Oral and gum health have long been associated with incidence and outcomes of cardiovascular disease. Periodontal disease increases myocardial infarction (MI) mortality by seven-fold through mechanisms that are not fully understood. The goal of this study was to evaluate whether lipopolysaccharide (LPS) from a periodontal pathogen accelerates inflammation post-MI through memory T-cell activation. We compared 4 groups (no MI, chronic LPS, day 1 post-MI, and day 1 post-MI with chronic LPS (LPS+MI); n=68 mice) using the mouse heart attack research tool 1.0 database and tissue bank coupled with new analyses and experiments. LPS+MI increased total CD8+ T-cells in the left ventricle versus the other groups (p<0.05 versus all). Memory CD8+ T-cells (CD44+CD27+) were 10-fold greater in LPS+MI compared to MI alone (p=0.02). Interleukin (IL)-4 stimulated splenic CD8+ T-cells away from an effector phenotype and towards a memory phenotype, inducing secretion of factors associated with the Wnt/β-catenin signaling that promoted monocyte migration and decreased viability. To dissect the effect of CD8+ T-cells post-MI, we administered a major histocompatibility complex-I blocking antibody starting 7 days before MI, which prevented effector CD8+ T-cell activation without affecting the memory response. The reduction in effector cells diminished infarct wall thinning but had no effect on macrophage numbers or MertK expression. LPS+MI+IgG attenuated macrophages within the infarct without effecting CD8+ T-cells suggesting these two processes were independent. Overall, our data indicate that effector and memory CD8+ T-cells at post-MI day 1 are amplified by chronic LPS to potentially promote infarct wall thinning.

During homeostasis, immune cells perform daily housekeeping functions to maintain heart health by acting as sentinels for tissue damage and foreign particles. Resident immune cells compose 5% of the cellular population in healthy human ventricular tissue. In response to injury, there is an increase in inflammation within the heart due to the influx of immune cells. Some of the most common immune cells recruited to the heart are macrophages, dendritic cells, neutrophils, and T-cells. In this review, we will discuss what is known about cardiac immune cell heterogeneity during homeostasis, how these cell populations change in response to a pathology such as myocardial infarction or pressure overload, and what stimuli are regulating these processes. In addition, we will summarize technologies used to evaluate cell heterogeneity in models of cardiovascular disease.

The purpose of this study was to generate a quantitative profile of electrocardiograms (ECG) 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 4 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 to 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 was 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 non-significant. Using a cut-off 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). Sex nor genotype were associated with false-predictions (p=0.18). Our data indicate the area under the QRS complex 1-min after ligation can improve reproducibility in MI surgeries.

Following a myocardial infarction (MI) monocytes and T-cells begin to infiltrate into the ischemic area in effort to remove necrotic debris and initiate formation of scar tissue. Interleukin (IL)-4 has been linked to improved cardiac wound healing via alterations in both the macrophage and T-cell populations. The goal of this study was determine if proteins secreted by IL-4 stimulated CD8+ T-cells would regulate monocyte physiology that would ultimately improve cardiac healing after an MI. Isolated splenic naïve CD8+ T-cells from day 0 (no MI) mice (n=5/sex/stimulation)were cultured in RPMI with either 0.1% FBS (unstimulated) or 0.1% FBS+ IL-4. After 24 hours of stimulation, the cells and media were collected and separated by centrifugation. The cell pellet was stained and analyzed for markers of activation (CD44) and memory (CD27) by flowcytometry. Conditioned media (secretome) was collected for stimulation of bone marrow monocytes (n=4; females only). After stimulation with the secretome, monocytes were analyzed for viability, phagocytosis, and macrophage phenotype by flow cytometry. Migration of the monocytes after stimulation was also measured using electric cell-substrate impedance sensing (ECIS). After IL-4 stimulation, there was a shift from effector (CD44+ CD27-) to the memory phenotype (CD44+ CD27+; p<0.05 vs unstimulated cells). Interestingly, bone marrow monocyte viability was decreased by 15% when stimulated with the secretome of IL-4 treated CD8+ T-cells compared to unstimulated CD8+ T-cells (0.1%). Phagocytosis was slightly elevated though not significant (p=0.07) in monocytes that were stimulated with the secretome from the IL-4 group compared to the unstimulated CD8+ T-cells. No differences were found in expression of macrophage markers F4/80 (p=0.532) or M1 marker CD86 (p=0.471). The secretome of IL-4 stimulated T-cells increased monocyte migration after wounding similar to levels of the positive control (monocytes in 10% FBS only). The data collected showed that IL-4 stimulated CD8+ T-cells were able to upregulate memory marker CD27. These memory-like CD8+ T-cells initiated monocyte phagocytosis and migration but decreased monocyte viability suggesting that they may play a role in regulating macrophage biology post-MI.

Introduction Electrocardiography measures the electrical activity of the heart to generate an electrocardiogram (ECG) through a non‐invasive process that involves placing electrodes on strategic points on the body. When an MI occurs, the lack of contractility induces changes to the ECG, visualized by a prolonged QRS wave and amplification of the R wave. Previously, we identified the area under the QRS wave, 1 min after permanent ligation in a mouse model of myocardial infarction (MI) determined surgical success. The purpose of this study was to further demonstrate in a second cohort that the previous ECG parameters identified accurately confirmed that an MI has been administered by left anterior coronary artery ligation in real‐time. Methods and Results Permanent coronary artery ligation was performed on WT and CD8atmak1 (CD8‐/‐) mice (n=9/genotype); an ECG was recorded using a surgical monitoring platform and was analyzed on iWorkx LabScribe Data Acquisition and Analysis software. Previous analysis of the first dataset indicated no differences between sexes so both males and females were used in the second cohort. A receiver operating characteristic (ROC) curve was used to link changes in the QRS complex at the 1‐min mark with MI surgical success. By ROC analysis, the area under the QRS complex had an area under the curve (AUC) of 0.94 (p=0.01) indicating a strong association with MI success. To remove any potential effect of surgical platform variability, we normalized the 1 min data to pre‐MI values to determine fold‐change 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 0.90 (p=0.02). Mice in the second cohort were divided into two groups based on a cut‐off point for either the area under the QRS complex (≥0.003 mV/sec) or the normalized data (≥ 3.1 fold change in the QRS complex) to distinguish the two groups. Using the non‐normalized data alone, there was an 84% success rate with 2 false positives and 1 false negative. Analysis using the normalized data alone had an 74% success rate, with 2 false positives and 3 false negatives. None of these false positives or negatives were linked to genotype or sex. Conclusion Our data indicates that while changes in the QRS complex 1‐min after ligation may be useful for confirming MI surgical success, there are potential limitations that could skew the data. One possible explanation for the false positives could be variation between ECG boards. To remove this, we normalized post‐MI data to pre‐MI values but, this seemed to decrease effectiveness. It is also important to note that many of the false negatives were due to anomalies within the ECG such as the presence of R‐prime waves. For this reason, users must be consistent when analyzing the ECG data points to minimize variation. In summary, using the ECG data 1 min after ligation had a fairly strong prediction rate for MI surgical success.