Effects of Pre- and Postconditioning on Arrhythmogenesis in the In Vivo Rat Model

Abstract

The antiarrhythmic potential of postconditioning in in vivo models remains poorly defined. We compared the effects of pre- and postconditioning on ventricular arrhythmogenesis against controls with and without reperfusion. Wistar rats (n = 40, 269 ± 3 g) subjected to ischemia (30 minutes)–reperfusion (24 hours) were assigned to the following groups: (1) preconditioning (2 cycles), (2) postconditioning (6 cycles), or (3) no intervention and were compared with (4) nonreperfused infarcts and (5) sham-operated animals. Infarct size was measured, and arrhythmogenesis was evaluated with continuous telemetric electrocardiographic recording, heart rate variability indices, and monophasic action potentials (MAPs). During a 24-hour observation period, no differences in mortality were observed. Reperfusion decreased infarct size and ameliorated sympathetic activation during the late reperfusion phase. Preconditioning decreased infarct size by a further 35% (P = .0017), but only a marginal decrease (by 18%, P = .075) was noted after postconditioning. Preconditioning decreased arrhythmias during ischemia and early reperfusion, whereas postconditioning almost abolished them during the entire reperfusion period. No differences were noted in MAPs or in the magnitude of sympathetic activation between the 2 interventions. Compared to postconditioning, preconditioning affords more powerful cytoprotection, but both interventions exert antiarrhythmic actions. In the latter, these are mainly evident during the ischemic phase and continue during early reperfusion. Postconditioning markedly decreases reperfusion arrhythmias during a prolonged observation period. The mechanisms underlying the antiarrhythmic effects of pre- and postconditioning are likely different but remain elusive.

Introduction

Acute myocardial infarction (MI) is a leading cause of morbidity and mortality worldwide. ¹ The main therapeutic target consists of timely restoration of coronary blood flow by thrombolysis and/or percutaneous coronary intervention. However, despite its proven benefit in limiting infarct size, reperfusion after prolonged ischemia is long known to be associated with deleterious effects in the ischemic myocardium ² ; these consist of a second wave of necrosis, accompanied by ventricular arrhythmogenesis. ³

Ischemic preconditioning decreases myocardial necrosis in animals ⁴ and humans ⁵ and has been shown to exert antiarrhythmic effects during ischemia ⁶ and reperfusion. ⁷ However, pharmacological strategies mimicking preconditioning have produced poor results in the clinical setting, mainly because they appear ineffective when administered after the onset of ischemia. ⁸ In contrast, postconditioning, consisting of very brief repetitive cycles of ischemia prior to the onset of reperfusion, has evolved as a clinically promising therapeutic approach. This intervention has been shown to elicit cytoprotection in animal models, ⁹ with favorable results in preliminary clinical studies.^10,11

Further to its cytoprotective effects, there is evidence in favor of an antiarrhythmic effect of postconditioning in various experimental settings^12–15; in fact, in the first demonstration of postconditioning in anesthetized cats, intermittent reperfusion markedly reduced the incidence of arrhythmogenesis after 20 minutes of coronary artery occlusion. ¹² These salutary effects were subsequently reproduced in anesthetized rats,^13,14 subjected to brief (5 minutes) ischemic periods, but no clear-cut antiarrhythmic effect could be demonstrated in anesthetized pigs. ¹⁶ In addition to species differences, these discrepant findings can be attributed to the confounding effects of anesthesia, thereby precluding firm inferences. The antiarrhythmic properties of postconditioning were reiterated in the ex vivo isolated working rat heart model, subjected to 15 minutes of ischemia ¹⁵ ; however, the arrhythmogenic pattern in this model differs markedly when compared to in vivo settings.^17,18 Thus, in view of the variability of experimental conditions and the reported results, the translational impact of the antiarrhythmic potential of postconditioning remains uncertain.

In the present work, we directly compared the antiarrhythmic effects of pre- and postconditioning during a prolonged observation period, and we evaluated potential underlying mechanisms. Specifically, we assessed the effects of postconditioning on arrhythmogenesis and infarct size and compared them with those elicited after preconditioning, using the in vivo rat model of ischemia–reperfusion (I/R). This model is suitable and widely utilized, as the rat exhibits a large number of ventricular tachycardia (VT) and fibrillation (VF) episodes in response to ischemia and reperfusion.^17–20 We opted a 30-minute ischemic period that presents higher relevance to the clinical setting of acute MI. ¹⁸ We used implantable miniature electrocardiography (ECG) telemetry devices, thereby avoiding the confounding effects of anesthesia ¹⁷ ; moreover, these recorders permit a substantial extension of the observation period post-MI. To provide further insight into the underlying mechanisms, we examined left ventricular (LV) monophasic action potentials (MAPs), and we evaluated sympathetic stimulation, using linear and nonlinear analysis of ECG signals.

Methods and Materials

Experimental Animal Population and Ethics

The animal study population consisted of 40 Wistar rats (all male, 12-14 weeks of age, and weighing 240-300 g). The animal care and procedures adhere to the Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research, National Research Council, Washington, DC, National Academy Press, 8th edition, 2011) as well as to European legislation (European Union directive for the protection of animals used for scientific purposes, 2010/63/EU). The animals were housed in Plexiglas cages in groups of 2 under optimal laboratory conditions (controlled temperature, humidity and 12:12-hour light:dark cycles) and were allowed free access to standard rodent pellet diet and water. The study protocol was approved by the responsible regulatory state authorities (Department of Veterinary Medicine and Animal Welfare, Prefecture of Eastern Attica, Athens, Greece).

The animals were assigned into 5 groups (n = 8 per group), namely (1) pre- and (2) postconditioning, (3) I/R, (4) ischemia without reperfusion, and (5) sham-operation. In the event of death due to surgical manipulations during the procedure, the animals were replaced. This sample size provides a ∼90% power to detect a ∼30% difference in infarct size and a ∼75% power to detect a (meaningful) 50% difference in arrhythmogenesis between pre- and postconditioning.

Implantation of ECG Telemetry Transmitters

Ventricular arrhythmias were recorded using miniature telemetry transmitters (TCA-F40, Dataquest; Data Sciences International, Transoma Medical, Arden Hills, Minnesota) that permit long-term, continuous ECG recordings in conscious, untethered animals.^17,19–21 The animals were placed in glass box and were anesthetized with isoflurane, prior to intubation with a 14-gauge catheter. They were mechanically ventilated with a rodent ventilator (7025; Ugo Basile, Comerio, Italy), and anesthesia was maintained with a mixture of oxygen and 2% isoflurane. The transmitters were implanted in the abdominal cavity as described previously^19–21; they were secured under the rectus abdominis muscle, the leads were tunneled under the skin and sutured to the underlying tissue to eliminate artifacts. The positive electrode was implanted under the right axilla and the negative electrode at the left hindlimb area. During recording, the rats were placed on a receiver (RCA-1020; DSI, Arden Hills, Minnesota) that continuously captured the signal, independent of the animal activity. The signal was displayed in real time with the use of a computer program (A.R.T.; DSI, Arden Hills, Minnesota) and stored for analysis.

Ischemia Induction

Ischemia was induced immediately after transmitter implantation, under continuous telemetry recording. The method used for ischemia induction in our laboratory has been described previously.^19–21 In brief, following left thoracotomy, the heart was exposed and exteriorized, and the left coronary artery was encircled approximately 4 mm from its origin, extending from the left atrial appendage to the pulmonary cone; following these anatomical landmarks ensures comparable size of the ischemic area.^19–21 For nonreperfused infarcts, permanent ligation was performed, whereas in sham-operated animals, the left coronary artery was encircled but not ligated. For I/R, the suture was threaded through a snare, the tightening and release of which induced ischemia and reperfusion, respectively. At the end of ischemia, the chest was rapidly closed in three layers (rib cage, pectoral muscles, and skin); anesthesia was discontinued, and the remaining air was aspirated from the thorax, allowing the rats to resume spontaneous respiration.

A 6-lead ECG was recorded (QRS-Card digital PC-ECG; Pulse Biomedical Inc, Norristown, Pennsylvania) after amplification by the software (QRS Card Cardiology Suite version 4.05; PBI, Norristown, Pennsylvania); ST-segment elevation in 2 or more leads was considered proof of induced ischemia. After a brief (∼2-3 minutes) monitoring period, the animals were extubated and returned to their cage, upon observation of stable spontaneous respiration.

Observation Phases

Myocardial ischemia was induced for 30 minutes, followed by 24 hours of reperfusion. The observation period was divided into 4 phases, based on the temporal differences in ventricular arrhythmogenesis observed during I/R.^17–22 Specifically, the following time intervals were defined (Figure 1): phase IA (first 10 minutes of ischemia), phase IB (10-30 minutes of ischemia), early reperfusion (0-30 minutes after the onset of reperfusion), phase IIA (60 minutes after the onset of ischemia to the 11th hour), and phase IIB (from 12th hour onward until the end of the observation period). The definition of phase IIB was based on previous reports,^19–24 indicating very low incidence of ventricular arrhythmias during this time frame. To avoid the confounding effects of medication, no drug regimen was administered during the 24-hour observation period.

Figure 1.

Study protocol. Gray and white areas represent ischemic and reperfusion time, respectively. Animal groups (from top to bottom): sham, no reperfusion, ischemia–reperfusion, preconditioning, and postconditioning.

Pre- and Postconditioning Protocols

Ischemic preconditioning consisted of 2 cycles, applied prior to ischemia induction; each cycle consisted of 5 minutes of ischemia, followed by 10 minutes of reperfusion. Postconditioning consisted of 6 cycles, applied immediately after the 30-minute ischemic period; each cycle included 10 seconds of ischemia, followed by 10 seconds of reperfusion. Since the number and duration of effective postconditioning cycles is species dependent, ²⁵ we chose repeated cycles of short duration, considered effective in the rat. ⁵

Mortality

The survival duration was accurately identified from the recorded ECG strips, and deaths were classified as tachy- or bradyarrhythmic, according to the previously set definitions.^19,20 Specifically, tachyarrhythmic death was defined as ventricular asystole, preceded by sustained ventricular tachyarrhythmia, whereas bradyarrhythmic death was defined as prolonged sinus tachycardia, followed by an abrupt onset of complete atrioventricular block and ventricular asystole. An example of the latter, indicative of death due to heart failure,^17,19,20 is shown in Figure 2A.

Figure 2.

Electrocardiographic recordings. Examples of recordings demonstrating bradyarrhythmia (A) and tachyarrhythmia (B).

Risk Area and Infarct Size Measurement

The risk and infarcted zones were measured at the end of the 24-hour observation period, using previously described methodology. ²⁶ Briefly, the heart was harvested and mounted on a perfusion apparatus; after perfusion with normal saline, the coronary ligature was tightened and a saline solution of green-fluorescent microspheres (Duke Scientific Corp, Palo Alto, California) was infused over 5 minutes. The heart was frozen at −20°C for 24 hours and sliced into 2-mm sections that were incubated in 1% triphenyltetrazolium chloride (diluted in isotonic phosphate buffer, at 37°C, pH 7.4) for 20 minutes; finally, the slices were immersed in 10% formaldehyde solution for further 24 hours.

After placing the slices between glass plates, the risk zone, the infarcted area, and the normal myocardium were identified under ultraviolet light (λ = 366 nm). These areas were traced on an acetate sheet and were scanned with a high-resolution scanner (Scanjet 4570c/5500c; Hewlett-Packard, Palo Alto, California). Measurements were performed with the use of a software program (Image Tool, University of Texas), and volumes were calculated by multiplying the corresponding areas by the slice thickness. The volumes of the infarcted zone and the area at risk were expressed in cm³, and their percentage ratio (%I/R) was calculated.

Heart Rate

Sinus heart rate (HR) is reported for the following time points: baseline, 5th, 10th, 15th, 30th, 35th, 40th, 45th, and 60th minutes after ischemia induction and hourly thereafter. The HR at each time point was measured as an average of 10 consecutive RR intervals, after exclusion of nonsinus beats.

Arrhythmia Analysis

During off-line analysis of stored ECG tracings, episodes of VT and VF were recorded, and the duration of each episode was measured using the time scale provided by the analysis software (Dataquest A.R.T. Analysis, version 3.10; DSI, Arden Hills, Minnesota). The VT was defined as 4 or more consecutive premature ventricular contractions, and VF as a signal with indistinguishable QRS deflections. Because separation between VT and VF is occasionally difficult in the rat,^{17,19–21,23} we report ventricular tachyarrhythmias collectively, as the number and total duration of VT/VF episodes as well as the average duration per episode (total duration divided by the number of episodes) for each time interval (ie, for phases IA and IB, early postreperfusion, and phases IIA and IIB). Reporting both the number and the duration of VT/VF episodes is meaningful, as they reflect different aspects of their pathogenesis, namely initiation and maintenance, respectively. ¹⁸ Examples of ECG strips displaying tachyarrhythmia episodes are shown in Figure 2B.

Monophasic Action Potential Recordings

Monophasic action potentials were recorded from the lateral LV epicardium, whereas recordings from the right ventricle (RV) served as reference. The method used in our laboratory has been described previously ²⁷ ; in brief, a probe (model 200; EP Technologies Inc, Boston Scientific Corporation, Sunnyvale, California) was placed on the epicardium, exerting mild, constant pressure to eliminate electrical artifacts. The signal was amplified (preamplifier model 300, EPT, Sunnyvale, California) and filtered using a bandpass filter (permitting a signal range between 0.05 and 500 Hz), after elimination of power line interference. A continuous data stream was directed into a computer equipped with an analog-to-digital converter (BNC 2110, National Instruments Corporation, Dallas, Texas). Two-minute steady state signal recordings were performed at baseline, 5 minutes, and 24 hours postischemia induction; care was taken to obtain signals from the peri-infarct region, according to prior guides^28,29 and our previous experience.^19,23 Specifically, the probe was positioned on the lateral LV epicardium and was gently moved toward the akinetic tissue, until signals of very low amplitude were encountered; subsequently, it was moved back and the recording was performed at the site where an abrupt change in signal morphology and amplitude reemerged.

The LV action potential duration (APD) was measured using a recording and analysis software, developed and validated in our institution. ³⁰ During analysis, nonsinus beats were excluded, and 50 consecutive sinus beats per recording were included. The LV APD at 75% (APD75) and 90% (APD90) of repolarization was measured, and the standard deviation of these measurements (per recording) was used as an indicator of electrical alternans. ²⁹ Finally, the maximum rate of MAP voltage rise (dV/dt_max) during phase 0 was calculated (Figure 1), which provides an index of depolarization rate and conduction velocity. ²⁸

Electrocardiography Indices of Sympathetic Activation

Autonomic tone was assessed using ECG indices of HR variability (HRV). Simple time domain analyses, such as standard deviation of RR interval (SDRR) and root mean square (RMS) of successive RR interval differences, have been widely employed, with reduced values reflecting decreased vagal tone. ³¹ Frequency domain analysis provides a more in-depth evaluation, ³² by isolating a high frequency (HF) band (reduced power of which is caused by impaired vagal tone) and a low-frequency (LF) band (modulated by sympathetic, but also by vagal tone); thus, sympathetic/vagal balance is thought to be accurately reflected by the ratio of LF/HF bands.^31,32

After exclusion of nonsinus beats, SDNN and RMS were calculated, followed by fast-Fourier transform calculation in the frequency domain, as mentioned previously. ²⁴ The power spectra of all segments were averaged, and the spectra of LF (0.25-0.75 Hz) and HF (0.75-3 Hz) bands were identified; LF/HF was calculated for the ischemic period (phases IA and IB), early reperfusion, and phases IIA and IIB.

The study protocol is depicted in Figure 1.

Statistical Analysis

All values are given as mean ± standard error of the mean (SEM). Kaplan-Meier survival curves were constructed for each group, and heterogeneity was examined by chi-square. Normal distribution of continuous variables was evaluated with the Kolmogorov-Smirnov test. Beat-to-beat variability of the APD and VT/VF duration per episode were not normally distributed, and the nonparametric Kruskal-Wallis analysis of variance was used in their evaluation, followed by median test. Normal distribution was present in all the remaining variables, and the observed differences between groups were compared using the analysis of variance, followed by the post hoc Duncan multistage test. Changes in continuous variables over time were assessed with analysis of variance for repeated measures. Statistical significance was set at P < .05.

Results

Animal Groups

The animal study population consisted of 40 animals (12.6 ± 0.1 weeks of age, weighing 269 ± 3 g); no differences were found between groups, in terms of age or weight.

Mortality

At the end of the observation period, mortality rates were 4 (50%) of 8 in I/R; 3 (37.5%) of 8 in postconditioned rats; 1 (12.5%) of 8 in preconditioned rats; and 3 (37.5%) of 8 in nonreperfused infarcts. These differences between groups did not reach statistical significance (P = .19). The type (brady- versus tachyarrhythmic) of each death is shown in Table 1, and the Kaplan-Meier survival curves, displaying the time of each death, are shown in Figure 3.

Figure 3.

Mortality. No differences were observed in the Kaplan-Meier survival curves between the 5 groups. I/R indicates ischemia/reperfusion; pre, preconditioning; post, postconditioning; no rep, no reperfusion.

Table 1.

Animal Study Cohort and Outcome.

Group	n Value at the End of Phase
	Study Entry	IA	IB	ER	IIA	IIB
Ischemia–reperfusion	8	6	6	6	5	4
Tachyarrhythmic deaths		2	0	0	0	0
Bradyarrhythmic deaths		0	0	0	1	1
Postconditioning	8	8	7	7	7	5
Tachyarrhythmic deaths		0	1	0	0	0
Bradyarrhythmic deaths		0	0	0	0	2
Preconditioning	8	8	8	8	7	7
Tachyarrhythmic deaths		0	0	0	0	0
Bradyarrhythmic deaths		0	0	0	1	0
No reperfusion	8	8	7	7	7	5
Tachyarrhythmic deaths		0	1	0	0	0
Bradyarrhythmic deaths		0	0	0	0	2
Sham	8	8	8	8	8	8
Tachyarrhythmic deaths		0	0	0	0	0
Bradyarrhythmic deaths		0	0	0	0	0

Abbreviation: ER, early reperfusion.

Infarct Size

No infarction was noted in sham-operated animals, but a significant F-statistic was present in the remaining 4 groups. This was due to smaller (P = .0017) infarcts after preconditioning, compared to I/R. The difference in infarct size between pre- and postconditioning failed to reach statistical significance (P = .062). Nonreperfused infarcts were larger (all P < .036) compared to the remaining groups. Values are depicted in Figure 4.

Figure 4.

Infarct size. Infarct size was smaller in the preconditioning group (n = 7) than in the I/R group (n = 4). Nonreperfused infarcts (n = 5) were larger compared to all remaining groups. I/R indicates ischemia/reperfusion; pre, preconditioning; post, postconditioning; no rep, no reperfusion.

Heart Rate

During ischemic phases IA and IB, HR increased in all groups, but this response was blunted in the preconditioning group, being comparable to sham-operated animals (Figure 5). Specifically, at the 30th and 60th minutes postischemia induction, HR was lower (all P < .016) in preconditioned animals compared to the remaining ischemia groups. During the early reperfusion period, HR in postconditioned animals reached comparable values with those in the preconditioning group. During phase IIA, HR increased in all but sham-operated animals and was similar in the I/R, pre- and postconditioning groups. During phase IIB, HR returned to baseline values in sham-operated animals but remained high in the 4 ischemia groups.

Figure 5.

Heart rate. Heart rate (in beats per minute, bpm) in the 5 groups, during ischemia (phases IA and IB), early (ER), and delayed (phases IIA and IIB) reperfusion. Note the lower values during ischemia in preconditioned rats (asterisk) and the rise in all ischemia groups after the first hour (arrow).

Arrhythmia Analysis

No episodes of VT/VF were recorded in sham-operated animals throughout the observation period. The VT/VF in the remaining 4 groups at different time intervals is shown in Table 2.

Table 2.

Arrhythmia Analysis.

Duration (seconds)	Phase	I/R	Post	Pre	No Rep
Total duration	IA	57.3 ± 18.8	70.7 ± 25.7	3.5 ± 2.2a	64.1 ± 10.3
Duration per episode		6.8 ± 4.6	12.3 ± 9.6	2.4 ± 1.4	5.3 ± 2.9
Total duration	IB	27.2 ± 5.6	21.0 ± 3.1	0.9 ± 0.4a	35.9 ± 5.9
Duration per episode		2.1 ± 0.3	2.3 ± 0.4	0.8 ± 0.3b	4.5 ± 1.6
Total duration	ER	52.2 ± 9.8	0c	14.2 ± 6.2	38.2 ± 11.4
Duration per episode		2.9 ± 0.5	–	7.4 ± 6.3	2.7 ± 0.4
Total duration	IIA	21.9 ± 11.8	0.7 ± 0.5	16.7 ± 7.8	112.2 ± 17.1a
Duration per episode		4.7 ± 1.5	0.7 ± 0.4	1.5 ± 0.9	4.5 ± 0.5
Total duration	IIB	0.5 ± 0.3	0	0.4 ± 0.3	3.3 ± 0.6a
Duration per episode		0.6 ± 0.2	–	0.8 ± 0.1	1.4 ± 0.2

Abbreviations: I/R, ischemia–reperfusion; post, postconditioning group; pre, preconditioning group; no rep, no reperfusion; ER, early reperfusion.

^aP < .05 compared to the remaining groups.

^bP < 0.05 compared to nonreperfused infarcts.

^cP < .05 compared to I/R group.

Phases IA and IB

During the initial 10 minutes of ischemia, there were fewer episodes (all P < .030) with a shorter total duration (all P < .035) in preconditioned animals, compared to the remaining ischemia groups. Between the 10 and 30th minutes of ischemia, there were fewer VT/VF episodes (all P < .045) with a shorter total duration (all P < .0035) in preconditioned animals, compared to the remaining ischemia groups. The VT/VF duration per episode was shorter (P = .0146) in preconditioned, compared to nonreperfused infarcts.

Early reperfusion

The incidence of VT/VF remained unchanged in nonreperfused infarcts between the 30th and 60th minute of ischemia, whereas reperfusion increased the number (P = .011) and duration (P = .035) of VT/VF episodes in the I/R group. In contrast, postconditioning decreased their number (P = .0058) and duration (P = .0026) compared to the immediately preceding interval; this effect resulted in less VT/VF episodes (P = .0038) with a shorter duration (P = .00048) in postconditioned rats than in the I/R group. No significant differences were found between pre- and postconditioning in the number or duration of VT/VF episodes.

Phase IIA

More VT/VF episodes with longer duration were observed in nonreperfused infarcts, compared to the I/R, postconditioning and preconditioning groups. No significant differences were found between the 4 ischemia groups in VT/VF duration per episode. The incidence of VT/VF was comparable in preconditioning, postconditioning, and I/R groups.

Phase IIB

More VT/VF episodes with longer duration were seen in nonreperfused infarcts, but phase IIB arrhythmogenesis was generally low in all the groups.

Monophasic Action Potentials

Five minutes after ischemia induction, LV dV/dt_max decreased to a similar degree in all ischemia groups, whereas RV dV/dt_max remained unchanged. The RV MAP duration remained stable, but LV MAP duration (at APD90 and at APD75) was shorter (compared to baseline) at the 5th minute of ischemia and at the end of the 24-hour observation period in all but sham-operated animals. However, no significant between-group differences were found between the 4 ischemia groups.

ECG Indices of Sympathetic Activation

Sympathetic activation was evident in all ischemia groups throughout the 24-hour observation period, but it was more prominent in nonreperfused infarcts and became significantly higher than the remaining ischemia groups during phase IIB (Figure 6).

Figure 6.

Heart rate variability. Changes (from baseline) in the standard deviation of NN interval and root mean square of successive RR interval differences in the time domain analysis (left panel) and in the ratio of low-to-high frequency spectra (right panel). Note the significantly different values in nonreperfused infarcts (asterisk) during phase IIB. I indicates ischemia, ER, early reperfusion.

Discussion

Main Findings and Comparison With Previous Studies

We report a prominent antiarrhythmic effect of postconditioning on VT/VF in the in vivo rat model of I/R. Our results are in accordance with the early findings of Na et al, ¹² who reported an over 10-fold reduction in VT/VF after intermittent reperfusion in anesthetized cats. Similar findings were reported in anesthetized rats during a 10-minute reperfusion interval, following 5 minutes of ischemia.^13,14 In contrast, no clear-cut antiarrhythmic effects were found in pigs (anesthetized with sodium thiopental) during a 120-minute reperfusion period following prolonged ischemia. ¹⁶ In addition to the confounding effects of anesthesia, the lack of significant reduction of VT/VF after postconditioning in swine may be explained by the wide variability in arrhythmic responses in this species ³³ and the inherent limitations of this model in the evaluation of VT/VF. ³⁴

Effects of Pre- and Postconditioning on Infarct Size

Preconditioning has been consistently shown to afford cytoprotection, ⁴ but the previously reported effects of postconditioning on infarct size have varied widely, ⁵ likely due to differences in experimental settings and I/R protocols ³⁵ ; hence, the relative efficacy of pre- and postconditioning remains poorly defined. ³⁶ In the present work, we directly compared infarct size after these interventions under identical experimental conditions; in agreement with the previous studies, ⁴ we found a powerful cytoprotective effect of preconditioning, leading to a ∼35% reduction in infarct size compared to the I/R group. In contrast, this effect was less prominent after postconditioning, leading to only a borderline reduction in infarct size. Thus, the cytoprotection afforded by preconditioning appears more potent than postconditioning in the in vivo rat model, but further head-to-head comparison may be required for firm conclusions. The latter statement is underscored by the equal reduction of sympathetic activation (during phase IIB) observed after either intervention in our experiments; indeed, sympathetic activation during this time frame correlates with the extent of myocardial necrosis and acute LV failure development.^17,19

Effects of Pre- and Postconditioning on Arrhythmogenesis

Preconditioning has been shown to decrease the incidence of ischemic VT/VF. ³⁷ In agreement with these findings, we demonstrated a marked reduction in arrhythmogenesis during the ischemic phases IA and IB, consisting of decreased number of VT/VF episodes, with a shorter mean duration. Preconditioning also blunted reperfusion arrhythmias during the early reperfusion phase, but this effect was attenuated during subsequent stages; this observation is consistent with previous results,^4,37 indicating a limited duration of preconditioning effects. On the other hand, postconditioning almost abolished reperfusion arrhythmias during the early phase in our experiments, and this effect was sustained during subsequent hours. Nonetheless, the importance of this observation is dubious, as the incidence of VT/VF was low in reperfused infarcts during phase IIA. This arrhythmia pattern is in line with clinical data, demonstrating a marked reduction in inhospital arrhythmogenesis beyond the initial phase of reperfusion. ³⁸

Mechanisms of Antiarrhythmic Action of Pre- and Postconditioning

The mechanisms of antiarrhythmic action of preconditioning have been subject of intense research during the past decade. ⁴ Using MAP recordings, we examined the electrophysiologic milieu of the peri-infarct area, which is thought to play an active role in the pathogenesis of ischemic arrhythmias. ¹⁸ However, no differences were detected between preconditioning and I/R groups with respect to either phase 0 upstroke or APD. Thus, we cannot confirm previous findings in dogs, indicating preserved MAP duration after preconditioning, ³⁹ although species differences in MAP morphology preclude direct comparison.

Autonomic modulation has been suggested as a mechanism underlying the antiarrhythmic action of preconditioning. ⁴⁰ In isolated, Langendorff-perfused rat hearts, decreased arrhythmogenesis after preconditioning was mediated by short-term stimulation of α1-adrenergic receptors by catecholamines (either endogenous or exogenously administered) ⁴⁰ ; these antiarrhythmic effects were reversed by the selective α1-adrenergic blocker prazosin and involved protein kinase C activation through a G-protein-mediated pathway. ⁴⁰

Although we did not find any differences in HRV, the finding of decreased number and duration of ischemic VT/VF episodes in our preconditioned rats favors autonomic modulation as a candidate mechanism. Moreover, α1-adrenergic receptor stimulation could provide an explanation for lower HR after preconditioning in our experiments, observed during ischemia and reperfusion. Indeed, α1-adrenergic receptors are present in the sinus node of various species, including rats ⁴¹ and humans, ⁴² and their activation exerts bradycardic effects. ⁴³ Taken together, these findings underline the previously demonstrated ⁴⁴ pathophysiologic role of α1-adrenergic receptors on ischemic and reperfusion arrhythmias and call for further research on this issue.

In contrast to preconditioning, no differences were found in the HR-response between postconditioning and I/R groups. This observation, along with the differing effects on the VT/VF duration (per episode) during early reperfusion, implies diversities in the antiarrhythmic mechanisms between the 2 interventions.

As arrhythmogenesis correlates with the extent of myocardial necrosis, ¹⁷ the antiarrhythmic effects of postconditioning could be merely attributed to its cardioprotective effects. ²³ However, this explanation seems unlikely, based on the modest reduction in infarct size found in our experiments; more importantly, potent antiarrhythmic effects were demonstrated after postconditioning following short (5 minute) ischemia, in the absence of myocardial necrosis.^13,14 Thus, alternate antiarrhythmic mechanisms are bound to be operative, but these remain elusive, despite hitherto research. ⁴⁵

Strengths and Limitations

The present study compared the cardioprotective and antiarrhythmic effects of pre- and postconditioning in the in vivo rat model of I/R. A major strength of this work is the evaluation of arrhythmogenesis and autonomic balance in conscious animals for an extended observation period. However, 2 limitations should be acknowledged; first, our study is underpowered to detect more subtle differences between pre- and postconditioning, with respect to infarct size and arrhythmogenesis. Given the sample size and the power of the study, the survival data presented here are relatively weak, and a type II error in the comparisons of infarct size is possible. Second, MAP recordings from several ventricular sites at more time points during the observation period would have enabled more solid inferences with respect to the mechanisms underlying the antiarrhythmic effects of pre- and postconditioning; however, this would be at the cost of increased invasiveness of the procedure, with potential interference with the arrhythmia responses.

Clinical Significance

The decreased arrhythmogenesis observed after postconditioning in our experiments adds to the current evidence favoring this intervention as a means of ameliorating reperfusion injury in the clinical setting.^10,11 In addition, our results are in line with those reported in a recent small-scale clinical study, ⁴⁶ indicating decreased QT dispersion and ventricular arrhythmias in patients with acute MI undergoing primary coronary intervention.

The clinical value of decreasing ventricular arrhythmogenesis after postconditioning is significant, despite the fact that they are invariably managed effectively in the setting of the catheterization laboratory or in the coronary care unit; indeed, prevention of inhospital VT/VF is desirable, as their occurrence is associated with increased short-term morbidity and mortality.^38,47

Conclusion

In the in vivo rat model of I/R, the cardioprotective effects of preconditioning appear more potent than those of postconditioning. Both interventions exerted prominent antiarrhythmic effects, independent of the final infarct size. Preconditioning decreased the incidence of VT/VF during ischemia and early reperfusion phase; postconditioning markedly decreased reperfusion arrhythmias during the early stage, and these effects were sustained during the entire (24 hours) observation period. Different mechanisms may underlie the antiarrhythmic potential of pre- and postconditioning, which appear operative in addition to their cardioprotective properties.