Development of Electrocardiography Standards for Evaluating Myocardial Infarction and Ischemia–Reperfusion Injury in Mice

Abstract

Background

Acute and chronic heart failure secondary to myocardial infarction (MI) and cardiac ischemia–reperfusion injury (IRI) are leading causes of death in ischemic heart disease. A mouse model is indispensable for investigating MI and IRI, and the development of reliable mouse MI and IRI models is essential for advancing research in this field. The clear early diagnostic criteria for confirming successful induction of MI and IRI in mice remain lacking.

Methods

Adult C57BL/6J background mice underwent left anterior descending coronary artery ligation to induce acute MI, or ligation followed by reperfusion to induce IRI. The success of the MI and IRI model establishment was confirmed by 2,3,5‐triphenyltetrazolium chloride staining and echocardiography. Electrocardiography was used to monitor the electric activity in the mice.

Conclusions

Electrocardiography demonstrated that ST‐segment elevation in ECG lead II and corrected QTc interval prolongation at 30 minutes following left anterior descending ligation as 2 key early indicators of successful MI. Echocardiography analysis revealed that the magnitude of ST‐segment elevation strongly correlated with the left anterior descending ligation site, where a more proximal ligation produced a greater ST‐segment elevation amplitude and more severe ischemia. In IRI models, ST‐segment elevation typically resolved and returned to baseline within 20 minutes of reperfusion. This study developed quantifiable early diagnostic criteria for successful MI and IRI induction based on characteristic ECG changes. These quantifiable ECG parameters provide early diagnostic standards that can significantly streamline and optimize modeling procedures.

Nonstandard Abbreviations and Acronyms

IRI

ischemia–reperfusion injury

QTc

corrected QT

TTC

2,3,5‐triphenyltetrazolium chloride

Heart failure caused by myocardial infarction (MI) accounts for >9 million deaths worldwide annually. ¹ , ² The pathophysiology involves abrupt coronary artery occlusion leading to myocardial ischemia and subsequent necrosis. ³ Although modern treatments such as thrombolytic therapy and percutaneous coronary intervention have significantly reduced acute death, many patients still experience ischemia–reperfusion injury (IRI) or heart failure, adversely affecting long‐term clinical outcomes. ⁴ , ⁵ To tackle these ongoing issues, animal models remain essential for investigating the mechanisms of MI and IRI and for developing new therapeutic strategies. ⁶ Mice are the most commonly used mammals in cardiovascular research, and the availability of diverse genetically modified strains makes them an ideal animal model for such studies. ⁷ , ⁸ However, the validity of this murine model for establishing early diagnostic criteria for MI/IRI remains inconclusive, which may lead to misinterpretation of experimental results and hinder translational research. ⁹

The lack of highly sensitive and standardized early diagnostic criteria presents a major bottleneck in cardiovascular translational studies. ⁸ While recent comprehensive hemodynamic and electrophysiological characterizations have emphasized the necessity of rigorous model validation, ¹⁰ these approaches still rely on terminal end points or delayed functional assessments. Without immediate and definitive confirmation of successful ischemia, researchers risk carrying failed surgical models (eg, due to insufficient ligation depth or anatomic variations) through long‐term functional or pharmacological studies. This delayed recognition not only violates the 3Rs (Replacement, Reduction, Refinement) principles of animal welfare ¹¹ by unnecessarily using animals that yield uninterpretable data but also profoundly confounds downstream readouts. For instance, an unrecognized modeling failure might be falsely attributed to the cardioprotective effect of a novel therapeutic agent, generating false‐positive results. Therefore, establishing highly sensitive early diagnostic parameters is critically needed to screen out surgical failures immediately.

As a fundamental tool, electrocardiography provides critical insights into myocardial electrical activity in both clinical and experimental settings. ¹² , ¹³ , ¹⁴ In human ST‐segment–elevation myocardial infarction (STEMI), the magnitude of ST‐segment elevation (STE) correlates with ischemic burden ¹⁵ and the corrected QT (QTc) interval, a measure of ventricular repolarization, is associated with the MI prognosis. ³ , ¹⁶ However, interpreting a murine ECG presents unique challenges due to species‐specific characteristics, such as heart rates >400 beats per minute and low‐voltage QRS complexes. ¹⁷ , ¹⁸ Although previous studies have suggested that STE in lead II after left anterior descending (LAD) coronary artery ligation may serve as a potential biomarker for murine MI modeling, ¹⁹ , ²⁰ several obstacles remain in using electrocardiography as an early diagnostic tool for successful MI and IRI induction. ²¹ These obstacles include an insufficient characterization of dynamic electrocardiographic changes, the accuracy of STE, and a lack of quantitative thresholds linking STE magnitude to ligation position. In addition, significant baseline ECG variability across mouse strains and age groups complicates postoperative assessment, underscoring the need for strain‐specific analytical approaches.

To address these questions, this study systematically characterized perioperative electrocardiographic changes in murine MI and IRI models, focusing on the dynamics of lead II STE and its spatial relationship to LAD ligation sites. We identified STE (>0.1 mV) in ECG lead II and QTc interval prolongation (>47 ms) at 30 minutes after surgery as 2 major early indicators for successful MI induction. Furthermore, the magnitude of STE showed a positive correlation with the proximity of LAD ligation site to cardiac apex. In the IRI model, we observed that the elevated ST segment gradually returned to baseline within 20 minutes of reperfusion in all mice. Therefore, we established quantifiable early diagnostic criteria for validating murine MI and IRI models within a half‐hour based on electrocardiographic profiles. This methodological advance enables researchers to objectively assess surgical outcomes while accounting for intrinsic electrophysiological variability, thereby significantly enhancing experimental reproducibility in cardiovascular research.

Methods

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Animals

All animal procedures complied with the guidelines approved by the Animal Ethics Committee of Shanghai First People's Hospital and by the institutional animal care and use committee of Shanghai Jiao Tong University. The experiments used male and female mice (C57BL/6J background), which were housed and cared for at the Experimental Animal Center of Shanghai First People's Hospital and Shanghai Jiao Tong University. The experimental groups were as follows: an MI group (n=51) induced by ligation of the LAD coronary artery; a sham surgery group (n=7), which underwent thoracotomy without coronary artery ligation to serve as the controls; and an ischemia–reperfusion group (n=22), subjected to 30 minutes of LAD ligation followed by reperfusion.

Mouse MI Model

Anesthesia was induced by placing mice in a sealed chamber under continuous flow of isoflurane (3%–4% for induction, 1%–2% for maintenance) and oxygen (0.5–1 L/min). Surgical anesthesia, confirmed by loss of righting reflex and abolition of toe‐pinch response within 2 to 3 minutes, was followed by prompt transfer to a thermostatically controlled surgical platform (37 °C). Mice were positioned in supine orientation for orotracheal intubation under direct laryngoscopic guidance. Anesthesia was maintained via endotracheal delivery of isoflurane (1%–1.5%) in oxygen (0.3–0.5 L/min), with mechanical ventilation (Harvard Apparatus, Holliston, MA) providing 160 breaths/min at a 1:1 inspiratory‐to‐expiratory ratio. Under surgical microscopy, the heart was exposed via a left lateral thoracotomy at the third or fourth intercostal space, ≈1 cm left of the sternum. The left atrial appendage was used as the anatomic landmark to locate the LAD artery. The critical occlusion was achieved by dual ligation of the proximal LAD (1–2 mm distal to appendage base) using 7–0 polypropylene suture. Immediate ischemia was confirmed by regional pallor and hypokinesis in the anterolateral left ventricular wall. Postoperatively, the mice were placed on a 37 °C heating pad to maintain body temperature and were administered antibiotics to prevent infection. All postoperative mice were closely monitored for at least 24 hours to ensure uneventful recovery and to treat any potential complications promptly.

Mouse Ischemia–Reperfusion Model

For IRI modeling, the surgical workflow mirrored that of MI induction except for a controlled reperfusion phase. Following anesthetic stabilization via intubation and mechanical ventilation (Harvard Apparatus; 160 breaths/min, I:E 1:1), ¹⁴ a thoracotomy was performed to expose the LAD using identical anatomic landmarks. The critical modification was a timed coronary occlusion: Similar to the MI model, proximal LAD occlusion was achieved by dual ligation using a releasable 7–0 polypropylene suture loop (2 loops around the vessel) to ensure complete ischemia. Successful occlusion was confirmed visually by sustained regional pallor, and electrocardiographically by STE and QTc interval prolongation on lead II. After 30 minutes of occlusion, the ligature was carefully released to restore perfusion, and successful reperfusion was confirmed by visible hyperemic blush over the previously ischemic area. Following model completion, the mice were immediately administered antibiotics to prevent infection and were placed on a 37 °C heating pad to aid recovery. Postoperative mice were closely monitored for at least 24 hours to assess their general condition and address any potential complications.

ECG Monitoring and Analysis

Equipment and Settings

ECGs were recorded from leads I, II, III, aVR, aVL, and aVF using a small animal electrocardiography monitoring system, model MED‐ECG‐2303B (Guangzhou Med Equipment Limited, Guangzhou, China). The ECG signals were digitized at a sampling rate of 1000 Hz. Signal processing was performed with a system frequency response of 0.05 to 160 Hz. To minimize baseline drift and high‐frequency noise while preserving murine ECG morphological features, the system's built‐in filters were applied, including an adaptive antidrift filter at 0.15 Hz, an AC filter (50/60 Hz), and an electromyogram filter (25/45 Hz).

Time Points

ECGs were recorded at baseline (presurgery) and at various postoperative time points, including 3, 10, 20, 30 and 60 minutes.

Data Processing

Electrocardiographic recordings were obtained from anesthetized and intubated mice using a 12‐lead system (ECG‐2303B platform) with AMP12‐V2.5 software. During recording, heart rates were between 400 and 500 beats/min. The QT interval was corrected for heart rate (QTc) using a formula for mice. ²² QTc=QT interval/(RR interval ^ 0.5).

Echocardiography

Cardiac function as assessed using the MYLAB Sigma Vet echocardiography system equipped with a 22‐MHz high‐frequency probe (SL3116). Following stabilization under 1% to 2% isoflurane anesthesia (target heart rate, 400–500 beats/min), mice were positioned supine on a thermoregulated platform (37 °C) for echocardiographic image acquisition. To optimize acoustic coupling, thoracic fur was removed with a chemical depilatory agent, and ultrasound transmission gel was applied. Left ventricular images were acquired in the long‐axis view. Functional indices, including ejection fraction (EF) and fractional shortening, were measured from these images using the Teichholz method. ²³ All echocardiographic data were independently analyzed by 2 experienced operators to minimize observer bias. For parameters such as EF, the average of measurements from multiple cardiac cycles was calculated to improve accuracy. Statistical analysis of data between the experimental and control groups was performed using appropriate methods (eg, permutation t test).

2,3,5‐Triphenyltetrazolium Chloride Staining

After euthanasia by cervical dislocation, the chest cavity was opened. The heart was excised, rinsed with ice‐cold saline to remove blood, and briefly frozen at −80°C for ≈5 minutes. After freezing, 1‐mm‐thick serial sections were prepared from the heart (longitudinally or transversely) for subsequent staining and analysis. These sections were stained in a 1% 2,3,5‐triphenyltetrazolium chloride (TTC) solution by incubation at 37 °C for 20 minutes. During incubation, the staining dish was gently rocked to ensure even exposure of the tissue sections to the dye. After staining, the sections were rinsed with 1× PBS to clear any unbound dye. Viable myocardium, which contains active dehydrogenase enzymes, stained a deep red, whereas the infarcted necrotic tissue, lacking active enzymes, remained white.

Statistical Analysis

All statistical analyses were performed by researchers blinded to the group allocation. Data are presented as mean±SD. Linear regression (with covariate adjustments as appropriate) was used to assess the association between variables. For group comparisons, given the small sample sizes, exact permutation tests were used: 2‐tailed permutation t tests for 2‐group comparisons, and permutation 1‐way ANOVA (10 000 permutations) for 3‐group comparisons implemented using the coin package in R (R Foundation for Statistical Computing, Vienna, Austria). Multiple testing was controlled using the maxT permutation method. Prism 10 (GraphPad Software, La Jolla, CA) was used for data visualization. A P value of <0.05 was considered statistically significant. Exact P values are reported to 2 significant figures, and statistically significant results are highlighted in bold.

Results

STE in ECG Lead II Is a Remarkable Early Criterion for Validating Murine MI Model Success

To establish the early criteria for successful modeling of mouse MI, we performed LAD coronary artery ligation and recorded ECGs preoperatively and 1 hour postoperatively. To validate the surgery subsequently, hearts were harvested for TTC staining 24 hours after the procedure (Figure 1A). In TTC‐stained sections, the presence of a pale, unstained region indicates myocardial ischemia, confirming successful MI modeling. Conversely, the absence of such a region suggests the model was not successfully established (Figure S1). Based on TTC staining, MI was successfully induced in 86% of mice (44/51) that underwent LAD ligation. These TTC‐confirmed MI mice exhibited significant STE in lead II on ECGs recorded at 1 hour after ligation (Figure 1B). Conversely, MI induction failed in 6% of the mice (3/51), as assessed by TTC staining. These 3 mice did not exhibit significant STE in lead II (Figure S1). Subsequently, we performed comprehensive echocardiography on mice exhibiting STE in lead II at 1 week after ligation. Echocardiography results confirmed pronounced left anterior wall thinning and dyskinesia in these STE‐positive mice (Figure 1C), whereas mice without STE demonstrated normal ventricular structure and function (Figure S1). The EF and fractional shortening of the unsuccessful models were not significantly different from those of the sham group (Figure 1D and 1E). Furthermore, ECG analysis in successful MI cases revealed that, in addition to STE >0.1 mV in lead II, 94% of the mice (48/51) also exhibited STE >0.1 mV in leads III and aVF.

Figure 1. STE >0.1 mV in lead II as a criterion for validating murine MI model success.

A, TTC staining at 24 hours after surgery revealed the infarct area in a representative mouse heart. White demarcations indicate the ischemic tissues. Scale bar, 2 mm. B, ECG recordings showed STE in lead II at 60 minutes after surgery, demonstrating a classic pattern of myocardial ischemia. C, Echocardiography performed 1 week after surgery revealed damaged cardiac function with regional contractile impairment in the mice represented in (B). D, EF in the sham group (n=7), failed MI model group (n=7), and successful MI model group (n=20). E, Fractional shortening in the sham group (n=7), failed MI model group (n=7), and successful MI model group (n=20). Statistical significance was evaluated via permutation 1‐way ANOVA followed by permutation t tests with maxT correction (D and E). EF indicates ejection fraction; MI, myocardial infarction; STE, ST‐segment elevation; and TTC, 2,3,5‐triphenyltetrazolium chloride.

QTc Interval Prolongation in ECG Lead II Serves as a Predictive Marker for Successful Establishment of the Murine MI Model

Interestingly, 8% of mice (4/51) exhibited STE in lead II at 1 hour after ligation (Figure 2A) yet were later classified as modeling failures by TTC staining at 24 hours (Figure 2B). To investigate potential distinguishing features, we performed a comparative analysis of the ECG patterns between these TTC‐negative mice and the TTC‐positive mice (Figure 2A through 2D). We found that successful STEMI models exhibited significant prolongation of the QTc interval in ECG lead II (Figure 2C). Quantitative analysis revealed a marked disparity in QTc interval prolongation between murine models. Failed MI models demonstrated a ΔQTc of 9.51±3.6 ms (pre‐ versus postoperative), whereas successful MI models exhibited a pronounced ΔQTc prolongation of 76.19±28.92 ms (Figure 2E). Echocardiographic assessment at 1 week after ligation further validated the functional difference between the 2 STE‐presenting groups. Mice with STE but short QTc intervals exhibited preserved left ventricular systolic function (Figure 2F), in stark contrast with those with both STE and prolonged QTc intervals, which showed severe impairment (Figure 1C).

Figure 2. QTc interval prolongation in lead II as a biomarker of murine MI model success.

A, Mice that exhibited STE without QTc interval prolongation in lead II represent unsuccessful MI models. B, TTC staining at 24 hours after surgery showed no infarct area in mice from the group represented in (A). Red demarcations indicate the viable myocardium. Scale bar, 2 mm. C, Mice exhibited both significant STE and QTc interval prolongation in postoperative ECGs compared with baseline. D, TTC staining at 24 hours after surgery revealed the infarct area in mice represented in (C). White demarcations indicate ischemic areas. Scale bar, 2 mm. (E) Successful MI models (n=9) exhibited QTc prolongation vs failed (n=3) and sham models (n=9). F, Echocardiography performed 1 week after surgery in mice, as shown in (A) revealed preserved ventricular function. Statistical significance was evaluated via permutation 1‐way ANOVA followed by permutation t tests with maxT correction (E). MI, myocardial infarction; QTc, corrected QT; STE, ST‐segment elevation; and TTC, 2,3,5‐triphenyltetrazolium chloride.

Therefore, our findings established 2 essential early diagnostic criteria for successful MI modeling in mice, 1 being persistent STE exceeding 0.1 mV in lead II, and the other being concomitant QTc interval prolongation.

Reliable Early Diagnosis of Successful MI Models Based on ECG Patterns Could be Achieved as Early as 30 Minutes After Surgery

To determine whether model success could be diagnosed on early ECG profiles, we recorded ECGs at 3, 10, 20, 30, and 60 minutes after surgery. STE and QTc prolongation were observed in lead II as early as 3 minutes after surgery in 85% of mice that underwent LAD ligation (23/27). Seventy‐eight percent of the total mice (21/27) exhibited both electrical abnormalities at all subsequent time points. However, in 7.4% of mice (2/27), T‐wave abnormalities and QTc interval prolongation were transient and gradually returned to near‐baseline levels by the 30‐ and 60‐minute time points (Figure 3A). TTC staining and echocardiographic results confirmed that these mice did not develop MI (Figure 3B and 3C).

Figure 3. Changes in postoperative ECG at 30 minutes as a time window for diagnosis.

A, Acute STE developed in lead II immediately after LAD ligation but resolved spontaneously within 60 minutes. B, TTC staining at 24 hours after surgery showed no evident ischemic regions in mice, as shown in (A). Scale bar, 2 mm. C, Echocardiography at 1 week after surgery in mice, as shown in (A), revealed preserved ventricular function. D, STE was not observed until 30 minutes after surgery. E, TTC staining at 24 hours after surgery showed evident ischemic regions in mice, as shown in (D). Scale bar, 2 mm. LAD indicates left anterior descending; STE, ST‐segment elevation; and TTC, 2,3,5‐triphenyltetrazolium chloride.

Among the 15% of LAD‐ligated mice (4/27) that lacked both STE and QTc prolongation at 3 minutes, half (2 of 4) later developed delayed electrophysiological changes. In these mice, T‐wave alterations and QTc interval prolongation developed progressively between 10 and 20 minutes after surgery. These mice showed stable STE and QTc prolongation at 30 and 60 minutes after surgery (Figure 3D), and the presence of MI was confirmed by TTC staining (Figure 3E).

Therefore, 30 minutes after surgery is the earliest and most reliable time window to diagnose a successful MI model on the basis of the combined criteria of STE and QTc prolongation in lead II.

Magnitude of STE in ECG Lead II Is Positively Correlated With the Ligation Site on the LAD Coronary Artery

When we applied our early diagnostic criteria, we observed significant interindividual variability in the magnitude of STE in lead II. This variability is likely attributable to anatomic differences, as the LAD coronary artery originates from the left main coronary artery and extends toward the cardiac apex. ²⁴ Although our standardized surgical protocol consistently targeted a highly proximal LAD occlusion, intrinsic anatomic variations and inherent microsurgical variability inevitably resulted in deviations of the actual ligation site. Accordingly, the confirmed ligation sites ranged from highly proximal (close to the left main coronary artery) to relatively more distal positions. We further aimed to determine whether the ligated positions account for the various magnitudes of STE. To investigate the relationship between STE magnitude and ligation site, we recorded ECGs and harvested the hearts 1 day after surgery. We measured the normalized distance from the ligated site to the cardiac apex and quantified the STE magnitude from the preoperative and 1‐hour postoperative ECGs (Figure 4A). We then compared ECG parameters between mice with marked STE (>0.2 mV) and those with low STE (0.1–0.2 mV) at 30 minutes after surgery (Figure 4B). A significant positive correlation was observed between the relative ligation distance and the magnitude of STE measurements at 30 minutes post‐surgery (Figure 4C). Assessment of infarct size by TTC staining at 24 hours demonstrated that the extent of myocardial ischemia corresponded to the postoperative STE magnitude, with higher STE predicting a larger infarct area (Figure 4D). Echocardiography at 1 week demonstrated that mice with higher STE presented transmural infarction of the left anterior wall, whereas those with lower STE exhibited only a limited apical infarction (Figure 4E). Moreover, we found a significant negative correlation between EF and the magnitude of STE (Figure 4F). Both the ligation distance and STE magnitude showed a significant inverse relationship with EF, indicating that more proximal ligations and a greater STE were associated with more severely impaired cardiac function (Figure 4G and 4H). Together, our findings establish that the magnitude of the STE in lead II is determined by the ligation site on the LAD. This relationship confirms that proximal occlusions induce a more severe ischemic insult, which in turn leads to worse ventricular functional recovery.

Figure 4. The relationship among the magnitude of STE, location of LAD coronary artery ligation, and EF.

A, Macroscopic analysis showing the ligation sites on the LAD coronary artery in mice with high and low STE, examined 1 day after surgery. B, ECGs at 30 minutes after ligation from mice in the high‐STE (STE >0.2 mV) and low‐STE (STE <0.2 mV) groups. C, Correlation between the magnitude of lead II STE at 30 minutes after surgery and the distance from the cardiac apex. D, TTC staining of hearts from mice with high STE or low STE in lead II at 24 hours after surgery. Ischemic areas are white. Scale bar, 2 mm. E, Echocardiography was performed 1 week after surgery in mice with high STE or low STE in lead II. F, Correlation between the magnitude of lead II STE at 30 minutes after surgery and EF. G, Gross morphology at 4 weeks after surgery, showing differences in ventricular remodeling between mice with high STE or low STE in lead II. H, The EF and fractional shortening were measured in mice with lead II STE greater than (n=6) or less than (n=7) 0.2 mV at 7 days after surgery. Statistical significance was evaluated using linear regression (C and F) and 2‐tailed permutation t test (H). EF indicates ejection fraction; LAD, left anterior descending; STE, ST‐segment elevation; and TTC, 2,3,5‐triphenyltetrazolium chloride.

Establishing Early ECG‐Based Diagnostic Criteria for a Mouse Model of Cardiac IRI

To establish a cardiac IRI model, we require the ligation and the reperfusion steps. The first ischemic phase, induced by ligating the LAD for 30 minutes, is identical to that of an MI model. Therefore, we can apply the same early ECG diagnostic criteria used for the mouse MI model, STE and QTc interval prolongation in lead II of the ECG, as the standard for the IRI model's ischemic step. The second step of the IRI model involves releasing the ligation to allow myocardial reperfusion. We then recorded mouse ECGs at various time points following 30 minutes of ischemia. ECG analysis revealed that 14% of mice (3/22) exhibited STE that returned to baseline within 3 minutes of reperfusion (Figure 5A), while another 14% of mice (3/22) displayed a mild reduction in STE compared with its level during the ischemic phase (Figure S2A). While the majority of mice (16/22) did not show a decrease in STE, they did exhibit a significant increase in QRS complex voltage along with significant T‐wave inversion at 3 minutes after reperfusion (Figure 5B). Furthermore, we found that STE returned to baseline in 77% of mice (17/22) by 10 minutes after reperfusion (Figure 5B), with all mice (22/22) showing the same recovery by 20 minutes (Figure S2B). Moreover, TTC staining confirmed the presence of MI in these mice, which is consistent with the successful IRI model (Figure 5C). Therefore, these data showed distinct reperfusion‐specific ECG signatures. The first is characterized by QRS/T‐wave abnormalities, signifying early reperfusion injury. The second is defined by delayed STE normalization (>3 minutes in 86% of mice), which may reflect microvascular obstruction but ultimately resolves within 20 minutes after reperfusion.

Figure 5. Validation of ECG‐based diagnostic criteria for assessing the mouse IRI model.

A, Representative lead II ECG traces showing STE resolution within 3 minutes after reperfusion. Red arrow, QRS wave; black arrow, T wave. B, Representative lead II ECG traces showing delayed STE resolution at 10 minutes after reperfusion. C, TTC staining at 24 hours after surgery confirmed anterolateral infarction, as shown in (A). White demarcations indicate ischemic myocardium. Scale bar, 2 mm. I indicates ischemia; IRI, ischemia–reperfusion injury; R, reperfusion; STE, ST‐segment elevation; and TTC, 2,3,5‐triphenyltetrazolium chloride.

Discussion

Murine models of MI and IRI are indispensable in cardiovascular research for elucidating ischemic pathophysiology and developing therapeutic strategies. This study systematically defined the early electrocardiographic patterns indicative of successful MI and IRI induction in mice and established a significant correlation between the magnitude of STE and the extent of ischemic injury. These findings provide a practical approach for the rapid and accurate validation of murine MI and IRI models, thereby enhancing the precision and reproducibility of experimental outcomes in cardiovascular research.

Through real‐time ECG monitoring and TTC staining, we confirmed successful induction of MI in most mice (44/51). Modeling failure, observed in a small subset (3/51) that did not exhibit STE, was likely attributable to insufficient depth of the ligation into the myocardial layer. STE (defined as >0.1 mV) was identified as a primary early feature in the majority of mice (48/51). However, STE alone was not a definitive indicator of successful modeling, as TTC staining revealed failed MI induction in 4 mice that presented with STE but without concomitant QTc interval prolongation. Therefore, the prolongation of the QTc interval (defined as ΔQTc >47 ms) should also be incorporated into the assessment of successful MI modeling. The concurrent presence of both STE and QTc prolongation served as a reliable criterion for confirming MI in mice. Notably, no cases exhibiting isolated QTc interval prolongation in the absence of concomitant STE were identified in our cohort, suggesting that QTc prolongation in this model is pathognomonically associated with ischemic myocardial injury rather than primary electrophysiological disturbances. Mechanistically, the critical role of QTc prolongation in distinguishing true infarction from superficial injury can be explained by the different electrophysiological origins of these ECG parameters. STE is primarily driven by localized injury currents generated between ischemic and nonischemic tissues. ²⁵ Consequently, a superficial or incomplete ligation may cause sufficient localized subepicardial stress to produce STE (yielding false‐positive models), while sparing the deeper myocardium. In contrast, the QTc interval represents the global duration of ventricular repolarization. A true, transmural MI leads to widespread ATP depletion, which subsequently triggers profound intracellular calcium overload, impaired potassium channel function, and structural uncoupling of gap junctions across the ventricular wall. ²⁶ , ²⁷ These extensive, transmural metabolic and structural disruptions significantly delay the overall repolarization process, thereby driving the pronounced QTc prolongation. ²⁸ Thus, while STE flags localized epicardial injury, concomitant QTc prolongation acts as the definitive mechanistic signature of deep, transmural ischemic burden essential for a successful MI model. Furthermore, we found that ECG monitoring at the 30‐minute postoperative time point provides a reliable and definitive window for the confirmation of a stable MI model.

The necessity of applying these highly sensitive early diagnostic parameters is underscored by our findings. As demonstrated in our cohort, the overall surgical error rate was ≈14% (7/51 mice failed to develop the intended infarction, including the 4 mice with false‐positive STE but no QTc prolongation). Without early diagnostic validation, this 14% error margin would inevitably confound long‐term functional or pharmacological studies. Such an oversight could lead to a severe misinterpretation of experimental results. For example, if a novel cardioprotective inhibitor is tested in a cohort containing these unconfirmed mice, the inherent absence of infarction in the failed models could be falsely attributed to the therapeutic efficacy of the drug, generating significant false‐positive outcomes. Therefore, identifying and excluding these surgical failures immediately via early ECG criteria is not only critical for saving resources and complying with animal welfare but is also essential for ensuring baseline disease uniformity and data integrity in cardiovascular research.

Our study further revealed a significant correlation between the LAD ligation position, the STE magnitude, and the extent of ischemic injury. Consequently, the early STE magnitude can serve as a practical intraoperative guide to standardize the ligation site, thereby improving the consistency of MI models. Additionally, it provides a useful metric for estimating the severity of the resulting ischemic injury. In clinical practice, STE in patients with acute MI is a well‐established indicator for assessing infarct size and the severity of coronary artery occlusion. ¹⁶ , ²⁹ These translational parallels underscore that, despite the known anatomic differences between murine and human hearts, the temporal evolution of STE in our model mirrors clinical STEMI patterns, where the STE magnitude correlates with ischemic burden.

For the IRI model, the procedure involves 2 sequential phases: ischemia and reperfusion. Because the initial ischemic phase is identical to the MI induction, it inherently carries the same surgical error rate of ≈14%. In contrast, the subsequent reperfusion phase (releasing the ligation) is highly reproducible; in our cohort, we observed a 100% success rate (22/22 mice), as confirmed by the complete resolution of STE within 20 minutes after reperfusion. Therefore, applying our early ECG criteria during the ischemic phase of the IRI model is crucial to avoid the initial ≈14% misinterpretation rate, ensuring that only mice with confirmed ischemia are allowed to proceed to reperfusion. Following successful reperfusion, the most prominent early reperfusion signature, observed within 3 minutes in 72% of mice (16/22), was a significant increase in QRS complex voltage accompanied by profound T‐wave inversion. Furthermore, the dynamic resolution of STE served as a highly informative marker. Although STE resolution indicates successful reperfusion, its time course reflects the extent of microvascular impairment. The majority of mice (17/22) achieved complete STE resolution within 10 minutes after reperfusion. The rapid normalization emerged as a functional biomarker of microvascular recovery. ²⁰ Conversely, delayed or incomplete ST‐segment normalization was associated with microcirculatory disturbances, calcium overload, and elevated oxidative stress, suggesting ECG dynamics can noninvasively track underlying reperfusion injury mechanisms. ¹³ In line with this, recent comprehensive electrophysiological characterizations of murine IRI models have used ECG to map the longitudinal progression of cardiac ischemia and reperfusion across multiple time points over 1 week, ¹⁰ and our study complements these findings by focusing on the immediate intraoperative window to directly validate the procedural success of the IRI model.

Conclusions

Our study establishes an optimized framework for standardizing murine MI and IRI models by defining key early electrocardiographic criteria, specifically the STE in lead II and the QTc prolongation at 30 minutes postoperatively, for the validation of model success and the estimation of ischemic injury severity.

Sources of Funding

P.L. is supported by grants from the National Natural Science Foundation of China (No. 82370339), the Shanghai Pujiang Talent Program (Category A; No. 23PJ1410500), and the Innovative Research Team of High‐Level Local Universities in Shanghai. Q.L. is supported by grants from the National Natural Science Foundation of China (No. 82470432) and the Innovative Research Team of High‐Level Local University in Shanghai (No. SHSMU‐ZDCX20212700). F.L. is supported by grants from the National Natural Science Foundation of China (Nos. 82470383 and 82170376). X.C. is supported by grants from the National Science Foundation for Young Scientists of China (No. 32400638). C.L. is supported by grants from the National Science Foundation for Young Scientists of China (No. 82500385), Basic Research Program of Jiangsu (No. BK20241624) and Natural Science Foundation of Shanghai (No. 25ZR1402439).

Disclosures

None.

Supporting information

Figures S1–S2