Effects of Dantrolene on Arrhythmogenicity in Isolated Regional Ischemia-Reperfusion Rabbit Hearts with or without Pacing-Induced Heart Failure

Abstract

Dantrolene was reported to suppress ventricular fibrillation (VF) in failing hearts with acute myocardial infarction, but its antiarrhythmic efficacy in regional ischemia-reperfusion (IR) hearts remains debatable. Heart failure (HF) was induced by right ventricular pacing. The IR rabbit model was created by coronary artery ligation for 30 min, followed by reperfusion for 15 min in vivo in both HF and non-HF groups (n = 9 in each group). Simultaneous voltage and intracellular Ca2+ (Cai) optical mapping was then performed in isolated Langendorff-perfused hearts. Electrophysiological studies were conducted and VF inducibility was evaluated by dynamic pacing. Dantrolene (10 μM) was administered after baseline studies. The HF group had a higher VF inducibility than the control group. Dantrolene had both antiarrhythmic (prolonged action potential duration (APD) and effective refractory period) and proarrhythmic effects (slowed conduction velocity, steepened APD restitution slope, and enhanced arrhythmogenic alternans induction) but had no significant effects on ventricular premature beat (VPB) suppression and VF inducibility in both groups. A higher VF conversion rate in the non-HF group was likely due to greater APD prolonging effects in smaller hearts compared to the HF group. The lack of significant effects on VPB suppression by dantrolene suggests that triggered activity might not be the dominant mechanism responsible for VPB induction in the IR model.

Chung-Chuan Chou 1,2 and Hui-Ling Lee 3 and Po-Cheng Chang 1,2 and Hung-Ta Wo 1 and Ming-Shien Wen 1,2 and San-Jou Yeh 1,2

Academic Editor:Mohamed Boutjdir

1, Division of Cardiology, Department of Medicine, Chang Gung Memorial Hospital, Linkou District, Taiwan
2, Chang Gung University College of Medicine, Taoyuan City, Taiwan
3, Department of Anesthesia, Chang Gung Memorial Hospital, Taipei, Taiwan

Received 10 October 2014; Revised 29 December 2014; Accepted 5 January 2015; 19 February 2015

This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1. Introduction

Reperfusion therapy by thrombolysis or immediate percutaneous coronary intervention is commonly performed for acute myocardial infarction (AMI). However, fatal arrhythmia may occur not only during ischemia but also during reperfusion. It has been reported that intracellular Ca²⁺ (Ca_i ) overload is involved in the development of fatal arrhythmias postischemia-reperfusion (IR) injury [1, 2]. The elevated Ca_i in IR myocardium may enhance triggered activity [3], thereby contributing to IR arrhythmias [4]. Both IR and heart failure (HF) impair sarcoplasmic reticulum (SR) Ca²⁺ ATPase pump activity [5, 6], which results in decreased SR Ca²⁺ load and elevated diastolic Ca²⁺ to facilitate afterdepolarizations induction and triggered activity [7]. It is reasonable to speculate that prevention of spontaneous SR Ca²⁺ leak may improve Ca_i homeostasis and reduce IR arrhythmias, especially in HF. Dantrolene, a stabilizer of skeletal muscle ryanodine receptor-1, has been reported to suppress diastolic Ca²⁺ leakage by stabilizing ryanodine receptor-2 of failing cardiomyocytes [8]. We previously reported that dantrolene is effective in preventing ventricular fibrillation (VF) storm by suppressing ventricular premature beats (VPBs) in isolated failing rabbit hearts with AMI [9]. Ortiz et al. reported that dantrolene protects the myocardium against myocardial stunning and IR arrhythmias in Wistar rat hearts [10]; however, other studies have reported that dantrolene has no significant effects on IR arrhythmia in canine hearts [11] and on IR injury in rabbits in vivo [12]. Thus, the effect of dantrolene on arrhythmogenesis in IR hearts remains debatable. The purpose of this study was to examine whether dantrolene has a primary antiarrhythmic effect and the mode of its action in isolated IR rabbit hearts with or without pacing-induced HF. We performed simultaneous membrane voltage ( [figure omitted; refer to PDF] ) and Ca_i mapping to investigate the electrophysiological and Ca_i changes during dantrolene infusion in Langendorff-perfused rabbit hearts with IR injury.

2. Materials and Methods

The research protocol was approved by the Institutional Animal Care and Use Committee of Chang Gung Memorial Hospital (approval number 2009092201) and conformed to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health. Twenty-two adult New Zealand white rabbits (2.8-4.2 kg) were used in the study. Two rabbits died within 4 weeks of pacing initiation, and the hearts from the remaining 20 rabbits (11 with pacing-induced HF (the HF group) and 9 without pacemaker implantation (non-HF group)) were harvested for IR induction. Two rabbits in the HF group developed VF storm during the ischemia period and were not used for optical mapping studies. Left ventricular (LV) function was assessed by echocardiography at baseline and after 4 weeks of pacing.

2.1. Pacing-Induced HF and IR Model Creation

Rapid right ventricular pacing was used to induce HF as described previously [9]. The IR model was created by the same method used in our previous study [13]. Briefly, the rabbits were premedicated with intramuscular injection of ketamine (35 mg/kg) and xylazine (5 mg/kg), intubated and anesthetized with isoflurane. When the rabbits were fully anesthetized and unresponsive to physical stimuli, the chests were opened via a left thoracotomy. An obtuse marginal branch of the left circumflex artery was then ligated halfway between the atrioventricular groove and the cardiac apex for 30 minutes, followed by reperfusion. The limb lead electrocardiogram (ECG) was monitored continuously.

2.2. Optical Mapping of Isolated Rabbit Hearts

After reperfusion for 15 minutes, the hearts were excised and Langendorff-perfused with 37°C Tyrode's solution (in mmol/L: NaCl 125, KCl 4.5, MgCl₂ 0.25, NaHCO₃ 24, NaH₂ PO₄ 1.8, CaCl₂ 1.8, glucose 5.5, and albumin 50 mg/L) and equilibrated with 95% O₂ and 5% CO₂ to maintain a pH of 7.4. The coronary perfusion pressure was regulated and maintained at 70-80 cmH₂ O. The hearts were stained with [figure omitted; refer to PDF] (Molecular Probes, Eugene, OR, USA) for Ca_i and RH237 (Molecular Probes) for [figure omitted; refer to PDF] mapping. The double-stained hearts were illuminated with a laser at 532 nm wavelength. The emitted fluorescence was filtered and acquired simultaneously with two charge-coupled device cameras (CA-D1-0128T; Dalsa Inc., Billerica, MA, USA) at 269 frames/second. Digital images (128 × 128 pixels) were gathered from the epicardium of the LV (25 × 25 mm² area), resulting in a spatial resolution of 0.2 × 0.2 mm² per pixel. Motion artifacts were suppressed by 5 μ M cytochalasin [figure omitted; refer to PDF] . The average fluorescence level ( [figure omitted; refer to PDF] ) of an individual pixel was first calculated for the duration of recording. The ratio on each pixel was then calculated as [figure omitted; refer to PDF] , color-coded with shades of red (depolarization) or blue (repolarization), and animated to show propagation patterns in the mapping field.

2.3. Experimental Protocols

A bipolar catheter was inserted into the right ventricular apex for pacing at twice the threshold. The effective refractory period (ERP) was measured by giving a premature stimulus after 8 beats at a 400 ms pacing cycle length (PCL). APD₈₀ (APD at 80% repolarization) restitution curve was constructed, and APD₈₀ and Ca_i alternans were induced by a dynamic pacing protocol [14]. VF inducibility was defined as the ability to provoke sustained VF (>2 min) with the dynamic pacing protocol [15]. If VF was not induced by dynamic pacing, burst pacing (PCL of 70-90 ms, 5 ms pulse width, and 5 mA current for 3-8 seconds) was used. Three episodes of VF (4 seconds each) were obtained consecutively for dominant frequency (DF) analysis. Defibrillation using epicardial patch electrodes was performed for sustained VF. After the baseline study, dantrolene (10 μ M) was administered for 20 minutes, and the experimental protocol was repeated.

2.4. Data Analysis

APD₈₀ restitution curves were constructed at selected evenly spaced sites [14]. The maximum slope of APD₈₀ restitution curve was determined after curve fitting by first-order exponential fitting. We used monoexponential fitting to compute the time constant [figure omitted; refer to PDF] of the decay portion of the Ca²⁺ transient [16] at a PCL of 400 ms. The thresholds of APD₈₀ and Ca_i alternans were defined as the longest PCL required to produce a 10 ms difference in APD₈₀ and a 10% difference in Ca_i amplitude between consecutive beats, respectively [17]. The phase was considered positive for a short-long APD and a small-large Ca_i amplitude sequence (color-coded in red) and was negative for a long-short APD and a large-small Ca_i amplitude sequence (color-coded in green). Spatially discordant alternans (SDA) was evidenced by the presence of both red and green regions separated by a nodal line. The SDA threshold was defined as the longest PCL required to reach the alternans threshold on both sides of a nodal line. To estimate conduction velocity (CV), we measured the distance and conduction time between the earliest activation point and two epicardial points in the non-IR and IR zones, respectively [13]. VPB burden (beats/5 min) was calculated by pseudo-ECG recordings at 0-5 min (taken as baseline), 5-10 min, 10-15 min, and 15-20 min during dantrolene infusion to evaluate its effects on VPB suppression.

2.5. Statistics

Continuous variables were expressed as mean ± standard deviation. One-way repeated measures ANOVA (with post hoc LSD analysis) was performed to evaluate the effects of dantrolene on VPB suppression during the 20-minute drug infusion period. Two-way repeated measures ANOVA was performed to evaluate the effects of dantrolene on APD₈₀ (PCL at 300, 200, and 150 ms) and CV (PCL at 300, 200, 150, and 120 ms). The McNemar test was used to compare the VF inducibility at baseline and after dantrolene infusion. A paired t -test was used to evaluate statistical significance in ERP, the mean maximum slope of APD restitution, Ca_i decay, the threshold of alternans and SDA, and the mean maximum DF ( [figure omitted; refer to PDF] ) of VF before and after dantrolene treatment in the same hearts. An unpaired t -test was performed to compare the CV, APD₈₀ , and Ca_i decay between the HF and non-HF groups and between the non-IR and IR zones. Generalized estimating equation was used to examine whether the effect of dantrolene on APD₈₀ was significantly different between the HF and non-HF groups. Differences were considered significant when [figure omitted; refer to PDF] .

3. Results

In the HF group, the mean LV ejection fraction was decreased from [figure omitted; refer to PDF] % to [figure omitted; refer to PDF] % after 4-week rapid pacing ( [figure omitted; refer to PDF] , [figure omitted; refer to PDF] ). In the non-HF group, the mean LV ejection fraction was [figure omitted; refer to PDF] % ( [figure omitted; refer to PDF] ). The wet weight of the hearts in the HF group ( [figure omitted; refer to PDF] g, [figure omitted; refer to PDF] ) was significantly heavier than the non-HF group ( [figure omitted; refer to PDF] g, [figure omitted; refer to PDF] , [figure omitted; refer to PDF] ).

3.1. Effects of Dantrolene on VPB

In the HF group, 6 of the 9 rabbit hearts had frequent VPBs at baseline ( [figure omitted; refer to PDF] beats/5 min); in the non-HF group, 4 of the 9 rabbits hearts had frequent VPBs at baseline ( [figure omitted; refer to PDF] beats/5 min). There was no significant difference of baseline VPB burden between two groups ( [figure omitted; refer to PDF] ). Dantrolene did not lower the frequency of VPB in either groups (Figures 1(a) and 1(b), [figure omitted; refer to PDF] for all comparisons). Figure 1(c) shows examples of pseudo-ECG tracings of a failing (upper) and a control (bottom) heart. The VPB burdens were 9, 8, 10, and 9 beats/5 min (HF) and 12, 8, 6, and 8 beats/5 min (non-HF) at 0-5, 5-10, 10-15, and 15-20 min, respectively.

Dantrolene had no significant effects on suppressing VPBs in the HF (a) and non-HF (b) groups. (c) pseudo-ECG showed no significant decrease of VPBs density (spikes) during dantrolene infusion in either group.

(a) [figure omitted; refer to PDF]

(b) [figure omitted; refer to PDF]

(c) [figure omitted; refer to PDF]

3.2. Electrophysiological Responses to Dantrolene Administration

3.2.1. APD₈₀ and ERP

The HF group had a significantly longer APD₈₀ than the control group at PCL = 300 ms ( [figure omitted; refer to PDF] ) (Table 1). In the HF group, dantrolene prolonged APD₈₀ at PCL = 300 ms ( [figure omitted; refer to PDF] ), 200 ms ( [figure omitted; refer to PDF] ), and 150 ms ( [figure omitted; refer to PDF] ) and the ERP ( [figure omitted; refer to PDF] ). In the non-HF group, dantrolene prolonged APD₈₀ at PCL = 300 ms ( [figure omitted; refer to PDF] ), 200 ms ( [figure omitted; refer to PDF] ), and 150 ms ( [figure omitted; refer to PDF] ) and the ERP ( [figure omitted; refer to PDF] ) (Table 1). Generalized estimating equation analysis showed that there was a trend that dantrolene exerted a greater effect on APD₈₀ prolongation in the non-HF group than in the HF group at PCL = 150 ms ( [figure omitted; refer to PDF] ). There was no significant difference in APD₈₀ between the non-IR and IR zones at baseline or with dantrolene at all PCLs in either group ( [figure omitted; refer to PDF] for all comparisons) (Table 2).

Table 1: Electrophysiological effects of dantrolene in isolated Langendorff-perfused hearts after IR injury in the HF and non-HF groups.

	[figure omitted; refer to PDF] (cm/s)	[figure omitted; refer to PDF] (cm/s)	[figure omitted; refer to PDF] (cm/s)	[figure omitted; refer to PDF] (cm/s)	[figure omitted; refer to PDF] (ms)	[figure omitted; refer to PDF] (ms)	[figure omitted; refer to PDF] (ms)	ERP(ms)	[figure omitted; refer to PDF] (Hz)	APDR slope	[figure omitted; refer to PDF] decay ( [figure omitted; refer to PDF] ) (ms)
HF ( [figure omitted; refer to PDF] )
Baseline	72 ± 12	68 ± 11	63 ± 11	55 ± 9	157 ± 16	130 ± 11	108 ± 10	161 ± 27	14.2 ± 1.3	1.14 ± 0.17	69 ± 12
Dantrolene	67 ± 10*	61 ± 11*	55 ± 10*	47 ± 6*	169 ± 20*	135 ± 12*	110 ± 8*	182 ± 12*	11.3 ± 1.5*	1.34 ± 0.24*	72 ± 10
Non-HF ( [figure omitted; refer to PDF] )
Baseline	81 ± 7	76 ± 9	70 ± 9	64 ± 8	148 ± 9	125 ± 9	107 ± 12	142 ± 11	13.0 ± 1.7	0.97 ± 0.19	66 ± 8
Dantrolene	73 ± 11*	67 ± 11*	61 ± 12*	55 ± 11*	160 ± 14*	134 ± 11*	112 ± 12*	170 ± 10*	10.4 ± 1.5*	1.19 ± 0.30*	67 ± 8

Values are mean ± SD. [figure omitted; refer to PDF] , [figure omitted; refer to PDF] , and [figure omitted; refer to PDF] , action potential duration at pacing cycle lengths of 300, 200, and 150 ms; [figure omitted; refer to PDF] , [figure omitted; refer to PDF] , [figure omitted; refer to PDF] , and [figure omitted; refer to PDF] , conduction velocity at pacing cycle lengths of 300, 200, 150, and 120 ms; [figure omitted; refer to PDF] : the maximal dominant frequency of ventricular fibrillation; Ca_i : intracellular [figure omitted; refer to PDF] ; ERP: effective refractory period; APDR slope: the mean maximum slope of APD restitution curves.

Asterisks denote values significantly different from the baseline.

Table 2: Electrophysiological effects of dantrolene in non-IR and IR zones in HF and non-HF groups.

	[figure omitted; refer to PDF] (cm/s)	[figure omitted; refer to PDF] (cm/s)	[figure omitted; refer to PDF] (cm/s)	[figure omitted; refer to PDF] (cm/s)	[figure omitted; refer to PDF] (ms)	[figure omitted; refer to PDF] (ms)	[figure omitted; refer to PDF] (ms)	[figure omitted; refer to PDF] (Hz)	Cai decay ( [figure omitted; refer to PDF] ) (ms)	APDR slope
HF ( [figure omitted; refer to PDF] )
Baseline
Non-IR	76 ± 11	72 ± 12	66 ± 11	57 ± 7	155 ± 13	130 ± 11	109 ± 10	14.4 ± 1.6	67 ± 11	1.14 ± 0.16
IR	70 ± 9	65 ± 9	60 ± 9	55 ± 9	158 ± 18	130 ± 11	108 ± 11	14.0 ± 1.3	70 ± 14	1.13 ± 0.20
Dantrolene
Non-IR	73 ± 9	66 ± 10	61 ± 9	51 ± 6	167 ± 19	135 ± 11	110 ± 6	11.7 ± 1.2	72 ± 10	1.34 ± 0.25
IR	67 ± 8	61 ± 10	54 ± 9	45 ± 6*	168 ± 22	134 ± 12	109 ± 8	10.9 ± 1.8*	73 ± 10	1.35 ± 0.26
Non-HF ( [figure omitted; refer to PDF] )
Baseline
Non-IR	84 ± 6	79 ± 7	73 ± 5	69 ± 5	148 ± 9	125 ± 9	107 ± 11	13.7 ± 1.3	63 ± 6	1.02 ± 0.16
IR	78 ± 7	73 ± 9	67 ± 7	63 ± 7	149 ± 9	125 ± 9	108 ± 12	12.3 ± 2.2*	69 ± 8*	0.91 ± 0.20
Dantrolene
Non-IR	78 ± 8	70 ± 8	66 ± 7	60 ± 8	162 ± 15	134 ± 11	113 ± 12	11.1 ± 1.5	65 ± 7	1.29 ± 0.38
IR	67 ± 11*	60 ± 9*	52 ± 10*	48 ± 9*	162 ± 15	134 ± 11	111 ± 11	9.7 ± 1.7*	70 ± 9*	1.09 ± 0.25

Values are mean ± SD. [figure omitted; refer to PDF] , [figure omitted; refer to PDF] , and [figure omitted; refer to PDF] , action potential duration at pacing cycle lengths of 300, 200, and 150 ms; [figure omitted; refer to PDF] , [figure omitted; refer to PDF] , [figure omitted; refer to PDF] , and [figure omitted; refer to PDF] , conduction velocity at pacing cycle lengths of 300, 200, 150, and 120 ms; [figure omitted; refer to PDF] : the maximal dominant frequency of ventricular fibrillation; Ca_i : intracellular [figure omitted; refer to PDF] ; CV: conduction velocity.

Asterisks denote values significantly different between non-IR and IR zones.

3.2.2. APD₈₀ Restitution Slope

There was a tendency of a steeper mean maximum slope of APD₈₀ restitution in the HF group than in the non-HF group ( [figure omitted; refer to PDF] , Table 1). There was no significant difference in the mean APD restitution slopes between IR and non-IR at baseline and after dantrolene infusion in either groups ( [figure omitted; refer to PDF] for all comparisons, Table 2). Dantrolene steepened the maximum slope of APD₈₀ restitution in the HF group ( [figure omitted; refer to PDF] ) and the non-HF group ( [figure omitted; refer to PDF] ) (Figure 2(a)). Figure 2(b) shows an example. In a failing heart (left subpanel), the mean maximal APD₈₀ restitution slopes were [figure omitted; refer to PDF] at baseline (blue) versus [figure omitted; refer to PDF] after dantrolene infusion (red) ( [figure omitted; refer to PDF] , [figure omitted; refer to PDF] points); in a non-HF heart (right subpanel), dantrolene also significantly steepened APD₈₀ restitution slope ( [figure omitted; refer to PDF] versus [figure omitted; refer to PDF] , [figure omitted; refer to PDF] , [figure omitted; refer to PDF] points).

Dantrolene steepened APD₈₀ restitution slopes in both groups. (a) Paired statistics for APD restitution (APDR) slopes. The data points (lines) are averages from each experiment. (b) representative examples of the spatial distribution of APDR curves at baseline (blue) and after dantrolene infusion (red) in both groups. The numbers under each subpanel are the maximum slopes of APD₈₀ restitution at the selected evenly-spaced sites. DI: diastolic interval.

(a) [figure omitted; refer to PDF]

(b) [figure omitted; refer to PDF]

3.2.3. CV

The HF group had a slower CV than the non-HF group at baseline ( [figure omitted; refer to PDF] , 0.02, 0.02, <0.001 at PCLs = 300, 200, 150, and 120 ms, resp.). Dantrolene decreased CV at all PCLs in both groups (Table 1), and the differences of CV between the HF and non-HF groups became less significant after dantrolene infusion ( [figure omitted; refer to PDF] , 0.22, 0.30, 0.02 at PCLs = 300, 200, 150, and 120 ms, resp.). In the HF group, there was no significant difference in CV between the IR and non-IR zones at baseline, but CV became significantly slower in the IR zone than the non-IR zone as the PCL decreased to 120 ms ( [figure omitted; refer to PDF] ) after dantrolene infusion (Table 2). In the non-HF group, there was a tendency of slower CV in the IR than in the non-IR zones at baseline ( [figure omitted; refer to PDF] , 0.1, 0.06, 0.06 at PCLs of 300, 200, 150, and 120 ms, resp.), and the differences became significant after dantrolene infusion at all PCLs ( [figure omitted; refer to PDF] , 0.04, 0.005, 0.009 at PCLs of 300, 200, 150, and 120 ms, resp.) (Table 2). Figure 3(a) shows an example in the HF group. Impulses propagated smoothly at PCLs >150 ms but took longer to activate the reperfused region at shorter PCLs (<150 ms), especially after dantrolene infusion. At baseline, CV (PCL = 300 ms versus 120 ms) was decreased from 77 cm/s to 62 cm/s in the non-IR zone and from 77 cm/s to 61 cm/s in the IR zone. After dantrolene infusion, CV (PCL = 300 ms versus 120 ms) was decreased from 72 cm/s to 50 cm/s in the non-IR zone and from 66 cm/s to 46 cm/s in the IR zone in this heart. Figure 3(b) shows an example of the non-HF group. At baseline, CV (PCL = 300 ms versus 120 ms) was decreased from 93 cm/s to 72 cm/s in the non-IR zone and from 82 cm/s to 68 cm/s in the IR zone. After dantrolene infusion, CV (PCL = 300 ms versus 120 ms) was decreased from 86 cm/s to 52 cm/s in the non-IR zone and from 70 cm/s to 47 cm/s in the IR zone in this heart. In addition, CV alternans was induced by rapid pacing, which played a role in facilitating SDA induction.

Effects of dantrolene on CV and anisotropic conduction. (a) and (b) are representative examples of isochronal maps in the HF and non-HF groups, respectively. Rate-dependent conduction slowing is shown in both groups. The number under each subpanel is the PCL. Dashed lines indicate the border of IR zone; red arrows indicate the directions of wavefront propagation. S: slow; F: fast.

(a) [figure omitted; refer to PDF]

(b) [figure omitted; refer to PDF]

3.2.4. Alternans and SDA

APD₈₀ and Ca²⁺ transient alternans were in synchrony in all mapped episodes (an example shown in Figure 5(c)). Dantrolene significantly lowered the pacing rate threshold for alternans induction in both groups (HF: [figure omitted; refer to PDF] ms (baseline) versus [figure omitted; refer to PDF] ms (dantrolene), [figure omitted; refer to PDF] , [figure omitted; refer to PDF] ; non-HF: [figure omitted; refer to PDF] ms (baseline) versus [figure omitted; refer to PDF] ms (dantrolene), [figure omitted; refer to PDF] , [figure omitted; refer to PDF] ). In addition, the threshold of SDA was lowered by dantrolene in both groups (HF: [figure omitted; refer to PDF] ms (baseline) versus [figure omitted; refer to PDF] ms (dantrolene), [figure omitted; refer to PDF] , [figure omitted; refer to PDF] ; non-HF: [figure omitted; refer to PDF] ms (baseline) versus [figure omitted; refer to PDF] ms (dantrolene), [figure omitted; refer to PDF] , [figure omitted; refer to PDF] ).

3.2.5. Ca_i Decay

Dantrolene had no significant effects on Ca_i reuptake in either group (Table 1). In the HF group, there was no significant difference in the [figure omitted; refer to PDF] values between the IR and non-IR zones at baseline or after dantrolene infusion ( [figure omitted; refer to PDF] for all comparisons) (Table 2). A representative example is provided in Figure 4(a). The mean [figure omitted; refer to PDF] values (non-IR versus IR) were [figure omitted; refer to PDF] ms and [figure omitted; refer to PDF] ms at baseline and [figure omitted; refer to PDF] ms and [figure omitted; refer to PDF] ms after dantrolene infusion in this failing heart. In the non-HF group, Ca_i decay was significantly slower in the IR zone than in the non-IR zone at baseline ( [figure omitted; refer to PDF] ) and after dantrolene infusion ( [figure omitted; refer to PDF] ) (Table 2). As shown in Figure 4(b), the mean [figure omitted; refer to PDF] values (non-IR versus IR) were [figure omitted; refer to PDF] ms and [figure omitted; refer to PDF] ms at baseline and [figure omitted; refer to PDF] ms and [figure omitted; refer to PDF] ms after dantrolene infusion in this non-HF heart.

Effects of dantrolene on Ca_i decay. (a) and (b) representative examples of the distribution of Ca²⁺ decay time in the non-IR and IR zones at baseline and during dantrolene infusion in the HF and non-HF groups, respectively. Dantrolene had no significant effects on Ca_i delay in either group. The number on each subpanel is the [figure omitted; refer to PDF] value (ms). The right subpanel illustrates the mapping field, and (A) to (H) indicate sites where the Ca_i decay time was measured. LAA: left atrial appendage; IVS: interventricular sulcus.

(a) [figure omitted; refer to PDF]

(b) [figure omitted; refer to PDF]

VF induction and VF conversion to VT during dantrolene administration in a failing heart. (a) [figure omitted; refer to PDF] tracings corresponding to the points labeled in panel (b) during rapid pacing (PCL = 90 ms) at baseline (left subpanel) and with dantrolene (right subpanel). The red arrows indicate wavefront propagation and the white lines indicate conduction block. (b) Schematic illustration of the mapped region (left subpanel) and isochronal maps (right subpanels) corresponding to two consecutive beats labeled in panel (a) (left subpanel, red bar). (c) Ca_i and APD alternated in synchrony. SDA was not induced at PCL = 140 ms until it was shortened to 120 ms at baseline but was induced at PCL = 140 ms with dantrolene infusion (panel (e), right subpanel). The black arrows indicate nodal lines. (d) and (e) isochronal maps of VF initiation and VF conversion to VT corresponding to the period labeled in panel (a) (right subpanel and red and blue bars, resp.). The white dashed lines indicate functional conduction block; white asterisks indicate focal breakthrough.

(a) [figure omitted; refer to PDF]

(b) [figure omitted; refer to PDF]

(c) [figure omitted; refer to PDF]

(d) [figure omitted; refer to PDF]

(e) [figure omitted; refer to PDF]

3.3. Effects of Dantrolene on VF Induction and Maintenance

The HF group had a higher VF inducibility than the non-HF group at baseline (7 of 9 versus 2 of 9, [figure omitted; refer to PDF] ). Almost all pacing-induced VF episodes (including burst pacing) were sustainable in the HF (9 of 9) and non-HF (8 of 9) groups at baseline. Dantrolene had no significant effects on VF inducibility (7 of 9 versus 7 of 9, [figure omitted; refer to PDF] ) or VF maintenance (9 of 9 versus 6 of 9, [figure omitted; refer to PDF] ) in the HF group. In the non-HF group, the VF inducibility was borderline increased (2 of 9 versus 6 of 9, [figure omitted; refer to PDF] ), but VF became less sustainable (8 of 9 versus 3 of 9, [figure omitted; refer to PDF] ) after dantrolene infusion.

Figure 5 shows an example of dantrolene effects on VF dynamics in the HF group. In this failing heart, VF was not induced during dynamic pacing at baseline (PCL = 90 ms, Figure 5(a), left subpanel), even if CV alternans (Figure 5(b)) and SDA (from PCL = 120 ms, Figure 5(c)) were induced. After dantrolene infusion, VF was induced by dynamic pacing (PCL = 90 ms) but converted to VT spontaneously (Figure 5(a), right subpanel). Figure 5(d) shows that rapid pacing induced functional conduction block to form figure-8 reentry (frames 320, 416, and 508 ms), and the conduction delay caused wavefronts to arise from the opposite side of the IR border to activate the IR zone and collide with the original wavefronts to perpetuate VF (frame 760 ms) (see online video 1 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/532820). However the VF was not sustainable. As shown in Figure 5(e), the breakthrough wavefronts could not propagate into the IR zone directly (frames 1768, 1884 ms). The functional conduction block simplified the wavefront propagation pattern and converted VF to VT (online video 2). SDA was induced at a longer PCL after dantrolene infusion (from 140 ms, Figure 5(e), right subpanel), and the nodal lines corresponded to the lines of functional conduction block as shown in Figures 5(d) and 5(e). In the HF group, VF was converted to ventricular tachycardia (VT) in 2 hearts and converted to sinus rhythm in 1 heart.

In the non-HF group, VF was converted to VT and then stopped ( [figure omitted; refer to PDF] ) or directly converted to sinus rhythm ( [figure omitted; refer to PDF] ) in 6 hearts with nonsustained VF after dantrolene infusion. Figure 6 shows an example. At baseline, sustained VF was induced as PCL decreased to 100 ms, and electrical defibrillation was needed to convert it (data not shown). After dantrolene infusion, VF was induced at a longer PCL but terminated spontaneously 49 seconds after induction. As shown in Figure 6(a) ( [figure omitted; refer to PDF] tracings), APD alternans was induced (at PCL = 120 ms) before VF induction. The corresponding isochronal maps (Figure 6(c)) show pacing-induced CV alternans and crowded isochronal lines (white arrows, frames 68 to 452 ms) at PCL = 120 ms. A longer APD was correlated to a slower CV, indicating that CV was controlled at least partly by APD (ERP) at faster pacing rates. When PCL was shortened to 110 ms, the pacing wavefront met a line of functional conduction block (frames 564 and 672 ms) to form reentry and perpetuated VF (frames 776 and 860 ms) (online video 3). Figure 6(b) ( [figure omitted; refer to PDF] tracings) and Figure 6(d) (snapshots) show spontaneous VF termination. A wavefront met a line of functional conduction block to form figure-8 reentry (snapshots 796 to 948 ms), but the new-formed wavefronts were blocked (snapshot 1008 ms), followed by a wavefront arising from the border of the IR zone (white arrow, snapshot 1036 ms) and then a wavefront from the border of the mapping field (snapshots 1204 to 1396 ms) before conversion to sinus rhythm (online video 4). Figure 6(e) shows that SDA was induced by rapid pacing (PCL 120 ms, left subpanel), and a new nodal line (red arrow, right subpanel) was formed as the PCL decreased to 110 ms. These nodal lines corresponded to the lines of functional conduction block shown in Figures 6(c) and 6(d) and played an important role in wavelet generation and annihilation.

VF induction and spontaneous termination during dantrolene administration in a non-HF heart. (a) and (b) [figure omitted; refer to PDF] tracings corresponding to the points labeled in panel (c) during VF induction and VF termination, respectively. The red arrows indicate wavefront propagation, and the white lines indicate conduction block. L: long; S: short. (c) Isochronal maps corresponding to the period labeled in panel (a) showing CV alternans followed by conduction block and VF initiation. The white lines indicate functional conduction block. (d) Frame shots corresponding to the period labeled in panel. (b) The white lines indicate lines of functional conduction block, and the white arrow indicates focal breakthrough. (e) APD difference maps showing SDA induction. The black and red arrows indicate nodal lines. (f) The dominant frequency (DF) maps of induced VF at baseline (A) and after dantrolene infusion (B). SR: sinus rhythm.

(a) [figure omitted; refer to PDF]

(b) [figure omitted; refer to PDF]

(c) [figure omitted; refer to PDF]

(d) [figure omitted; refer to PDF]

(e) [figure omitted; refer to PDF]

(f) [figure omitted; refer to PDF]

The [figure omitted; refer to PDF] of VF was significantly decreased by dantrolene infusion (Table 1). In the HF group, the difference of [figure omitted; refer to PDF] between the IR and non-IR zones was not significant at baseline ( [figure omitted; refer to PDF] , [figure omitted; refer to PDF] , Table 2); however, it became significant with dantrolene infusion ( [figure omitted; refer to PDF] , [figure omitted; refer to PDF] , Table 2). In the non-HF group, the IR zone had a lower [figure omitted; refer to PDF] than the non-IR zone at baseline ( [figure omitted; refer to PDF] , [figure omitted; refer to PDF] ) and after dantrolene infusion ( [figure omitted; refer to PDF] , [figure omitted; refer to PDF] , Table 2). As shown in Figure 6(f), the mean values of the [figure omitted; refer to PDF] of VF were [figure omitted; refer to PDF] Hz at baseline and [figure omitted; refer to PDF] Hz with dantrolene. The non-IR zone had a higher mean [figure omitted; refer to PDF] of VF than that in the IR zone at baseline ( [figure omitted; refer to PDF] Hz versus [figure omitted; refer to PDF] Hz) and with dantrolene infusion ( [figure omitted; refer to PDF] Hz versus [figure omitted; refer to PDF] Hz). This suggests a driven rather than a driver role of the IR zone in VF dynamics in this model.

4. Discussion

We studied the effects of dantrolene on suppression of VPB and VF inducibility in post-IR injury hearts with and without pacing-induced HF. The major results were that dantrolene did not suppress spontaneous VPBs and VF inducibility in either group. Dantrolene exerted antiarrhythmic action via prolonging APD and ERP, which may underlie a higher VF conversion rate in smaller hearts in the non-HF group. But the APD and ERP lengthening effect also intensified the rate-dependent conduction delay; in addition, dantrolene steepened APD restitution properties and facilitated both spatially concordant and discordant alternans induction, all of which may oppose its antiarrhythmic effects.

4.1. VPB Suppression in IR Hearts

We previously reported that dantrolene was effective in suppressing VPBs via decreasing diastolic SR Ca²⁺ oscillations in an AMI with HF model [9]. In post-IR injury hearts, Sharma et al. reported that reperfusion for 30 minutes resulted in a significant increase in tissue Ca²⁺ in the IR zone [18]. Since a reduced binding affinity of calmodulin to ryanodine receptor-2 fitted for dantrolene's action on SR is found in failing hearts rather than normal hearts [19], dantrolene would be predicted to suppress VPBs in the IR model, especially in the HF group. However, our data did not show a decreased VPB burden during dantrolene infusion neither in HF group nor in non-HF group. There are some possible explanations. First, as Vera et al. reported [20], triggered activity may not be the dominant mechanism responsible for VPBs in IR hearts. Yee et al. noted only small-amplitude oscillatory afterpotentials during reperfusion but no triggered activity, which cast doubt on whether triggered activity is a mechanism of reperfusion arrhythmias [21]. On the other hand, enhanced automaticity has been reported to be the dominant mechanism of VPBs in post-IR injury hearts [22]. The lack of a VPB suppressing effect by dantrolene could be due to its inability to suppress automaticity [23, 24]. Second, if triggered activity is the dominant mechanism of VPBs, it implies that dantrolene could not ameliorate the Ca_i overload condition effectively in these IR hearts. Temsah et al. reported that the attenuated recovery of contractile function and Ca_i overload post-IR injury are due to reduced SR Ca²⁺ uptake, Ca²⁺ release, and ryanodine-binding activities [25]. Therefore, suppression of diastolic SR Ca²⁺ leakage alone may not be sufficient to improve Ca_i overload conditions.

4.2. Dantrolene on Conduction Delay in the IR Rabbit Model

Penkoske et al. reported that reperfusion arrhythmias are initiated by enhanced pacemaker activity and maintained by localized and slowed conduction within IR regions [26]. Both the IR-induced downregulation of Na⁺ channel [27] and reduced gap junction conductance as a result of the elevated Ca_i of the IR myocardium may account for a depressed conduction in the IR region [28]. Salata et al. reported that dantrolene has no significant effect on upstroke velocity of phase 0 but prolongs APD mediated in part by a decrease in the intracellular free Ca²⁺ concentration [23, 29]. Therefore, dantrolene can either ameliorate conduction depression by decreasing intracellular free Ca²⁺ concentration (to improve gap junction conductance) or further decelerate CV via prolonging APD (as impulses traveling through partially refractory tissue). Our data showed that dantrolene deteriorated conduction delay and led to frequency-dependent functional conduction block, which may play a role in facilitating VF induction (as shown in Figures 5 and 6). However, the worsening of conduction delay was caused by APD prolongation, which neutralized its arrhythmogenicity by hindering wavelet propagation to stop VF (as shown in Figure 6).

4.3. Anti- and Proarrhythmic Effects of Dantrolene on IR Hearts

Dantrolene prolongs APD, which by itself can decrease the [figure omitted; refer to PDF] of VF and influence the persistence of VF through its effects on wavelength [30]. However, the electrophysiological studies and mapping data also showed that dantrolene has proarrhythmic effects (i.e., slowing CV, steepening APD restitution slope, and lowering the pacing threshold of arrhythmogenic alternans induction) in the IR model. These effects have been demonstrated to create functional electrophysiological dispersion that destabilizes wave propagation and enhances the tissue vulnerability to initiate reentry [31, 32]. The lack of significant effects of dantrolene on the VF inducibility and maintenance in the HF group might be due to a counterbalance of its antiarrhythmic and proarrhythmic effects. On the other hand, dantrolene had a higher VF conversion rate in the non-HF group, which is likely due to a greater APD prolonging effect in smaller hearts in the non-HF group compared with the HF group. Salata et al. demonstrated that dantrolene prolongs APD, possibly mediated by inhibiting L-type Ca²⁺ channels, subsequently depressing Ca²⁺ -sensitive potassium channels [29]. Because L-type Ca²⁺ channels are impaired in pacing-induced HF [33], it is possible that fewer L-type Ca²⁺ channels are available to respond to the inhibitory effect of dantrolene, resulting in a less degree of APD prolongation in the HF group.

4.4. Clinical Implications

Dantrolene has been shown to prevent VF storm in failing hearts with AMI [9]. However, the presented data suggest that dantrolene has no significant effects on VF inducibility and maintenance in failing hearts post-IR injury. The clinical benefits of continuous dantrolene use in HF patients with AMI and VF storm postreperfusion therapy are not obvious and may require further investigations.

4.5. Study Limitations

Given that the electrophysiological studies were performed with Langendorff perfusion, it is possible that electrophysiological parameters may be different when the hearts are in situ and also caution is advised when extrapolating our model to clinical IR cardiac injury. Because the Ca²⁺ amplitude of optical signals was a ratio derived from the difference of Ca²⁺ dye fluorescence intensity between the peak and the trough of acquired beats rather than the absolute value of intracellular Ca²⁺ concentration changes, we could not make comparisons of the absolute Ca²⁺ amplitude between the optical signals acquired at different moments (baseline versus after dantrolene). Therefore, whether the amplitude of Ca²⁺ transient was affected by dantrolene was not shown in this study. The estimated CV slowing in the IR zone might be underestimated since the initial portion of IR distance measurement (from the earliest activation point to the IR zone border) was in the non-IR zone. We did not perform patch clamp studies and measure gap junction conductance; therefore, whether dantrolene exerted a direct inhibition effect on sodium current and/or gap junction coupling was not shown in this study.

Acknowledgment

This work was supported by Chang Gung Memorial Hospital Medical Research Programs [CMRPG38154] and [CMRPG3C1651] to Chung-Chuan Chou.

Conflict of Interests

The authors have no potential conflict of interests to disclose.

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