Sum of effects of myocardial ischemia followed by electrically induced tachycardia on myocardial function

Background

OBJECTIVE:

The aim of the study was to analyze the physiopathology of cardiac dysfunction in an acute experimental model of a supraphysiological increase in heart rate preceded by ischemia, considering the influence of the type and duration of ischemia.

Material and Methods

EXPERIMENTAL SERIES:

A total of 24 young pigs of both sexes (7 males and 17 females) with a mean weight of 24.5±3 kg were used. The experiments were conducted respecting the Spanish legislation on “Protection of animals used for experimental and other scientific purposes” and the directives of the European Community (Royal Decree 1201/2005).

Four experimental series were formed by direct allocation of animals to different groups by a random method of the animal house staff, using no specific procedure to mask distribution. The series were: (S1), ventricular pacing was performed in 5 pigs following the protocol discussed later; (S2: A and B), series of a single ischemia and ventricular pacing, S2A consisted of 5 pigs that underwent complete occlusion of the left anterior descending coronary artery (LAD) for 10 min before applying the stimulation protocol, and S2B included 5 pigs with occlusion of LAD for 20 min prior to the stimulation protocol; S3, in 5 pigs, 20 processes of ischemia with a duration of 2 min followed by 3 min of reperfusion for each process were performed; (S4), control series in which the experimental preparation was performed in 4 pigs without causing ischemia or applying ventricular pacing.

EXPERIMENTAL PREPARATION:

The animals were anesthetized with thiopental (15 mg/kg. weight) bolus IV followed by endotracheal intubation and channelling of the external jugular. Maintenance of anesthesia was achieved with sevoflurane (2.5%) in a mixture of 40% oxygen and 60% nitrous oxide. Analgesia and relaxation were obtained with vecuronium bromide (0.08 mg/kg. weight) and 20 mg of morphine chloride bolus IV and maintained at the same dose in 50 cc of physiological saline in an infusion pump at 12 ml/h. Gasometric tests were conducted to monitor and correct the respiration parameters; rectal temperature was monitored and maintained at 38–39°C with an electric blanket; and continuous peripheral ECG recording was performed with subcutaneous electrodes in the extremities.

Opening the thorax was performed through a sternotomy and pericardial opening to expose the heart. After dissection of the LAD at its upper and middle thirds, a flowmeter (Transonic Systems, NY, USA) was placed at the top, and in the middle third a reference was located to carry out coronary occlusions. A flowmeter was also implanted around the aortic root for calculation of stroke volume and cardiac output. Two pairs of ultrasonic microcrystals (Biopac Systems Santa Barbara, CA, USA) were introduced in both the subepicardium in the area of the left ventricular apex (subsidiary of LAD) and the subepicardium of the base of the left ventricle (subsidiary of the circumflex coronary artery [LCx]). A catheter was introduced through the free wall of the left ventricle to record ventricular pressure.

ISCHEMIC PROTOCOL:

In series 2A and B, there was a single occlusion of the LAD in its middle third with a vascular clamp, in S2A for 10 min and in S2B for 20 min. In series 3, there were 20 occlusions of the LAD of 2 min each followed by 3 min of reperfusion.

STIMULATION PROTOCOL:

An electro-catheter was fixed by 1 point of suture to the anterior face of the upper third of the left ventricular epicardium in all the animals. Electrical stimulation (Arrhythmia Investigation System Type 4279 DEVICES, Hertfordshire, UK.) was performed with an intensity twice the threshold calculated for each experiment and 60% above its baseline rate for 2 h. The 4 control animals of the S4 remained with the thorax open and instrumented for 5 h without performing coronary ischemia or ventricular pacing.

DATA COLLECTION:

All parameters of cardiac function were digitized and stored in an electronic memory system (BIOPAC systems Inc., Santa Barbara, CA, USA): ECG (LII or LIII); left ventricular peak systolic pressure (LVP in mm Hg); end-diastolic left ventricular pressure (EDLVP in mm Hg); the first derivative of the pressure in relation to time (dP/dt of LVP in mm/sec.); aortic flow shown as stroke volume (ml) and LAD flow (ml/min). Segmental myocardial function parameters, ie, end-diastolic and end-systolic lengths (mm), and the shortening fraction (% over the end-diastolic length) of the apex and base of the left ventricle were recorded with a Sonometrics Corporation Digital Ultrasonic Measurement System (London, Ontario, Canada).

The following measures were selected for analysis: 1) baseline after experimental preparation; 2) at the end of ischemia in series 2A, 2B and 3; 3) before pacing; 4) during pacing at 30, 60, 90, and 120 min; and 5) at 5, 30, and 60 min after cessation of pacing. In the control series (S4), these parameters were recorded after preparation (1^st h) and 4 more hourly monitorings.

The role that oxidative stress may play in this model of tachymyocardiopathy and ischemia was evaluated by quantification of serum levels of glutathione (GSH GSSG; Cayman Chemical Company, USA) as an antioxidant marker, and lipoperoxide (LPO) levels (Lipid hydroperoxide Assay Kit Cayman Chemicals Company) as an oxidative marker.

Blood samples for analysis of parameters of oxidative stress were obtained: 1) at baseline (before opening of thorax); 2) before pacing; 3) after 60 min of pacing; 4) immediately after cessation of pacing; and 5) 60 min after cessation of pacing. In S4, blood samples were drawn: 1) at baseline; 2) after instrumentation; and 3) at 60, 120, and 180 min after manipulation.

A total of 15 ml of blood was extracted each time in sterile tubes containing EDTA, centrifuged at 3000 rpm at 4°C for 10 min and the plasma obtained was stored at −80°C in 3 cryopreservation tubes of 2 ml each until their study.

STATISTICAL ANALYSIS:

To determine the differences between means, sample size was calculated based on those used in other studies [14–16]. All data are represented as mean and standard deviation. Normal distribution was verified with the Kolmogorov-Smirnov test. To determine differences between mean values along the protocol with respect to baseline, ANOVA and Student’s t test were used for paired and unpaired samples when comparisons were made between series. Mann-Whitney or Kruskal-Wallis tests were performed in the parameters whose distribution was not normal. It was assumed that differences between means were statistically significant when the p value was <0.05. Statistical analysis was performed using SPSS 9.0 for Windows.

Results

OVERALL FUNCTION:

Stroke volume did not differ significantly during ischemia and reperfusion (S2A-B), presenting similar values in all series. Ventricular pacing led to a decline in stroke volume and its recovery at the cessation of pacing. There were no significant differences among series in any of the phases of the study. S3 showed a significant decrease until the end of follow-up compared to baseline (26.9±5.3 vs. 16±6.2 ml; p<0.05) (Table 3).

Cardiac output did not differ among series in any phase of the study, although in S1 there was an elevation together with increased heart rate and stroke volume after ventricular pacing with respect to baseline (Table 1), and in S3 there was a significant decline after ventricular pacing with respect to baseline (2.7±0.6 vs. 1.5±0.9 l/min, p<0.05) (Table 3).

During ventricular pacing, LVP decreased significantly in all cases and recovered after cessation except in S3, which was already affected by the ischemia and did not recuperate after pacing, remaining significantly lower at the end of the study compared to baseline (70.6±6.6 vs. 46±8.4 mm Hg, p<0.05, Table 3) and other series (Figure 1).

The EDLVP showed no significant differences during the study from baseline in any series (Tables 1–4) or between series. Both maximum +dP/dt and minimum −dP/dt of the left ventricle in S3 showed a significant impairment relative to baseline (Table 3) and they were significantly reduced with respect to the other series at 60 min after pacing (p<0.05 S3 vs. S1, S2A, S2B and S4).

CORONARY FLOW:

The flow of the LAD in S2A and B, in which continuous ischemia was induced, showed significant hyper flux after ischemia compared to baseline (Table 2) and to the other series (Figure 2). During ventricular pacing it decreased in all series and recovered in all except for S3, which remained significantly lower at the end of the study when compared to baseline (13.1±1.5 vs. 8.4±1.6 ml/min, p<0.05) (Table 3).

REGIONAL FUNCTION:

Tables 1–4 show the mean and standard deviation values of the myocardial-shortening fraction at the base and apex of the left ventricle in each series throughout the study. To compare regional function parameters among series, the percentage of change of shortening fraction compared to baseline has been used (Figures 3 and 4).

Left ventricle base shortening fraction (Base SF): During ischemia, significant differences between series were observed. In the series with short, repeated ischemia (S3), there was a reduction of the shortening fraction compared with an increase in the series with 10 min of ischemia (S2A) (p=0.01). In the 2 h of ventricular pacing, the shortening fraction was significantly decreased in S3 and S2B compared to S1 (p=0.04), S2A (p=0.04), and S4 (p<0.001). These differences disappeared in the recovery period, although there was a downward trend in S3 (Figure 3) with significantly lower values when recovering with respect to baseline (5.6±2.8 vs. 2.2±0.7%, p<0.05, Table 3).

Left ventricle apex shortening fraction (SF apex): In the variable percentage of change in the shortening fraction, differences among series were statistically significant (p<0.001) during the course of ischemia. These differences were expected and were due to the fact that the series with ischemia showed a reduction in shortening fraction compared with control series (S4) and the series without ischemia (S1) in measurements made in the first hour, and before pacing, respectively. However, among the series submitted to ischemia, there were no differences during ischemia or reperfusion. During ventricular pacing, all series showed significantly lower values in the shortening fraction compared to S4, with no differences between them, but in the recovery, differences were lost (Figure 4). The 3 series with ischemia prior to pacing showed impairment of the shortening fraction of this region at the end of follow-up (Tables 2 and 3).

PARAMETERS OF OXIDATIVE STRESS:

The measurements of LPO levels show that in the extraction performed prior to ventricular pacing there were no significant changes compared to baseline in any series or differences between series. Table 5 shows the average values of each series in each extraction. During ventricular pacing, all series reduced LPO levels, without significant differences between them. After cessation of pacing, LPO values were increased compared to values obtained during ventricular pacing in all series. After 1 h of recovery, S2B and S3 presented higher levels than the initial LPO (Figure 5A), with significantly higher levels in S3 than the less aggressive S1 (p<0.05), S4 (p<0.05), and S2A (p=0.02).

During the experimental protocol, the total glutathione (GSH + GSSG) activity and the GSH/GSSG ratio of the plasma underwent fluctuations that differed depending on the series. In the blood extraction conducted before ventricular pacing, it was observed that glutathione activity was reduced in all series when compared with the values prior to opening the thorax (Table 5), with values of GSH/GSSG below baseline values. Only series 1 showed recovery of the ratio during and after stimulation; the other series presented significantly lower endpoint values of the ratio GSH/GSSG and total glutathione compared to baseline (Figure 5), with significant differences in S1 compared to S2A (p<0.05) and S3 (p<0.05) series with previous ischemia.

Discussion

LIMITATIONS OF THE STUDY:

Since this experimental model involves opening the thorax, there is an increase in oxidative stress (see control series, S4) that adds to the oxidative stress due to ischemia and cardiac pacing. In spite of this, series with more aggressive ischemic protocol had higher oxidative activation.

Conclusions

Ischemia and high cardiac rate occurring simultaneously have additive effects, resulting in increased cardiac involvement. The type of ischemia influenced the involvement of global and regional cardiac function; the model of brief, repeated ischemia and reperfusion followed by tachycardia was more likely to induce heart dysfunction than the models in which ischemia was continuous (at least 20 min). This could be useful for the development of new animal models that reproduce mechanisms of acute cardiac failure in the context of ischemic heart disease and tachyarrhythmia.