Riding the wave: a quantitative report of electrocardiogram utilization for myocardial infarction confirmation

Abstract

The purpose of this study was to generate a quantitative profile of electrocardiograms (ECGs) for confirming surgical success of permanent coronary artery ligation. An ECG was recorded at baseline, and 0, 1, and 5 min after ligation and analyzed using iWorkx LabScribe software. Cohort 1 (C57Bl6/J, n = 8/sex) was enrolled to determine ECG characteristics that were confirmed in cohort 2 (C57Bl6/J, n = 6/sex; CD8^-/-n = 6 males/4 females). Of the 16 mice in cohort 1, 12 (6/sex) had an infarct ≥35% and four mice (2/sex) had <35% based on 2,3,5-triphenyltetrazolium chloride staining. After ligation, the QRS complex and R-S amplitude were significantly different compared with baseline. No differences were observed in the R-S amplitude between mice with infarcts ≥35% versus <35% at any time point, whereas the QRS complex was significant 1 min after ligation. Receiver operating characteristic (ROC) curve linked changes in the QRS complex but not the R-S amplitude at 1 and 5 min with surgical success. Data were normalized to baseline values to calculate fold change. ROC analysis of the normalized QRS data indicated strong sensitivity and specificity for infarcts ≥35%; normalized R-S amplitude remained nonsignificant. With a cutoff generated by ROC analysis of cohort 1 (>80% sensitivity; >90% specificity), the non-normalized QRS complex of cohort 2 had an 86% success rate (2 false positives; 1 false negative). The normalized data had a 77% success rate (2 false positives; 3 false negatives). Neither sex nor genotype was associated with false predictions (P = 0.18). Our data indicate that the area under the QRS complex 1 min after ligation can improve reproducibility in MI surgeries.NEW & NOTEWORTHY Our study describes a quantitative method for using an electrocardiogram (ECG) to determine which animals have infarcts that reflect coronary artery ligation. Using a quantitative ECG, investigators will have the benefit of having real-time feedback during the procedure, which will ultimately decrease the amount of time investigators spend performing experiments. This overall increase in efficiency will help investigators decrease animal numbers used due to better surgical outcomes.

Keywords: electrocardiogram; heart; myocardial infarction; reproducibility.

Figure 1.

Representative images of the electrocardiogram (ECG) demonstrate widening of the QRS complex and amplification of the R to S amplitude after the ligation of the left anterior coronary artery (LAD). The QRS complex and R-S amplitude were analyzed as an average for 10 consecutive cycles from each time point collected before ligation (pre-MI) and 0-, 1-, and 5-min postligation. A: area shaded in gray for the QRS symbolizes the area under the curve, which was measured to see the difference of pre-MI to post-MI to determine association to surgical success. B: gray arrow demonstrates the amplitude from R to S, which was used to determine successful LAD ligation. C and D: representative ECG image of the same mouse on the Indus surgical monitor (C) and iWorks LabScribe analysis software (D). MI, myocardial infarction.

Figure 2.

ECG changes after ligation resulted in QRS widening and decreased R-S amplitude but did not correlate with infarct size. A: measurements of the QRS complex demonstrated significant changes in the area under the QRS complex at 0-, 1-, and 5-min postligation compared with pre-MI values. B: normalization to the pre-MI values showed around a threefold or higher increase at each of the time points postligation. C: changes in the QRS complex were not correlated with infarct size as demonstrated by an R² = 0.001 (0 and 1 min) and 0.075 (5 min). D: R-S amplitude was significantly lower immediately after ligation and continued to decrease over time with 5 min having the smallest drop compared with pre-MI. E: normalization of the post-MI data to the pre-MI values also demonstrated a drop in the R-S amplitude after ligation compared with pre-MI values. F: infarct size was not correlated with R-S amplitude at any time point measured. Temporal data were analyzed using a paired one-way ANOVA with Student–Newman–Keuls posttest. Simple linear regression was used to correlate infarct size with ECG parameters. Blue data points signify male mice (n = 6/time point), and yellow data points signify female mice (n = 6/time point). *P < 0.05 vs. pre-MI; #P < 0.05 vs. 0 min; ^P < 0.05 vs. 1 min. ECG, electrocardiogram; MI, myocardial infarction.

Figure 3.

Comparisons of ECG parameters for each time point showed definitive differences in the QRS complex between mice with infarcts ≥35% compared with those with <35% infarct size. A: area under the QRS complex was not significantly different at the 0-min mark between the two groups. The widening of the QRS complex after ligation was significantly elevated at both 1 min (B) and 5 min (C) in the group with infarcts ≥35% compared with the mice with infarct <35%. D–F: R-S amplitude was not significantly different between groups at the 0 (D)-, 1 (E)-, or 5 (F)-min time points. Blue circles and squares visualized are males (n = 6 for ≥35%; n = 2 for <35%) and yellow circles and squares visualized are females (n = 6 for ≥35%; n = 2 for <35%). Unpaired t test was used to compare groups. *P < 0.05 vs. ≥35%. ECG, electrocardiogram.

Figure 4.

Normalization to pre-MI data demonstrated a similar predictive index in the QRS complex between mice with infarcts ≥35% compared with those with <35% infarct size. A: at the 0-min mark, there was no significance between groups. B and C: after 1 (B)- and 5 (C)-min postligation, significant differences were identified in the QRS complex of mice with infarcts ≥35% compared with the mice with infarct <35%. D–F: normalization of the R-S amplitude did not improve predictive index with no significant differences being observed at the 0 (D)-, 1 (E)-, or 5 (F)-min time points. Unpaired t test was used to compare groups. Blue circles and squares visualized are males (n = 6 for ≥35%; n = 2 for <35%) and yellow circles and squares visualized are females (n = 6 for ≥35%; n = 2 for <35%). *P < 0.05 vs. ≥35%. MI, myocardial infarction.

Figure 5.

A receiver operating characteristic (ROC) curve demonstrates accuracy for predictive test of LAD ligation at the ECG at the 1 and 5 min. A: area under the curve (AUC) for the QRS complex of the first cohort was 0.938 (P = 0.011) at both 1- and 5-min postligation indicating a strong association with MI success. To remove any potential effect of surgical platform variability, we normalized the post-MI data to pre-MI values in the area under the QRS complex. ROC analysis of the normalized data also indicated a strong sensitivity and specificity for MI success with an AUC value of 0.896 (P = 0.021) at 1-min and 0.875 (P = 0.029) at 5-min postligation. B: R-S amplitude did not strongly predict infarct sizes ≥35% with an AUC value of 0.56 (P = 0.72) at 1-min and 0.77 (P = 0.12) at the 5-min mark. Normalized R-S amplitude data were also not predictive of MI success with an AUC of 0.58 (P = 0.63) 1 min after ligation. This improved after 5 min though still not significant with an AUC of 0.81 (P = 0.07). n = 12 (6/sex) for ≥35% infarct; n = 4 (2/sex) for <35% infarct. ECG, electrocardiogram; LAD, left anterior descending; MI, myocardial infarction.

Figure 6.

ECG data from CD8^−/− mice predicted coronary artery ligation efficiency similar to WT mice. A: ROC analysis of nonnormalized (AUC-0.96; P = 0.02) and normalized QRS complex data (AUC-0.92; P = 0.03) from CD8^−/− mice show strong predictive index for infarcts ≥35%. Similar to WT mice, the change in the R-S amplitude did not predict surgical success with (AUC-0.67; P = 0.44) or without normalization (AUC-0.55; P = 0.80) to pre-MI values. B: CD8^−/− mice with infarcts ≥35% had significant elevation in the area under the QRS complex before and after normalization compared with infarcts <35%. This was not observed with the R-S amplitude even after normalizing to pre-MI values. No significant differences were observed in the QRS complex or R-S amplitude data between WT and CD8^−/− mice with (P = 0.29 and P = 0.16, respectively) or without normalization (P = 0.59 and 0.22, respectively). Genotype comparisons were analyzed using a one-way ANOVA with Student–Newman–Keuls posttest. *P < 0.05 vs. ≥35%; for WT n = 6 (3/sex) for ≥35% infarct; n = 6 (3/sex) for <35% infarct; for CD8^−/− n = 6 (3/sex) for ≥35% infarct; n = 4 (3 males and 1 female) for <35% infarct. ECG, electrocardiogram; MI, myocardial infarction; ROC, receiver operating characteristic; WT, wild-type.

Figure 7.

Mice in the second cohort were divided into two groups based on a cutoff point determined by ROC analysis (>80% sensitivity; >90% specificity) for the area under the QRS complex (≥0.003 mV/s; left) and the normalized data (≥3.1 -fold change; right). Using the nonnormalized data alone, there were 2 false positives and 1 false negative (86% success). Analysis using the normalized data had 2 false positives and 3 false negatives (77% success). None of these false positives or negatives could be explained solely due to genotype or sex. Red dots indicative of infarct size <35% and black dots >35% based on TTC staining; ● = WT males (n = 6); + = WT females (n = 6); ■ = CD8^−/− males (n = 6); x = CD8^−/− females (n = 4). ROC, receiver operating characteristic.

Figure 8.

Analysis of the ECG data showed anomalies that could be described as an R-prime (R′) wave. Representative images of the two different genotypes [WT (top) and CD8^−/− (bottom)] taken 5 min after LAD ligation were used to show that the R′ phenomenon was not genotype specific. Presence of this double R could affect analysis, making the width of the QRS seem smaller than it actually is depending on where the measurement point is placed. ECG, electrocardiogram; LAD, left anterior descending; WT, wild-type.