Bullfrog heart model reproducing electrocardiographic changes of lateral wall myocardial infarction

Abstract

In previous experiments using bullfrog hearts, we successfully replicated both the elevation and reciprocal depression of the ST segment observed in electrocardiograms of human ischemic heart disease. In this study, we induced subepicardial burn injuries in the lateral part of the frog heart ventricle, which allowed us to mimic typical electrocardiographic changes associated with human lateral wall myocardial infarction. These changes included a marked ST segment elevation in the high lateral limb leads (I, augmented Voltage Left; aVL) and reciprocal ST segment depression in the opposite limb leads (III, augmented Voltage Foot; aVF). We surmise that the electrical disparity between injured and intact cardiomyocytes generates currents of injury, leading to the observed ST segment alterations.

Lateral wall myocardial infarction represents 15–20% of all cases of acute myocardial infarction [21]. Although it generally has a better prognosis than anterior wall myocardial infarction, serious complications such as ventricular arrhythmias, sudden cardiac arrest, rupture of the free wall or papillary muscle, and severe hypotension due to left ventricular dysfunction can lead to worse outcomes [8, 18, 22]. The rapid diagnosis of acute myocardial infarction is crucial for improving patient outcomes, with electrocardiography being the most effective method in this regard [25]. However, in lateral wall myocardial infarction, the characteristic elevation of the ST segment may not be easily detectable, as these changes are often subtle and noncontiguous in electrographic limb leads [6, 19, 20]. Moreover, ST segment elevation alone does not confirm acute myocardial infarction, as it can also occur in other cardiac conditions such as early repolarization, acute pericarditis, right bundle-branch block, and Takotsubo cardiomyopathy [7, 10, 13]. A “reciprocal” ST segment change refers to ST segment depression on electrocardiographic leads opposite to those showing ST segment elevation [26], a common finding in acute myocardial infarction. The presence of this reciprocal change is a distinctive feature of acute myocardial infarction, confirming its diagnosis [3, 28]. In our previous studies, we replicated both the ST segment elevation and reciprocal ST segment depression characteristic of human ischemic heart disease by inducing burn injuries on the surface or opposite side of bullfrog hearts [11, 15]. Additionally, by inducing subepicardial burn injuries on the inferior part of the frog heart ventricle, we reproduced electrocardiographic changes similar to those observed in human inferior wall myocardial infarction [12]. In the present study, we successfully replicated electrocardiographic changes similar to those observed in human lateral wall myocardial infarction by inducing burn injuries on the lateral part of the frog heart ventricle.

Fifteen adult bullfrogs, each weighing between 450 and 550 g, were purchased from Ohuchi Shōten (Saitama, Japan) for the experiments. The frogs were anesthetized using isoflurane (Pfizer Inc., New York, NY, USA), followed by an intramuscular injection of the long-lasting anesthetic alfaxalone (10mg/kg; Meiji Animal Health Co., Ltd., Tokyo, Japan), as previously described [2, 14, 23]. Then, silver wire needle electrodes (coated with silver chloride and soldered to the output pins) were inserted into the upper and lower limbs of the frogs under deep anesthesia (Fig. 1). These electrodes were linked to an electrocardiogram (ECG) amplifier to capture electrical signals from the limb leads (leads I, II, III, augmented Voltage Right; aVR, augmented Voltage Left; aVL, and augmented Voltage Foot; aVF), as previously described [12]. In the bipolar limb leads, such as lead I, the left-arm electrode functioned as the exploring electrode (positive pole), whereas the right-arm electrode served as the reference electrode (negative pole), detecting the potential difference between the right and left arms [4, 12, 17] (Fig. 1). Similarly, lead II measured the potential difference between the right arm and left leg, whereas lead III assessed the potential difference between the left arm and left leg (Fig. 1). The right-leg electrode served as an electronic reference to reduce unwanted noise (Fig. 1). In the augmented unipolar limb leads aVR, aVL, and aVF, the electrodes in the right arm, left arm, and left leg, respectively, served as the positive pole, whereas the negative pole was formed by averaging the readings from the electrode in the other limbs [4, 12, 17] (Fig. 1). ECG waveforms from these limb leads were then recorded using a data logger (midi LOGGER HV GL2000, GRAPHTEC Corp., Yokohama, Japan). The heart was surgically exposed to create a subepicardial injury on the lateral wall of the ventricle. As previously described [11, 12, 15], the tip of a glass capillary tube (1.5 mm in diameter) was heated to over 600°C and quickly pressed onto the lateral ventricular surface (Fig. 1). Repeating this process several times resulted in overlapping burn injuries in the subepicardial myocardium on the lateral wall of the heart. After the experiments, the bullfrogs were euthanized via alfaxalone overdose. All experimental procedures were approved by the Ethics Review Committee for Animal Experimentation of Miyagi University (No. 2025-01). We analyzed the experimental data by Microsoft Excel (Microsoft Corp., Redmond, Washington, DC, USA) and reported them as means ± SD.

Fig. 1.

Detection of electrocardiogram (ECG) signals from the limb leads and induction of lateral wall burn injury in a bullfrog heart. Needle electrodes inserted into the upper and lower frog limbs were connected to an electrocardiographic amplifier, and the electrical signals were detected using a data logger. In the bipolar limb leads, such as lead I, the left arm (L) electrode serves as an exploring electrode (the positive pole) and the right arm (R) electrode as a reference electrode (the negative pole). Similarly, lead II detects the potential difference between the right arm and left leg (LF), whereas lead III detects the difference between the left arm and left leg. The right-leg (RF) electrode serves as an electronic reference to improve the rejection of unwanted noise. In the augmented unipolar limb leads augmented Voltage Right; aVR, augmented Voltage Left; aVL, and augmented Voltage Foot; aVF, the electrodes in the left arm, right arm, and left leg serve as the positive pole, whereas the negative pole formed from the average of the measurements from the other limb electrodes. To induce subepicardial burn injuries on the lateral wall of the myocardium, a heated glass capillary tube was repeatedly placed on the lateral part of the frog heart ventricle.

By placing electrodes directly on the ventricular surface of the heart (precordial leads) (Fig. 2A and 2B, top), ECG waveforms consisting of a series of QRS complexes followed by T waves, similar to those in humans, were obtained (Fig. 2A and 2B, bottom). Upon the induction of burn injuries on the lateral ventricular surface where the electrocardiographic electrode was placed (Fig. 2A, top), the ECG immediately displayed a marked elevation of the ST segments from the isoelectric line (625 ± 181 mV increase from the isoelectric line, n=6; Fig. 2A, bottom), signifying the occurrence of myocardial injury. Conversely, when the electrographic electrode was placed on the ventricular surface opposite to the side where the burn injuries were inflicted (Fig. 2B, top), the ECG showed pronounced depression of the ST segment from the isoelectric line (331 ± 171 mV decrease from the isoelectric line, n=6; Fig. 2B, bottom). The morphological patterns of the depressed ST segments mirrored those of the elevated ones (Fig. 2B, bottom right vs. Fig. 2A, bottom). This resembled the “reciprocal” ST segment changes often seen in human acute myocardial infarction [26], indicating myocardial injury on the side opposite to where the electrocardiographic electrode was placed.

Fig. 2.

Changes in precordial lead electrocardiogram (ECG) signals before and after burn injuries were induced on the lateral wall of the frog heart ventricle. (A) Top: An electrocardiographic electrode was placed on the lateral wall of the ventricle where subepicardial burn injuries were induced. Bottom: ECG signals detected from the precordial lead before (left) and after (right) burn injuries were induced. (B) Top: An electrocardiographic electrode was placed on the side opposite to the lateral wall of the ventricle where the subepicardial burn injuries were induced. Bottom: ECG signals detected from the precordial lead before (left) and after (right) burn injuries were induced.

Compared with the ECG waveform amplitudes recorded from the precordial leads (Fig. 2), those obtained from the limb leads were significantly smaller (Fig. 3). Nonetheless, these waveforms were sufficient for displaying a series of distinct QRS complexes followed by T waves (Fig. 3), with the ST segments recorded along the isoelectric lines. Shortly after the burn injuries had been inflicted on the lateral part of the ventricular surface (Fig. 1), the ECG from leads I and aVL showed obvious elevations in the ST segments (I, 24.7 ± 15.3 mV increase from the isoelectric line, n=6; aVL, 24.6 ± 11.5 mV increase from the isoelectric line, n=6; Fig. 3). As we have previously shown, such changes in the ST segment suggest that myocardial injuries had been induced in the frog heart ventricle [11, 12, 15]. Moreover, the pattern of changes in these limb leads resembled those typically observed in acute lateral myocardial infarction in humans [6, 21]. Conversely, the ECG from leads III and aVF exhibited obvious depressions in the ST segments from the isoelectric lines (III, 20.2 ± 10.6 mV decrease from the isoelectric line, n=6; aVF, 15.6 ± 9.50 mV decrease from the isoelectric line, n=6; Fig. 3). These observations also mirrored the reciprocal ST segment changes often observed in human lateral wall myocardial infarction [20, 21]. Quantitative data of the ST segment changes in precordial and limb leads were summarized and shown in graphs (Fig. 4). Generally, the presence of reciprocal changes in ECG signals indicates the extent of myocardial ischemia [9] and supports the diagnosis of acute myocardial infarction [3, 28]. Notably, in patients with high lateral myocardial infarction, the presence of such reciprocal ST segment depression in leads III and aVF is known as the South African flag sign [1, 20, 21], which clinically suggests occlusion of the first diagonal branch of the left anterior descending coronary artery as the culprit lesion [5, 21, 24]. Therefore, the typical ECG findings replicated in our frog heart model can assist in identifying the target vessel for revascularization, thereby facilitating the prompt initiation of coronary angioplasty.

Fig. 3.

Changes in limb lead electrocardiogram (ECG) signals before and after burn injuries were induced on the lateral wall of the frog heart ventricle. ECG signals detected from the bipolar limb leads (I, II and III) and unipolar limb leads (augmented Voltage Right; aVR, augmented Voltage Left; aVL and augmented Voltage Foot; aVF) before (left) and after (right) burn injuries were induced.

Fig. 4.

ST segment changes before and after burn injuries were induced on the lateral wall of the frog heart ventricle. (A) Quantitative data of ST segment changes in precordial lead electrocardiogram (ECG) signals. (B) Quantitative data of ST segment changes in limb lead ECG signals. Values are means ± SEM (n=6).

In the present study, cardiac burn injury did not affect the sinus rhythm (Figs. 2 and 3). In human hearts, right coronary artery is the major source of the blood supply to the sinoatrial (SA) and atrioventricular (AV) nodes that are responsible for the heart rate and AV conduction [27]. Therefore, inferior wall myocardial infarction, which is generally caused by the occlusion of the right coronary artery, often accompanies with sinus bradycardia or AV conduction block [27]. On the other hand, since lateral wall myocardial infarction is generally caused by the occlusion of the left circumflex artery or the diagonal branch of the left anterior descending [21], it does not usually accompany with bradyarrhythmia.

In our previous studies, burn injuries reduced the protein expression of sodium/potassium (Na⁺/K⁺)-ATPase in cardiomyocytes, thereby diminishing the activity of the pump [12, 15]. Consequently, this hindered the transport of extracellular K⁺ ions back into the cardiomyocytes, increasing the ratio of extracellular to intracellular K⁺ concentrations. According to the Nernst equation [11, 15], this results in a shift in the resting membrane potential of injured cardiomyocytes toward depolarization. Such electrical difference in the resting membrane potential induced between the injured and adjacent intact cardiomyocytes would generate “currents of injury” within the myocardium [16]. Among the limb leads, I and aVL are referred to as “high lateral limb leads” because they detect electrical heart vectors directed toward the left superior part of the ventricle (Fig. 5, middle), with the electrode in the left arm acting as the positive pole. In other words, these high lateral limb leads can be described as primarily “observing” the heart from the left superior wall of the ventricle (Fig. 5, middle). From the perspectives of leads I and aVL (Fig. 5, white arrows), the currents of injury flowed away when myocardial injury was induced on the lateral wall of the ventricular surface (Fig. 5, middle). During the diastolic phase of the cardiac cycle, the flow of these currents resulted in a negative deflection of the ECG vector from the isoelectric line (Fig. 5, top), which in turn caused the ST segment to appear elevated during systole. In contrast to the high lateral limb leads, leads III and aVF capture electrical heart vectors directed toward the right inferior section of the ventricle (Fig. 5, middle). These leads essentially provide an observation perspective of the heart from the right inferior part of the ventricle (Fig. 5, middle), which is almost opposite to the orientation of leads I and aVL. Consequently, when myocardial injury was induced on the lateral wall of the ventricle (Fig. 5, middle), the injury currents flowed almost directly toward the perspectives of leads III and aVF (white arrows). This resulted in a positive deflection of the ECG vector during diastole (Fig. 5, bottom), causing the ST segment to appear depressed during the systolic phase.

Fig. 5.

Mechanisms underlying reciprocal ST segment changes in the frog model of lateral wall burn injury. Leads I and aVL are expressed as if they “observe” (view) the heart from the left superior wall of the ventricle. From the perspectives of leads I and aVL (white arrow), the currents of injury flow away when myocardial injury is induced on the lateral wall of the ventricular surface. Thus, the electrocardiogram (ECG) vector negatively deflected from the isoelectric line (arrows) during diastole, making the ST segment appear elevated during the systolic phase (gray waveform). By contrast, leads III and aVF are expressed as if they observe the heart from the right inferior part of the ventricle, which is almost opposite to the direction of leads I and aVL. When myocardial injury is induced on the lateral wall of the ventricle, the currents of injury flow nearly toward the views of leads III and aVF (white arrows). This causes the ECG vector to be positively deflected during diastole (arrows), resulting in the ST segment appearing depressed during the systolic phase (gray waveform).

In conclusion, by creating subepicardial burn injuries on the lateral part of the frog heart ventricle, we were able to replicate typical electrocardiographic changes similar to those seen in human lateral wall myocardial infarction, such as a marked elevation of the ST segments in the high lateral limb leads (I, aVL) and their reciprocal depression in the opposite limb leads (III, aVF).

CONFLICTS OF INTEREST

The authors have no conflicts of interest to declare