, we additionally administered the ICl(Ca) inhibitor, 4,4⬘-diisothiocyanostilbene-2,2⬘-disulfonic acid (DIDS) (Sigma Chemical) (3.6 mg/kg) (10), in another 6 experiments in the presence of propranolol. The thapsigargin and DIDS were dissolved in 70% ethanol. This study conformed to the Guideline for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Statistical Analysis Results are presented as mean ⫾ SE, and the Mann-Whitney test was used for statistical analysis.
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A value of P ⬍ .05 was considered statistically significant.
Results Characteristics of Electrical and Mechanical Alternans Electrical and mechanical alternans occurred after an abrupt change in the pacing cycle length to 350 ms. Figure 1 shows some representative experiments. T wave alternans, MAP alternans, and LVP alternans occurred simultaneously. A stronger beat was associated with a shorter plateau of the MAP (ie, a short MAPD30), and a weaker beat was associated with a MAP with a longer plateau (ie, a longer MAPD30). These alternans were distinct for the first several beats, but gradually became obscure with rapid stimulation and disappeared at around the 50th beat in the controls. Effect of Abrupt Shortening of the Cycle Length on the Serum Catecholamine Levels So far, the mechanism of the gradual attenuation of electrical and mechanical alternans with rapid stimulation has remained largely uninvestigated, but it is important to examine the mechanism of this phenomenon in order to understand the mechanisms of alternans. In the beating heart, neurohumoral systems have profound effects on the cardiac electrophysiology. In Figure 1, the LVP was 125 mm Hg at the basic cycle length, but it decreased to ⬃100 mm Hg by an abrupt shortening of the pacing cycle length at around the 30th paced beat. After shortening the cycle length for 1 minute, the norepinephrine levels obtained from the coronary sinus significantly increased from 317 ⫾ 55 pg/mL to 507 ⫾ 56 pg/mL (P ⬍ .01, n ⫽ 6). To examine whether catecholamines modulate electrical and mechanical alternans, we performed the following experiments. Modulation of Electrical and Mechanical Alternans by the -adrenergic Signaling Pathway Because the effects of catecholamines on the heart are mediated by 1 receptors, we investigated whether the -receptor antagonist propranolol inhibited the gradual attenuation of alternans with
128 Journal of Electrocardiology Vol. 36 No. 2 April 2003
Fig. 1. Temporary alternans observed in the monophasic action potential (MAP) and left ventricular pressure (LVP) after a change in the cycle length from 1000 to 350 ms. The MAPD30 (the MAP duration at the 30% repolarization level) was measured as an index of the MAP configuration. T wave alternans was also noted.
mm Hg to 30 ⫾ 5 ms and 28 ⫾ 4 mm Hg, respectively (P ⬍ .01) (Table 1). Figure 2 shows the results of representative experiments showing the ECG, MAP, and LVP from the 30th to 32nd paced beat. Among the controls (Fig. 2A), MAP alternans and LVP alternans were observed. Figure 2B indicates that propranolol enhanced the alternans of the MAP and LVP. The
rapid stimulation. Propranolol caused no significant change in comparison with the control animals in respect to the MAPD30 or LVP, but it prolonged to 84 ⫾ 6 ms at a basic cycle length of 1000 ms (P ⬍ .01), indicating a decreased diastolic function (Table 1). In the presence of propranolol, the magnitude of the MAP alternans and that of the LVP alternans increased significantly, from 11 ⫾ 2 ms and 13 ⫾ 2
Table 1. Effect of Drugs on Left Ventricular Pressure, Monophasic Action Potential Duration at 30% Repolarization, ⫾dp/dt, Time Constant of Left Ventricular Pressure Decay, and Magnitude of Mechanical and Electrical Alternans Basic Cycle Length of 1000 ms Drug
n
LVP, mm Hg
MAPD30, ms
⫹dP/dt, mm Hg/s
⫺dP/dt, mm Hg/s
, ms
Control Isoproterenol (ISP) ISP ⫹ thapsigargin Propranolol Propranolol ⫹ Milrinone Propranolol ⫹ DIDS
18 6 6 12 6 6
113 ⫾ 5 127 ⫾ 8 107 ⫾ 7** 99 ⫾ 4 102 ⫾ 2 107 ⫾ 5
239 ⫾ 5 220 ⫾ 10 240 ⫾ 7 248 ⫾ 6 231 ⫾ 11 246 ⫾ 8
1607 ⫾ 117 2053 ⫾ 271 1648 ⫾ 181 1088 ⫾ 71† 2242 ⫾ 256# 1421 ⫾ 209
⫺1547 ⫾ 120 ⫺2312 ⫾ 279† ⫺1533 ⫾ 147** ⫺1123 ⫾ 113‡ ⫺1418 ⫾ 178 ⫺1249 ⫾ 70
60 ⫾ 3 46 ⫾ 2‡ 77 ⫾ 11* 84 ⫾ 6† 44 ⫾ 3§ 84 ⫾ 11
Magnitude of Alternans Drug
n
⌬LVP, mm Hg
⌬MAPD30, ms
Control Isoproterenol (ISP) ISP ⫹ thapsigargin Propranolol Propranolol ⫹ Milrinone Propranolol ⫹ DIDS
18 6 6 12 6 6
13 ⫾ 2 2 ⫾ 1† 19 ⫾ 1* 28 ⫾ 4† 1 ⫾ 0# 29 ⫾ 6¶
11 ⫾ 2 1 ⫾ 0† 24 ⫾ 7* 30 ⫾ 5† 1 ⫾ 1§ 5 ⫾ 2§
Values are mean ⫾ SE. LVP indicates the left ventricular pressure; MAPD30, the MAP duration at 30% repolarization; , the time constant of LVP decay; ⌬LVP, the magnitude of the LVP alternans; ⌬MAPD30, the magnitude of the MAPD30 alternans; and DIDS, 4,4⬘-diisothiocyanostilbene-2,2⬘-disulfonic acid. † P ⬍ 0.01, ‡ P ⬍ 0.05, vs Control; * P ⬍ 0.01, ** P ⬍ 0.05, vs ISP; and # P ⬍ 0.001, § P ⬍ 0.01, ¶ P ⬎ 0.9999, vs. Propranolol.
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Fig. 2. Effects of propranolol and milrinone on the alternans. (A) MAP alternans and LVP alternans in the controls. (B) Propranolol augmented the alternans. The magnitude of the MAP alternans and LVP alternans increased to 32 ms and 38 mm Hg, respectively. (C) Milrinone attenuated the electrical and mechanical alternans.
magnitude of the MAP alternans and LVP alternans increased to 32 ms and 38 mm Hg, respectively, and a distinct T wave alternans was observed during rapid stimulation. It is likely that propranolol bound to the 1 receptors and inhibited the response of the heart to the reflex increase in the sympathetic tone or in the catecholamines production during rapid stimulation. 1 receptor stimulation results in the activation of the cAMP-dependent phosphorylation pathway. To confirm the contribution of this signaling pathway to the attenuation of the alternans during rapid stimulation, we administered milrinone, a phophodiesterase III inhibitor, in the presence of propranolol. Compared with propranolol (Table 1), at a basic cycle length of 1000 ms in 6 experiments, milrinone shortened from 84 ⫾ 6 ms to 44 ⫾ 3 ms (P ⬍ .01); and it prevented the occurrence of MAP alternans and LVP alternans during rapid stimulation. The magnitudes of the MAP and LVP alternans decreased to 1 ⫾ 1 ms and 1 ⫾ 0 mm Hg, respectively (Table 1). Figure 2C shows a representative experiment. After we administered milrinone in the presence of propranolol, the T wave alternans, MAP alternans and LVP alternans were markedly attenuated. To further examine the contribution of the -adrenergic signaling pathway, we examined the effect of isoproterenol on the alternans. In comparison with the control, isoproterenol shortened the MAPD30 from 239 ⫾ 5 to 220 ⫾ 10 ms and
increased the LVP from 113 ⫾ 5 mm Hg to 127 ⫾ 8 mm Hg, but the difference was not statistically significant (Table 1). was significantly shortened from 60 ⫾ 3 ms to 46 ⫾ 2 ms (P ⬍ .05), indicating an increase in the diastolic function. The occurrence of electrical and mechanical alternans was significantly attenuated by isoproterenol. The magnitude of the MAP alternans and that of the LVP alternans were 1 ⫾ 0 ms and 2 ⫾ 1 mm Hg, respectively (P ⬍ .01) (Table 1). Figure 3B shows a representative experiment. After we administered isoproterenol, the magnitude of the MAP alternans and LVP alternans decreased to 1 ms and 1 mm Hg, respectively. Isoproterenol mimicked the suppressive effects of milrinone on the alternans. Effects of Thapsigargin on the Alternans in the Presence of Isoproterenol We administered thapsigargin in the presence of isoproterenol in 6 experiments. Compared with isoproterenol at a basic cycle length of 1000 ms, prolonged from 46 ⫾ 2 to 77 ⫾ 11 ms (P ⬍ .01), suggesting that thapsigargin inhibited the SR Ca2⫹ pump function (Table 1). In 6 experiments thapsigargin enhanced both the electrical and mechanical alternans, even in the presence of isoproterenol. In comparison with those after administration of isoproterenol alone, the magnitude of the MAP alternans and that of the LVP alternans increased signif-
130 Journal of Electrocardiology Vol. 36 No. 2 April 2003
Fig. 3. Effects of isoproterenol or thapsigargin on alternans. (A) MAP alternans and LVP alternans in the controls. (B) Isoproterenol significantly attenuated both alternans. (C) Additional administration of thapsigargin in the presence of isoproterenol augmented the electrical and mechanical alternans.
icantly from 1 ⫾ 0 ms and 2 ⫾ 1 mm Hg to 24 ⫾ 7 ms and 19 ⫾ 1 mm Hg, respectively (P ⬍ .01). Figure 3 shows a representative experiment. Under the control conditions, temporary alternans of the MAP and LVP was induced by rapid stimulation, but isoproterenol completely suppressed both the MAP and LVP alternans (Figs. 3A and B). However, both the electrical and mechanical alternans reappeared, and a distinct T wave alternans was observed after the administration of thapsigargin (Fig. 3C). Effects of DIDS on the Electrical Alternans To enhance the magnitude of alternans, the following experiments were performed in the presence of propranolol. After we used DIDS, an inhibitor of ICl(Ca) (10), the MAPD30 decreased from 248 ⫾ 6 ms to 246 ⫾ 8 ms, but the difference did not reach a level of statistical significance. The LVP and also did not change significantly. The magnitude of the MAP alternans markedly decreased, to 5 ⫾ 2 ms, without influencing the LVP alternans (P ⬍ .01) (Table 1). Figure 4 shows the results in a representative experiment. Compared to the controls (Fig. 4A), the MAP alternans and LVP alternans were enhanced by propranolol (Fig. 4B). The magnitude of the MAP alternans was 51 ms and the T wave alternans was easily discernable during
rapid stimulation. In a propranolol-treated heart, we administered DIDS (Fig. 4C). The magnitude of the MAP alternans markedly decreased to 4 ms. From those results we concluded that the ICl(Ca) contributes to the electrical alternans.
Discussion The major findings in this study were as follows: (1) The magnitude of the electrical and mechanical alternans was enhanced by thapsigargin and by a -blocker but were suppressed by isoproterenol and milrinone. (2) DIDS attenuated the magnitude of the MAP alternans without affecting the LVP alternans. We proposed the Ca2⫹ cycling hypothesis to explain the electrical and mechanical alternans (3). Our data supported this hypothesis and provided new insights into the cellular mechanisms of alternans. In the following paragraphs we will discuss the significance of the SR Ca2⫹ pump and ICl(Ca) on the alternans and their modulation by the -adrenergic signaling pathway revealed in the beating hearts. Ca2ⴙ Cycling Hypothesis Cytosolic Ca2⫹ released from the SR is taken up again by the Ca2⫹ pump of the longitudinal SR
Role of the Ca2⫹ Pump Function in Alternans •
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Fig. 4. Effects of 4,4⬘-diisothiocyanostilbene-2,2⬘disulfonic acid (DIDS) on alternans. (A) MAP alternans and LVP alternans in the controls. (B) Propranolol augmented alternans. (C) DIDS attenuated the electrical alternans without affecting the LVP alternans.
(uptake site) and transported to the junctional SR, where it can be released with some “delay” (release site), and is then released again by the next depolarization (Fig. 5A) (14). However, when the pacing cycle length is shortened more than the “delay,” the amount of Ca2⫹ transported to the release site becomes smaller, resulting in a lowering of the amount of Ca2⫹ released from the SR (Fig. 5B, left). The remaining large amount of Ca2⫹ in the process of uptake and transportation within the SR will reach the release site by the next stimulation, producing a greater Ca2⫹ release from the SR (Fig. 5B, right). Thus, rapid stimulation can cause alternate changes in the amount of Ca2⫹ released from the SR. We previously reported that the ICa is a major cause of the electrical alternans, since the MAP alternans probably results from cross-signaling between the L-type Ca2⫹ channels and the ryanodine receptors in the SR (3). A small increase in the [Ca2⫹]i potentiated the L-type Ca2⫹ channel activity within the same beat, which produced a lower LVP and a longer action potential duration. But a greater increase in the [Ca2⫹]i suppressed the Ltype Ca2⫹ channel activity, which raised the LVP and shortened the action potential duration (6,15). The Ca2⫹-sensitive transient outward current, identified as the ICl(Ca), seems to be another important current which produces the electrical alternans. The ICl(Ca) is activated by an increase in the [Ca2⫹]i associated with a Ca2⫹-induced Ca2⫹ release mech-
anism (10), and the amplitude of this current was increased in a [Ca2⫹]i-dependent manner (10). A large amount of Ca2⫹ transient tends to shorten the MAPD by increasing the ICl(Ca), and vice versa (Fig. 5B). Intracellular Ca2⫹ has been reported to modulate several other currents such as a delayed rectifier K⫹ current (IK) (16), Na⫹/Ca2⫹ exchange current and Ca2⫹-activated nonselective current, which have been suspected of causing the electrical alternans (11). However these currents appeared to play a minor role in our experimental conditions because the electrical alternans was markedly suppressed by DIDS. In support of this concept, Kawano and Hiraoka have reported that IK did not fluctuate during rapid stimulation (9). The Role of the -adrenergic Signaling Pathway in Protecting the Heart from Alternans during Heart Rate Acceleration In our experiment, the electrical and mechanical alternans were temporary (Fig. 1), and the plasma catecholamines increased with rapid stimulation. From this response of the temporary alternans to propranolol, milrinone, or isoproterenol, we concluded that the activation of the -adrenergic/ cAMP dependent phosphorylation pathway resulted in the attenuation of the electrical and mechanical alternans. In the beating heart, a de-
132 Journal of Electrocardiology Vol. 36 No. 2 April 2003 Fig. 5. The Ca2⫹ cycling hypothesis producing alternans. ICl(Ca) represents the Ca2⫹ activated Cl⫺ current; ICa, the L-type Ca2⫹ current; SR, the sarcoplasmic reticulum; PL, phospholamban; and P, phosphorylation. The white arrows represent ion currents and black arrows represent the electrical feedback to the ion channels. (A) Cytosolic Ca2⫹ released from the SR is taken up again by the SR Ca2⫹ pump (SERCA) and transported to the release site with a “delay,” and is then released again by the next depolarization. (B) When the pacing cycle length becomes shorter than the “delay,” the amount of the Ca2⫹ transported to the release site becomes smaller, resulting in a lower amount of Ca2⫹ released from the SR (left panel). The remaining large amounts of Ca2⫹ within the SR reach the release site by the next stimulation, producing a larger amount of Ca2⫹ released from the SR (right panel). (C) -adrenergic stimulation activates the Ca2⫹ pump and the ryanodine channels of the SR. The SR can become loaded and can release more Ca2⫹ during short diastolic intervals. (D) Thapsigargin decreased the Ca2⫹ pump function and induced the slowing of the Ca2⫹ recycling, resulting alternating changes of Ca2⫹ transient.
crease in the LVP after an abrupt shortening of the cycle length stimulates a reflex increase in the sympathetic tone and in the catecholamine production from the adrenal medulla, which result in the activation of the 1 receptors on the cardiac myocytes. -adrenergic stimulation can accelerate the Ca2⫹ recycling process and reduce the alternate changes in the Ca2⫹ released from the SR between beats, because the -adrenergic stimulation phosphorylates phospholamban to relieve the inhibition
of the SR Ca2⫹ pump (5) (Fig. 5C), and the SR can become loaded to release more Ca2⫹, in spite of the short diastolic intervals during rapid stimulation. -adrenergic stimulation phosphorylates the ryanodine channels and accelerates the Ca2⫹ release from the SR (8). The modulation of the alternans by the autonomic nervous system is controversial (17–19). Our data, obtained using intact, beating canine hearts, clearly indicated the importance of the sympathetic
Role of the Ca2⫹ Pump Function in Alternans •
stimulation in the inhibition of the development of alternans. -stimulation increases heart rate, which can induce alternans, thus it does not always inhibit alternans but sometimes induces it depending on the conditions. Impaired Ca2ⴙ Pump Function of the SR Causes Alternans While extensive studies to examine the mechanism of the alternans have been presented, this is the first report to show the importance of the SR Ca2⫹ pump function. Our results obtained by the enhancement of the electrical and mechanical alternans with thapsigargin indicate a possible mechanism that the impaired SR Ca2⫹ pump function causes the alternans. Thapsigargin decreased the Ca2⫹ pump activity of the SR, even in the presence of isoproterenol, and probably induced a slowing of the Ca2⫹ recycling process, which caused an alternating change in the amount of Ca2⫹ released from the SR and produced alternans (Fig. 5D). Thapsigargin has been well characterized as a specific inhibitor for the SR Ca2⫹-ATPase and delays the Ca2⫹ recycling process in cytosol (20,21). Kirby et al. (20) reported that 100 nM of thapsigargin decreased the magnitude of the Ca2⫹ transient by 80% (22). Thapsigargin does not affect the sarcolemmal Ca2⫹ pump, Na⫹-K⫹-ATPase or actomyosin ATPase activity (21), all of which could regulate the myocardial contractility. Moreover, thapsigargin does not affect the properties of the ionic membrane currents or the ryanodine channels in the SR (20 –22). Importance of the Restitution/Preceding Diastolic Interval on Alternans Restitution (23,24) and the preceding diastolic interval are very important for the MAPD. Gettes et al. reported that the amplitude and the rate-of-rise of the action potensial upstroke depended on the recovery of the inward currents (INa and ICa) and decreased with decreasing S1–S2 intervals (25). Saitoh H et al. examined the role of the memory and restitution in the process of the APD alternans induced by an abrupt shortening of the cycle length in the canine Purkinje and ventricular muscle fibers (26). They also found that the APD change during alternans was dependent on the preceding diastolic interval in the Purkinje fibers, but not in the ventricular muscle fibers. Furthermore, we previously reported that the MAPD30/100 was not dependent on the preceding diastolic interval in the
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intact canine beating heart even before reaching a steady state condition (3). In this experiment, the MAPD30 alternans was not always accompanied by MAPD100 alternans (ie, there can be no difference in the preceding diastolic interval between a long MAPD30 and a short MAPD30 during alternans). However, we could not deny the partial contribution of the preceding diastolic interval on the alternans in this experiment, especially when the preceding diastolic interval was less than 50 ms. Contribution of the Ryanodine Channel to the Alternans Some investigators have previously reported an effect of ryanodine on the alternans previously (27–30). Saitoh et al. (31) also reported that ryanodine (10 M) and caffeine (2 mM) completely suppressed both the electrical and mechanical alternans in the dog ventricular muscle fibers and that caffeine (10 mg/kg) completely suppressed both the electrical and mechanical alternans in intact beating heart (3). Caffeine initially enhances the release of Ca2⫹ from the SR and subsequently inhibits the Ca2⫹ uptake, thereby depleting this intracellular organelle of releasable Ca2⫹. Lessening fluctuations in the amount of Ca2⫹ released from the SR by ryanodine and caffeine may cause this concomitant suppression of both the MAP and LVP alternans. Thus, the Ca2⫹ release channel of the SR, ryanodine receptor, is supposed to play an important part in the mechanism of the alternans. One possible explanation is that slow recovery of the ryanodine receptor may cause intracellular Ca2⫹ oscillations between odd and even beats and thus produce the electrical and mechanical alternans. But again, thapsigargin is a specific inhibitor of the SR Ca2⫹ATPase, and does not affect the properties of the membrane ionic currents or the ryanodine channels in the SR (20 –22). However, we could not deny the possibility that an altered concentration of the intra-SR Ca2⫹ by thapsigargin may have change the recovery of the ryanodine receptor and thus produced the oscillations of the Ca2⫹ release from the SR between the odd and even beats. Clinical Implications In the failing human myocardium, expression of the genes encoding the Ca2⫹ uptake pump is impaired, and the rate of Ca2⫹ uptake in the SR is depressed (32,33). This may explain why T wave alternans was frequently observed in patients with contractile dysfunction (2,34). The pathological
134 Journal of Electrocardiology Vol. 36 No. 2 April 2003 conditions that produce the spatial heterogeneity of the Ca2⫹ pump activity as well as the membrane ionic properties between cells may facilitate the electrical gradient during alternans between neighboring cells and may form an electrophysiological substrate responsible for arrhythmias. Microvoltlevel T wave alternans associated with ventricular arrhythmias has been reported in the absence of structural heart disease (2,7,34). Positive results with microvolt-level T wave alternans may indicate the presence of subclinical heart disease in which the Ca2⫹ pump function of the SR is impaired.
Limitations The significance of the ICl(Ca) in producing the electrical alternans is demonstrated in the canine heart, but may not apply to human because the existence of the ICl(Ca) has not been demonstrated in the human ventricular myocytes. MAP recording using suction electrodes reflects action potentials in the epicardium of the heart. In this experiment, we kept the negative pressure of the suction electrodes between 25 and 30 mm Hg to minimize the myocardial cell damage by the combined effects of the pressure and resulting ischemia. We think this is the lowest pressure under which a stable MAP can be recorded. This experiment should have been conducted by using a motioncompensated holder (35) for the true intracellular potential recording. Extrapolation from this data to other regions including the mid-myocardium or the endocardium should be done with caution because electrical transmural heterogeneities have been reported (11). In the beating heart, alternating changes in the Ca2⫹ sensitivity of the myofilaments or in the end-diastolic pressure may influence LVP alternans (36), and further studies will be needed to clarify the importance of these factors.
Acknowledgment We thank Ms Nobuko Yoshida for her expert secretarial help. We also thank Mr John Martin for his linguistic assistance.
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Possible Contribution of the Sarcoplasmic Reticulum Ca2ⴙ Pump Function to Electrical and Mechanical Alternans
Mikihiko Kameyama, MD, Yoshiyuki Hirayama, MD, PhD, Hirokazu Saitoh, MD, PhD, Mitsunori Maruyama, MD, Hirotsugu Atarashi, MD, PhD, and Teruo Takano, MD, PhD
Abstract: We investigated the role of the sarcoplasmic reticulum’s (SR) Ca2⫹ pump function of the in the mechanism of alternans. We recorded the surface ECG, monophasic action potential (MAP) and left ventricular pressure (LVP) in the canine beating heart. Alternans was induced with an abrupt shortening of the cycle length from 1000 to 350 ms. After the control studies, we administered propranolol or isoproterenol. In the presence of propranolol, we administered milrinone or 4,4⬘-diisothiocyanostilbene-2,2⬘-disulfonic acid (DIDS). In the presence of isoproterenol, we administered thapsigargin. Isoproterenol and milrinone attenuated both the electrical and mechanical alternans. Thapsigargin, a specific SR Ca2⫹ pump inhibitor, and propranolol magnified both types of alternans. DIDS, a Ca2⫹-activated Cl⫺ current (ICl(Ca)) inhibitor, attenuated the MAP alternans without an affect on the LVP alternans. Thus, the delayed intracellular Ca2⫹ cycling caused by the impaired SR Ca2⫹ pump function might produce electrical and mechanical alternans. -adrenergic stimulation eliminated these alternans. The ICl(Ca) contributed to the appearance of the electrical alternans. Key words: Alternans, arrhythmia, calcium, Ca2⫹ pump, sarcoplasmic reticulum (SR), Cl⫺ channel.
Electrical alternans can precipitate serious ventricular arrhythmias (1), and sensitive ECG processing techniques have been established to detect T wave alternans at the microvolt-level (2). Despite
the clinical recognition of the importance of T wave alternans, the mechanism of alternans is not yet fully understood. We proposed that a delayed intracellular Ca2⫹ cycling mediated by the sarcoplasmic reticulum (SR) plays an important role in the concomitant occurrence of electrical and mechanical alternans (3), but the precise mechanism of how the intracellular Ca2⫹ cycling is delayed has not been examined. We reported that a high stimulation frequency combined with a lowering of the blood temperature produced an increase in the magnitude of the alternans (3). It is reported that a reduced temperature decreases the Ca2⫹ ATPase activity of SR (4)
From the First Department of Internal Medicine, Nippon Medical School, Tokyo, Japan. Presented in part at the 66th Annual Scientific Meeting of the Japanese Circulation Society, Sapporo, Japan, April 24, 2002. Reprint requests: Yoshiyuki Hirayama, MD, PhD, First Department of Internal Medicine, Nippon Medical School, 1-1-5 Sendagi Bunkyo-ku, Tokyo 113-8603, Japan; e-mail: [email protected]. © 2003 Elsevier Inc. All rights reserved. 0022-0736/03/3602-0007$30.00/0 doi:10.1054/jelc.2003.50021
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126 Journal of Electrocardiology Vol. 36 No. 2 April 2003 and may inhibit the SR Ca2⫹ pump function. In the myocardium, the Ca2⫹ pump constitutes nearly 40% of the protein component of the SR (5). The Ca2⫹ pump of the SR reduces the cytosolic Ca2⫹ level and plays a central role in Ca2⫹ recycling (6). We hypothesized that the impaired Ca2⫹ pump function in the SR causes a delayed intracellular Ca2⫹ cycling, and thus produces alternans. Several authors have investigated the mechanism of alternans by using excised hearts or the Langendorf heart model (7). But these models have several potential limitations. For example, sympathetic stimulation is absent in isolated heart preparations. -adrenergic stimulation activates the ryanodine channels (8) and the Ca2⫹ pump function of the SR (5), and seems to accelerate the intracellular Ca2⫹ cycling. Our second hypothesis was that -adrenergic stimulation suppresses electrical and mechanical alternans. The transient outward currents play an important role in the action potential repolarization (9). The Ca2⫹ sensitive component of this current has been identified as the Ca2⫹-activated Cl⫺ current (ICl(Ca)) (10). The amplitude of the ICl(Ca) changes depending on the intracellular Ca2⫹ concentration ([Ca2⫹]i) (10), and there has been speculation by several authors on the importance of the ICl(Ca) in electrical alternans (3,11). However, this hypothesis remains unproven. To confirm these hypotheses and extend our understanding, we conducted the following experiments on a beating canine heart.
Materials and Methods In Vivo Canine Beating Heart We anesthetized 18 mongrel dogs of both sexes (weight, 20 to 30 Kg) with sodium pentobarbital 30 mg/kg, i.v., and made a midsternal incision in each dog. Supplemental pentobarbital (1–2 mg/kg i.v.) was given every 1 to 2 hours to maintain general anesthesia. The dogs were ventilated with a respirator (model SN-480, Shimano Inc., Tokyo, Japan). Each dog’s heart was suspended in a pericardial cradle and a complete atrioventricular block was induced with an injection of 40% formaldehyde through the right ventricular free wall into the bundle of His region. The arterial pH and PCO2 were measured periodically and maintained at normal levels by adjusting the ventilatory volume or frequency or by administering intravenous sodium bicarbonate. Electrical stimuli with a 2 ms pulse
duration and twice the diastolic threshold were delivered through bipolar platinum wire electrodes positioned in both the right atrial appendage and left ventricular apex to produce simultaneous atrioventricular pacing to avoid the effects of the atrial contraction on the alternans. Simultaneous Recording of ECG, Monophasic Action Potential, and Left Ventricular Pressure We used a suction electrode to record the monophasic action potential (MAP) by the technique described previously (3). In this experiment, we kept the negative pressure of the suction electrode between 25 and 30 mm Hg to minimize the myocardial cell damage from the combined effects of the pressure and resultant ischemia. The MAP configurations were checked to satisfy the MAP quality criteria described by Franz (12) before and after the recordings. The Left ventricular pressure (LVP) was also measured with a tip-manometer angiocatheter. We simultaneously recorded one MAP, ECG limb lead II and the LVP with a multichannel recorder (MP100 system; BIOPAC Systems, Inc, Gioleta, CA). These signals were digitized at 5 ms intervals with an on-line analog-to-digital converter, then displayed and stored on the hard drive of a computer. Monophasic action potential durations at 30% and 100% repolarization levels (MAPD30, MAPD100, respectively) were measured (12) with the investigator blinded to the pharmacological conditions under which the analysis recordings were obtained. The time constant of the isovolumic LV relaxation () was calculated from a semilogarhythmic plot of pressure against time using the first 40 ms after the peak negative dP/dt (13). Induction of Alternans We measured the surface temperature of the heart directly with a thermistor probe and lowered it to 32°C with ice packs on both the cervical and femoral regions (3). We paced the heart at a basic cycle length of 1000 ms for 30 seconds and abruptly changed the cycle length to 350 ms. The coupling interval of the first stimulus at a cycle length of 350 ms to the last stimulus at a cycle length of 1000 ms was fixed at 350 ms. The magnitude of the MAP or LVP alternans was defined as the difference between the 30th and 31st MAPD30 or LVP after changing the cycle length, respectively.
Role of the Ca2⫹ Pump Function in Alternans •
Serum Catecholamine Levels To evaluate the effect of the -adrenergic signaling pathway on the alternans, venous blood samples from the coronary sinus were obtained to measure the catecholamine levels during basic stimulation at 1 Hz and after rapid stimulation at a cycle length of 350 ms for 1 minute. Study Protocols After the control studies, we administered propranolol (with a loading dose of 0.1 mg/kg followed by a maintenance infusion of 0.02 mg/kg/h) in 12 experiments and isoproterenol (0.01 g/kg/min) in 6 experiments to investigate the effects of the -adrenergic stimulation on the alternans of the MAP and LVP. In the presence of propranolol, we additionally administered milrinone (with a loading dose of 5 g/kg/min for 10 minutes followed by a maintenance infusion of 0.75 g/kg/min) in 6 experiments to investigate the contribution of the cAMP-dependent phosphorylation pathway to the magnitude of the alternans. We hypothesized that an impaired Ca2⫹ pump function in the SR causes a delayed intracellular Ca2⫹ cycling and thus produces alternans. To confirm our hypothesis, we additionally administered the specific inhibitor of the SR Ca2⫹ pump, thapsigargin (10 g/kg), in the presence of isoproterenol in 6 experiments. We previously reported that electrical alternans was eliminated by verapamil and that the L-type Ca2⫹ current (ICa) contributed to the formation of the MAP alternans (3). Ca2⫹ entry through the L-type Ca2⫹ channel induces Ca2⫹ release from the SR, which activates several ion channels including the ICl(Ca). We hypothesized that the ICl(Ca) is another major current which forms the MAP alternans. To test this hypothesis, we additionally administered the ICl(Ca) inhibitor, 4,4⬘-diisothiocyanostilbene-2,2⬘-disulfonic acid (DIDS) (Sigma Chemical) (3.6 mg/kg) (10), in another 6 experiments in the presence of propranolol. The thapsigargin and DIDS were dissolved in 70% ethanol. This study conformed to the Guideline for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Statistical Analysis Results are presented as mean ⫾ SE, and the Mann-Whitney test was used for statistical analysis.
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A value of P ⬍ .05 was considered statistically significant.
Results Characteristics of Electrical and Mechanical Alternans Electrical and mechanical alternans occurred after an abrupt change in the pacing cycle length to 350 ms. Figure 1 shows some representative experiments. T wave alternans, MAP alternans, and LVP alternans occurred simultaneously. A stronger beat was associated with a shorter plateau of the MAP (ie, a short MAPD30), and a weaker beat was associated with a MAP with a longer plateau (ie, a longer MAPD30). These alternans were distinct for the first several beats, but gradually became obscure with rapid stimulation and disappeared at around the 50th beat in the controls. Effect of Abrupt Shortening of the Cycle Length on the Serum Catecholamine Levels So far, the mechanism of the gradual attenuation of electrical and mechanical alternans with rapid stimulation has remained largely uninvestigated, but it is important to examine the mechanism of this phenomenon in order to understand the mechanisms of alternans. In the beating heart, neurohumoral systems have profound effects on the cardiac electrophysiology. In Figure 1, the LVP was 125 mm Hg at the basic cycle length, but it decreased to ⬃100 mm Hg by an abrupt shortening of the pacing cycle length at around the 30th paced beat. After shortening the cycle length for 1 minute, the norepinephrine levels obtained from the coronary sinus significantly increased from 317 ⫾ 55 pg/mL to 507 ⫾ 56 pg/mL (P ⬍ .01, n ⫽ 6). To examine whether catecholamines modulate electrical and mechanical alternans, we performed the following experiments. Modulation of Electrical and Mechanical Alternans by the -adrenergic Signaling Pathway Because the effects of catecholamines on the heart are mediated by 1 receptors, we investigated whether the -receptor antagonist propranolol inhibited the gradual attenuation of alternans with
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Fig. 1. Temporary alternans observed in the monophasic action potential (MAP) and left ventricular pressure (LVP) after a change in the cycle length from 1000 to 350 ms. The MAPD30 (the MAP duration at the 30% repolarization level) was measured as an index of the MAP configuration. T wave alternans was also noted.
mm Hg to 30 ⫾ 5 ms and 28 ⫾ 4 mm Hg, respectively (P ⬍ .01) (Table 1). Figure 2 shows the results of representative experiments showing the ECG, MAP, and LVP from the 30th to 32nd paced beat. Among the controls (Fig. 2A), MAP alternans and LVP alternans were observed. Figure 2B indicates that propranolol enhanced the alternans of the MAP and LVP. The
rapid stimulation. Propranolol caused no significant change in comparison with the control animals in respect to the MAPD30 or LVP, but it prolonged to 84 ⫾ 6 ms at a basic cycle length of 1000 ms (P ⬍ .01), indicating a decreased diastolic function (Table 1). In the presence of propranolol, the magnitude of the MAP alternans and that of the LVP alternans increased significantly, from 11 ⫾ 2 ms and 13 ⫾ 2
Table 1. Effect of Drugs on Left Ventricular Pressure, Monophasic Action Potential Duration at 30% Repolarization, ⫾dp/dt, Time Constant of Left Ventricular Pressure Decay, and Magnitude of Mechanical and Electrical Alternans Basic Cycle Length of 1000 ms Drug
n
LVP, mm Hg
MAPD30, ms
⫹dP/dt, mm Hg/s
⫺dP/dt, mm Hg/s
, ms
Control Isoproterenol (ISP) ISP ⫹ thapsigargin Propranolol Propranolol ⫹ Milrinone Propranolol ⫹ DIDS
18 6 6 12 6 6
113 ⫾ 5 127 ⫾ 8 107 ⫾ 7** 99 ⫾ 4 102 ⫾ 2 107 ⫾ 5
239 ⫾ 5 220 ⫾ 10 240 ⫾ 7 248 ⫾ 6 231 ⫾ 11 246 ⫾ 8
1607 ⫾ 117 2053 ⫾ 271 1648 ⫾ 181 1088 ⫾ 71† 2242 ⫾ 256# 1421 ⫾ 209
⫺1547 ⫾ 120 ⫺2312 ⫾ 279† ⫺1533 ⫾ 147** ⫺1123 ⫾ 113‡ ⫺1418 ⫾ 178 ⫺1249 ⫾ 70
60 ⫾ 3 46 ⫾ 2‡ 77 ⫾ 11* 84 ⫾ 6† 44 ⫾ 3§ 84 ⫾ 11
Magnitude of Alternans Drug
n
⌬LVP, mm Hg
⌬MAPD30, ms
Control Isoproterenol (ISP) ISP ⫹ thapsigargin Propranolol Propranolol ⫹ Milrinone Propranolol ⫹ DIDS
18 6 6 12 6 6
13 ⫾ 2 2 ⫾ 1† 19 ⫾ 1* 28 ⫾ 4† 1 ⫾ 0# 29 ⫾ 6¶
11 ⫾ 2 1 ⫾ 0† 24 ⫾ 7* 30 ⫾ 5† 1 ⫾ 1§ 5 ⫾ 2§
Values are mean ⫾ SE. LVP indicates the left ventricular pressure; MAPD30, the MAP duration at 30% repolarization; , the time constant of LVP decay; ⌬LVP, the magnitude of the LVP alternans; ⌬MAPD30, the magnitude of the MAPD30 alternans; and DIDS, 4,4⬘-diisothiocyanostilbene-2,2⬘-disulfonic acid. † P ⬍ 0.01, ‡ P ⬍ 0.05, vs Control; * P ⬍ 0.01, ** P ⬍ 0.05, vs ISP; and # P ⬍ 0.001, § P ⬍ 0.01, ¶ P ⬎ 0.9999, vs. Propranolol.
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Fig. 2. Effects of propranolol and milrinone on the alternans. (A) MAP alternans and LVP alternans in the controls. (B) Propranolol augmented the alternans. The magnitude of the MAP alternans and LVP alternans increased to 32 ms and 38 mm Hg, respectively. (C) Milrinone attenuated the electrical and mechanical alternans.
magnitude of the MAP alternans and LVP alternans increased to 32 ms and 38 mm Hg, respectively, and a distinct T wave alternans was observed during rapid stimulation. It is likely that propranolol bound to the 1 receptors and inhibited the response of the heart to the reflex increase in the sympathetic tone or in the catecholamines production during rapid stimulation. 1 receptor stimulation results in the activation of the cAMP-dependent phosphorylation pathway. To confirm the contribution of this signaling pathway to the attenuation of the alternans during rapid stimulation, we administered milrinone, a phophodiesterase III inhibitor, in the presence of propranolol. Compared with propranolol (Table 1), at a basic cycle length of 1000 ms in 6 experiments, milrinone shortened from 84 ⫾ 6 ms to 44 ⫾ 3 ms (P ⬍ .01); and it prevented the occurrence of MAP alternans and LVP alternans during rapid stimulation. The magnitudes of the MAP and LVP alternans decreased to 1 ⫾ 1 ms and 1 ⫾ 0 mm Hg, respectively (Table 1). Figure 2C shows a representative experiment. After we administered milrinone in the presence of propranolol, the T wave alternans, MAP alternans and LVP alternans were markedly attenuated. To further examine the contribution of the -adrenergic signaling pathway, we examined the effect of isoproterenol on the alternans. In comparison with the control, isoproterenol shortened the MAPD30 from 239 ⫾ 5 to 220 ⫾ 10 ms and
increased the LVP from 113 ⫾ 5 mm Hg to 127 ⫾ 8 mm Hg, but the difference was not statistically significant (Table 1). was significantly shortened from 60 ⫾ 3 ms to 46 ⫾ 2 ms (P ⬍ .05), indicating an increase in the diastolic function. The occurrence of electrical and mechanical alternans was significantly attenuated by isoproterenol. The magnitude of the MAP alternans and that of the LVP alternans were 1 ⫾ 0 ms and 2 ⫾ 1 mm Hg, respectively (P ⬍ .01) (Table 1). Figure 3B shows a representative experiment. After we administered isoproterenol, the magnitude of the MAP alternans and LVP alternans decreased to 1 ms and 1 mm Hg, respectively. Isoproterenol mimicked the suppressive effects of milrinone on the alternans. Effects of Thapsigargin on the Alternans in the Presence of Isoproterenol We administered thapsigargin in the presence of isoproterenol in 6 experiments. Compared with isoproterenol at a basic cycle length of 1000 ms, prolonged from 46 ⫾ 2 to 77 ⫾ 11 ms (P ⬍ .01), suggesting that thapsigargin inhibited the SR Ca2⫹ pump function (Table 1). In 6 experiments thapsigargin enhanced both the electrical and mechanical alternans, even in the presence of isoproterenol. In comparison with those after administration of isoproterenol alone, the magnitude of the MAP alternans and that of the LVP alternans increased signif-
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Fig. 3. Effects of isoproterenol or thapsigargin on alternans. (A) MAP alternans and LVP alternans in the controls. (B) Isoproterenol significantly attenuated both alternans. (C) Additional administration of thapsigargin in the presence of isoproterenol augmented the electrical and mechanical alternans.
icantly from 1 ⫾ 0 ms and 2 ⫾ 1 mm Hg to 24 ⫾ 7 ms and 19 ⫾ 1 mm Hg, respectively (P ⬍ .01). Figure 3 shows a representative experiment. Under the control conditions, temporary alternans of the MAP and LVP was induced by rapid stimulation, but isoproterenol completely suppressed both the MAP and LVP alternans (Figs. 3A and B). However, both the electrical and mechanical alternans reappeared, and a distinct T wave alternans was observed after the administration of thapsigargin (Fig. 3C). Effects of DIDS on the Electrical Alternans To enhance the magnitude of alternans, the following experiments were performed in the presence of propranolol. After we used DIDS, an inhibitor of ICl(Ca) (10), the MAPD30 decreased from 248 ⫾ 6 ms to 246 ⫾ 8 ms, but the difference did not reach a level of statistical significance. The LVP and also did not change significantly. The magnitude of the MAP alternans markedly decreased, to 5 ⫾ 2 ms, without influencing the LVP alternans (P ⬍ .01) (Table 1). Figure 4 shows the results in a representative experiment. Compared to the controls (Fig. 4A), the MAP alternans and LVP alternans were enhanced by propranolol (Fig. 4B). The magnitude of the MAP alternans was 51 ms and the T wave alternans was easily discernable during
rapid stimulation. In a propranolol-treated heart, we administered DIDS (Fig. 4C). The magnitude of the MAP alternans markedly decreased to 4 ms. From those results we concluded that the ICl(Ca) contributes to the electrical alternans.
Discussion The major findings in this study were as follows: (1) The magnitude of the electrical and mechanical alternans was enhanced by thapsigargin and by a -blocker but were suppressed by isoproterenol and milrinone. (2) DIDS attenuated the magnitude of the MAP alternans without affecting the LVP alternans. We proposed the Ca2⫹ cycling hypothesis to explain the electrical and mechanical alternans (3). Our data supported this hypothesis and provided new insights into the cellular mechanisms of alternans. In the following paragraphs we will discuss the significance of the SR Ca2⫹ pump and ICl(Ca) on the alternans and their modulation by the -adrenergic signaling pathway revealed in the beating hearts. Ca2ⴙ Cycling Hypothesis Cytosolic Ca2⫹ released from the SR is taken up again by the Ca2⫹ pump of the longitudinal SR
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Kameyama et al. 131
Fig. 4. Effects of 4,4⬘-diisothiocyanostilbene-2,2⬘disulfonic acid (DIDS) on alternans. (A) MAP alternans and LVP alternans in the controls. (B) Propranolol augmented alternans. (C) DIDS attenuated the electrical alternans without affecting the LVP alternans.
(uptake site) and transported to the junctional SR, where it can be released with some “delay” (release site), and is then released again by the next depolarization (Fig. 5A) (14). However, when the pacing cycle length is shortened more than the “delay,” the amount of Ca2⫹ transported to the release site becomes smaller, resulting in a lowering of the amount of Ca2⫹ released from the SR (Fig. 5B, left). The remaining large amount of Ca2⫹ in the process of uptake and transportation within the SR will reach the release site by the next stimulation, producing a greater Ca2⫹ release from the SR (Fig. 5B, right). Thus, rapid stimulation can cause alternate changes in the amount of Ca2⫹ released from the SR. We previously reported that the ICa is a major cause of the electrical alternans, since the MAP alternans probably results from cross-signaling between the L-type Ca2⫹ channels and the ryanodine receptors in the SR (3). A small increase in the [Ca2⫹]i potentiated the L-type Ca2⫹ channel activity within the same beat, which produced a lower LVP and a longer action potential duration. But a greater increase in the [Ca2⫹]i suppressed the Ltype Ca2⫹ channel activity, which raised the LVP and shortened the action potential duration (6,15). The Ca2⫹-sensitive transient outward current, identified as the ICl(Ca), seems to be another important current which produces the electrical alternans. The ICl(Ca) is activated by an increase in the [Ca2⫹]i associated with a Ca2⫹-induced Ca2⫹ release mech-
anism (10), and the amplitude of this current was increased in a [Ca2⫹]i-dependent manner (10). A large amount of Ca2⫹ transient tends to shorten the MAPD by increasing the ICl(Ca), and vice versa (Fig. 5B). Intracellular Ca2⫹ has been reported to modulate several other currents such as a delayed rectifier K⫹ current (IK) (16), Na⫹/Ca2⫹ exchange current and Ca2⫹-activated nonselective current, which have been suspected of causing the electrical alternans (11). However these currents appeared to play a minor role in our experimental conditions because the electrical alternans was markedly suppressed by DIDS. In support of this concept, Kawano and Hiraoka have reported that IK did not fluctuate during rapid stimulation (9). The Role of the -adrenergic Signaling Pathway in Protecting the Heart from Alternans during Heart Rate Acceleration In our experiment, the electrical and mechanical alternans were temporary (Fig. 1), and the plasma catecholamines increased with rapid stimulation. From this response of the temporary alternans to propranolol, milrinone, or isoproterenol, we concluded that the activation of the -adrenergic/ cAMP dependent phosphorylation pathway resulted in the attenuation of the electrical and mechanical alternans. In the beating heart, a de-
132 Journal of Electrocardiology Vol. 36 No. 2 April 2003 Fig. 5. The Ca2⫹ cycling hypothesis producing alternans. ICl(Ca) represents the Ca2⫹ activated Cl⫺ current; ICa, the L-type Ca2⫹ current; SR, the sarcoplasmic reticulum; PL, phospholamban; and P, phosphorylation. The white arrows represent ion currents and black arrows represent the electrical feedback to the ion channels. (A) Cytosolic Ca2⫹ released from the SR is taken up again by the SR Ca2⫹ pump (SERCA) and transported to the release site with a “delay,” and is then released again by the next depolarization. (B) When the pacing cycle length becomes shorter than the “delay,” the amount of the Ca2⫹ transported to the release site becomes smaller, resulting in a lower amount of Ca2⫹ released from the SR (left panel). The remaining large amounts of Ca2⫹ within the SR reach the release site by the next stimulation, producing a larger amount of Ca2⫹ released from the SR (right panel). (C) -adrenergic stimulation activates the Ca2⫹ pump and the ryanodine channels of the SR. The SR can become loaded and can release more Ca2⫹ during short diastolic intervals. (D) Thapsigargin decreased the Ca2⫹ pump function and induced the slowing of the Ca2⫹ recycling, resulting alternating changes of Ca2⫹ transient.
crease in the LVP after an abrupt shortening of the cycle length stimulates a reflex increase in the sympathetic tone and in the catecholamine production from the adrenal medulla, which result in the activation of the 1 receptors on the cardiac myocytes. -adrenergic stimulation can accelerate the Ca2⫹ recycling process and reduce the alternate changes in the Ca2⫹ released from the SR between beats, because the -adrenergic stimulation phosphorylates phospholamban to relieve the inhibition
of the SR Ca2⫹ pump (5) (Fig. 5C), and the SR can become loaded to release more Ca2⫹, in spite of the short diastolic intervals during rapid stimulation. -adrenergic stimulation phosphorylates the ryanodine channels and accelerates the Ca2⫹ release from the SR (8). The modulation of the alternans by the autonomic nervous system is controversial (17–19). Our data, obtained using intact, beating canine hearts, clearly indicated the importance of the sympathetic
Role of the Ca2⫹ Pump Function in Alternans •
stimulation in the inhibition of the development of alternans. -stimulation increases heart rate, which can induce alternans, thus it does not always inhibit alternans but sometimes induces it depending on the conditions. Impaired Ca2ⴙ Pump Function of the SR Causes Alternans While extensive studies to examine the mechanism of the alternans have been presented, this is the first report to show the importance of the SR Ca2⫹ pump function. Our results obtained by the enhancement of the electrical and mechanical alternans with thapsigargin indicate a possible mechanism that the impaired SR Ca2⫹ pump function causes the alternans. Thapsigargin decreased the Ca2⫹ pump activity of the SR, even in the presence of isoproterenol, and probably induced a slowing of the Ca2⫹ recycling process, which caused an alternating change in the amount of Ca2⫹ released from the SR and produced alternans (Fig. 5D). Thapsigargin has been well characterized as a specific inhibitor for the SR Ca2⫹-ATPase and delays the Ca2⫹ recycling process in cytosol (20,21). Kirby et al. (20) reported that 100 nM of thapsigargin decreased the magnitude of the Ca2⫹ transient by 80% (22). Thapsigargin does not affect the sarcolemmal Ca2⫹ pump, Na⫹-K⫹-ATPase or actomyosin ATPase activity (21), all of which could regulate the myocardial contractility. Moreover, thapsigargin does not affect the properties of the ionic membrane currents or the ryanodine channels in the SR (20 –22). Importance of the Restitution/Preceding Diastolic Interval on Alternans Restitution (23,24) and the preceding diastolic interval are very important for the MAPD. Gettes et al. reported that the amplitude and the rate-of-rise of the action potensial upstroke depended on the recovery of the inward currents (INa and ICa) and decreased with decreasing S1–S2 intervals (25). Saitoh H et al. examined the role of the memory and restitution in the process of the APD alternans induced by an abrupt shortening of the cycle length in the canine Purkinje and ventricular muscle fibers (26). They also found that the APD change during alternans was dependent on the preceding diastolic interval in the Purkinje fibers, but not in the ventricular muscle fibers. Furthermore, we previously reported that the MAPD30/100 was not dependent on the preceding diastolic interval in the
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intact canine beating heart even before reaching a steady state condition (3). In this experiment, the MAPD30 alternans was not always accompanied by MAPD100 alternans (ie, there can be no difference in the preceding diastolic interval between a long MAPD30 and a short MAPD30 during alternans). However, we could not deny the partial contribution of the preceding diastolic interval on the alternans in this experiment, especially when the preceding diastolic interval was less than 50 ms. Contribution of the Ryanodine Channel to the Alternans Some investigators have previously reported an effect of ryanodine on the alternans previously (27–30). Saitoh et al. (31) also reported that ryanodine (10 M) and caffeine (2 mM) completely suppressed both the electrical and mechanical alternans in the dog ventricular muscle fibers and that caffeine (10 mg/kg) completely suppressed both the electrical and mechanical alternans in intact beating heart (3). Caffeine initially enhances the release of Ca2⫹ from the SR and subsequently inhibits the Ca2⫹ uptake, thereby depleting this intracellular organelle of releasable Ca2⫹. Lessening fluctuations in the amount of Ca2⫹ released from the SR by ryanodine and caffeine may cause this concomitant suppression of both the MAP and LVP alternans. Thus, the Ca2⫹ release channel of the SR, ryanodine receptor, is supposed to play an important part in the mechanism of the alternans. One possible explanation is that slow recovery of the ryanodine receptor may cause intracellular Ca2⫹ oscillations between odd and even beats and thus produce the electrical and mechanical alternans. But again, thapsigargin is a specific inhibitor of the SR Ca2⫹ATPase, and does not affect the properties of the membrane ionic currents or the ryanodine channels in the SR (20 –22). However, we could not deny the possibility that an altered concentration of the intra-SR Ca2⫹ by thapsigargin may have change the recovery of the ryanodine receptor and thus produced the oscillations of the Ca2⫹ release from the SR between the odd and even beats. Clinical Implications In the failing human myocardium, expression of the genes encoding the Ca2⫹ uptake pump is impaired, and the rate of Ca2⫹ uptake in the SR is depressed (32,33). This may explain why T wave alternans was frequently observed in patients with contractile dysfunction (2,34). The pathological
134 Journal of Electrocardiology Vol. 36 No. 2 April 2003 conditions that produce the spatial heterogeneity of the Ca2⫹ pump activity as well as the membrane ionic properties between cells may facilitate the electrical gradient during alternans between neighboring cells and may form an electrophysiological substrate responsible for arrhythmias. Microvoltlevel T wave alternans associated with ventricular arrhythmias has been reported in the absence of structural heart disease (2,7,34). Positive results with microvolt-level T wave alternans may indicate the presence of subclinical heart disease in which the Ca2⫹ pump function of the SR is impaired.
Limitations The significance of the ICl(Ca) in producing the electrical alternans is demonstrated in the canine heart, but may not apply to human because the existence of the ICl(Ca) has not been demonstrated in the human ventricular myocytes. MAP recording using suction electrodes reflects action potentials in the epicardium of the heart. In this experiment, we kept the negative pressure of the suction electrodes between 25 and 30 mm Hg to minimize the myocardial cell damage by the combined effects of the pressure and resulting ischemia. We think this is the lowest pressure under which a stable MAP can be recorded. This experiment should have been conducted by using a motioncompensated holder (35) for the true intracellular potential recording. Extrapolation from this data to other regions including the mid-myocardium or the endocardium should be done with caution because electrical transmural heterogeneities have been reported (11). In the beating heart, alternating changes in the Ca2⫹ sensitivity of the myofilaments or in the end-diastolic pressure may influence LVP alternans (36), and further studies will be needed to clarify the importance of these factors.
Acknowledgment We thank Ms Nobuko Yoshida for her expert secretarial help. We also thank Mr John Martin for his linguistic assistance.
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