Atherosclerosis exacerbates arrhythmia following myocardial infarction: Role of myocardial inflammation

Abstract

Background: Atherosclerotic animal models show increased recruitment of inflammatory cells to the heart after myocardial infarction (MI), which impacts ventricular function and remodeling.

Objective: The purpose of this study was to determine whether increased myocardial inflammation after MI also contributes to arrhythmias.

Methods: MI was created in 3 mouse models: (1) atherosclerotic (apolipoprotein E deficient [ApoE(-/-)] on atherogenic diet, n = 12); (2) acute inflammation (wild-type [WT] given daily lipopolysaccharide [LPS] 10 μg/day, n = 7); and (3) WT (n = 14). Sham-operated (n = 4) mice also were studied. Four days post-MI, an inflammatory protease-activatable fluorescent probe (Prosense680) was injected intravenously to quantify myocardial inflammation on day 5. Optical mapping with voltage-sensitive dye was performed on day 5 to assess electrophysiology and arrhythmia susceptibility.

Results: Inflammatory activity (Prosense680 fluorescence) was increased approximately 2-fold in ApoE+MI and LPS+MI hearts vs WT+MI (P<.05) and 3-fold vs sham (P<.05). ApoE+MI and LPS+MI hearts also had prolonged action potential duration, slowed conduction velocity, and increased susceptibility to pacing-induced arrhythmias (56% and 71% vs 13% for WT+MI and 0% for sham, respectively, P<.05, for ApoE+MI and LPS+MI groups vs both WT+MI and sham). Increased macrophage accumulation in ApoE+MI and LPS+MI hearts was confirmed by immunofluorescence. Macrophages were associated with areas of connexin43 (Cx43) degradation, and a 2-fold decrease in Cx43 expression was found in ApoE+MI vs WT+MI hearts (P<.05). ApoE+MI hearts also had a 3-fold increase in interleukin-1β expression, an inflammatory cytokine known to degrade Cx43.

Conclusion: Underlying atherosclerosis exacerbates post-MI electrophysiological remodeling and arrhythmias. LPS+MI hearts fully recapitulate the atherosclerotic phenotype, suggesting myocardial inflammation as a key contributor to post-MI arrhythmia.

Keywords: Arrhythmia; Atherosclerosis; Inflammation; Myocardial infarction; Optical mapping.

Figure 1

Post-MI electrophysiology. (A–D) Maps of activation, repolarization (Repol₈₀), action potential duration (APD₈₀) and action potential (AP) rise time (TRise). Slowing of activation and prolongation of repolarization and APD are observed in the ApoE+MI and LPS+MI hearts. (E) Example optical APs from the infarct region (red box in D). (F) APD₈₀ from remote and infarct regions (green and red boxes, respectively in D). (G) TRise from remote and infarct regions. (H) Schematic showing location of LAD ligation and regions of interest for electrophysiological and molecular imaging analysis. RV: right ventricle, LV: left ventricle, LAD: left anterior descending coronary artery. *p<0.05, **p<0.01, ***p<0.001.

Figure 2

Reentrant arrhythmias and conduction velocity (CV). (A) An example of the S1–S2 pacing protocol showing no arrhythmia in a WT+MI heart. (B) The same S1–S2 protocol produces non-sustained ventricular tachycardia (VT) in an ApoE+MI heart. (C) Activation maps at two different S2 intervals (110 ms and 60 ms) showing a pronounced slowing of CV in the ApoE+MI heart at shorter S2 coupling intervals. (D) Proportion of hearts in which VT was induced. (E) Mean CV at S1 (150 ms) and S2 (at the effective refractory period [ERP]). (F) CV restitution curves for all groups showing blocked conduction at an S2 of approximately 60 ms for WT+MI and Sham and continued slow conduction at shorter S2 coupling intervals for ApoE+MI and LPS+MI hearts. ECG: electrocardiogram, OAP: optical action potential, VT: ventricular tachycardia. *p<0.05, **p<0.01, ***p<0.001 vs WT. ^†p<0.05, ^††p<0.01, ^†††p<0.001 vs Sham.

Figure 3

PVCs in post-MI hearts. (A) Number of spontaneously occurring PVCs in 20 min. (B) Representative optical AP (OAP, red) and ECG (black) showing spontaneous PVCs in an LPS+MI heart. Location of the OAP is indicated with an asterisk in the first sinus rhythm activation map in C. (C) Activation maps of sinus rhythm and PVCs corresponding to the traces in B. The PVCs appear focal in origin and arise from the basal infarct region. *p<0.05.

Figure 4

Ex-vivo fluorescence reflectance imaging of macrophage protease activity (ProSense680 fluorescence). (A) Prosense680 intensity on the anterior epicardial surface of the heart showing increased fluorescence in the infarct region of ApoE+MI and LPS+MI hearts (images identically scaled). (B) Representative 2 mm short-axis images showing transmural protease activity. Slice locations correspond to dashed white lines in A. (C) Mean fluorescence intensity from the infarct region (white box in A). (D–F) Correlations between inflammation in the infarct region (ProSense fluorescence) and corresponding electrophysiological parameters from the same region when data from all groups are pooled (color of data point indicates group). *p<0.05, ***p<0.001.

Figure 5

Fluorescence microscopy of residual ProSense680 fluorescence and the macrophage marker CD68. (A) Residual ProSense680 fluorescence (top, purple) from the infarct region of a WT+MI (left) and ApoE+MI (right) heart. These same tissue sections were then labeled for CD68 (red, bottom) to assess co-localization of ProSense680 and macrophages. V: vessel used for anatomical landmark. (B) CD68 immunofluorescence of infarct regions show increased macrophage infiltration in ApoE+MI and LPS+MI hearts.

Figure 6

Fluorescence microscopy of macrophage infiltration and corresponding Cx43 expression. (A) Uninjured myocardium remote from the infarct shows uniform staining of Cx43 (green) and few macrophages (CD68, red) in an ApoE+MI heart. (B) The region of mock ligation in a sham heart shows Cx43 degradation at the site of injury, which corresponds to increased macrophage infiltration (CD68). Inset reveals Cx43 internalization and degradation in myocytes within and neighboring CD68 positive regions. (C) The infarct region of an ApoE+MI heart shows marked reduction in Cx43 expression corresponding to an increase in macrophage infiltration. Inset reveals degradation and internalization of Cx43. (D) Western blots (each sample run in duplicate) and corresponding quantification showing decreased expression of Cx43 and increased expression of IL-1β in ApoE+MI versus WT+MI. HEK: Hek293 cell lysates, which do not express Cx43 or IL-1β were used as a negative control, Mark: molecular weight marker. *p<0.05, **p<0.01.