Background: Life-threatening ventricular tachycardia (VT) frequently occurs in the early phase after acute myocardial infarction (MI). Therapeutic approaches are mainly limited to the administration of antiarrhythmic drugs or defibrillation as VT occur. The gold standard therapy, implantable cardioverter defibrillator implantation, is recommended 40 days post-MI, leaving patients vulnerable to arrhythmia during the transition from in-hospital specialized care to definitive treatment. We and others have recently demonstrated that cardiac immune cells such as neutrophil granulocytes (hereafter neutrophils) contribute to VT pathogenesis in patients with acute MI. Accordingly, neutrophil depletion has been shown to reduce VT burden post-MI in mice. Since a broad depletion of neutrophils is clinically not sensible in critically ill patients, we tested here, whether a targeted approach against specific neutrophilic proteins is sufficient to reduce arrhythmia burden. Specifically, we hypothesized that neutrophil elastase (NE) contributes to cardiac remodeling and damage post-MI via Connexin43 (Cx43) breakdown, a gap junction protein important for orderly cardiac conduction, and that pharmacological inhibition of NE lowers VT burden and alleviates tissue damage post-MI.
Methods: 10-12 weeks old C57BL/6J mice were fed a potassium-deficient diet (≤0.02%) for 21 days to establish hypokalemia. On day 14, mice were implanted with an ETA-F10 telemetric ECG device. Mice were subjected to either NE-inhibition (NEi, n=8; 2mg/kg GW311616A, p.o. on day 19). MI-mice without additional treatment were used as controls (n=8). On day 21, acute MI was induced by permanent ligation of the left anterior descending artery in all experimental groups. After surgery, MI-mice were subjected to 24h of continuous ECG monitoring to assess VT burden. Infarct size was quantified 24h post-MI by measurement of blood troponin levels and cardiac tissue staining with triphenyltetrazoliumchloride. In separate mouse cohorts, we harvested hearts and blood from MI-mice treated with NEi (n=9) and controls (n=5) for multi-color flow cytometry to assess neutrophil counts and phenotypes. For further mechanistical analyses, cardiac slices (CS) from explanted human hearts were incubated with NE or vehicle solution. CS were fixated in paraffin and stained for Cx43 using a standard protocol. Confocal microscopy images of CS were analyzed using FIJI software.
Results: Both NEi and control MI-mice showed comparable infarct sizes and troponin levels. ECG recordings revealed reduced VT burden in NEi-MI-mice compared to controls, evident as reduced time under VT, total number of VT episodes and cardiac cycles spent in VT during the 24h ECG monitoring period. Flow cytometry of cardiac tissue revealed unchanged leukocyte, neutrophil, monocyte and macrophage counts between control and NEi-MI-mice. Flow cytometry analysis of blood did not show significant differences in neutrophil or monocyte counts. Preliminary histological analyses of CS indicate a reduction of Cx43 area fraction after NE incubation compared to vehicle incubation.
Conclusion: NE inhibition was associated with reduced arrythmia burden early after MI, highlighting NE as a promising target for future therapies. In ongoing analyses, we investigate the underlying mechanisms.
