Myeloperoxidase causes both arrhythmogenic structural and electrical remodelling in neonatal rat ventricular myocyte monolayers

FUNDING ACKNOWLEDGEMENTS: Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This study was supported by Netherlands Organization for Health Research and Development BACKGROUND: Myeloperoxidase (MPO), an enzyme most abundantly expressed by neutrophils, is increased in the atrial tissue of AF patients and is associated with treatment failure. Moreover, MPO increases fibrosis and AF susceptibility in mouse atria. However, how MPO promotes AF has yet to be fully established. PURPOSE: To investigate the role of MPO in electrophysiological and structural remodelling. METHODS: We incubated cultured neonatal rat ventricular myocyte (NRVM) monolayers with 1 μg/mL MPO or vehicle for 24 hours. We then performed electrophysiological mapping using an 8x8 multielectrode array (MEA) setup with 100 μm-sized electrodes and 700 μm inter-electrode distance (n=8 MPO, n=7 control) and recorded action potentials (AP) using a microelectrode during pacing. Additionally, we quantified the number of fibroblasts using vimentin staining (n=5/group) and the expression of extracellular matrix (ECM) genes (COL1A1, COL8A2, FN1, CTGF, TNC, THBS2, MMP2, MMP12) using qPCR (n=4/group). Multielectrode data were analyzed with custom-made software in MATLAB. Statistical analysis was performed in R. Data presented as median [interquartile range]. RESULTS: NRVM monolayers incubated with MPO were unresponsive to pacing (5/8 vs 0/7 control) or had a higher pacing threshold (1300 [1000-2500] vs 500 [400-600] μA) (p=0.02). The percent of excitable tissue was lower in the MPO than in the control group, and the propagation of spontaneous activations was slower and more heterogeneous (index: 5 [3.6-9.5] vs 2 [1.5-3], p=0.004). More fractionated electrograms were present in MPO than in the control (29 [25-40] vs 11 [4-23] electrodes per grid, p=0.02). Re-entry was observed in 3/8 MPO-incubated monolayers vs 0/7 controls. Intracellular recordings revealed a less negative resting membrane potential (-50 [-24- -52] vs -69 [-56- -79] mV, p=0.04) and lower AP upstroke velocity (4 [2.5-14] vs 26 [22-28] V/s, p=0.01) in MPO-incubated cardiomyocytes than in the control. Moreover, relative to the control, MPO significantly increased the number of fibroblasts (126 [120-128] vs 87 [70-106] cells/0.26 mm^2, p=0.02) and the expression of MMP12 (1.17 (fold-change), p=0.04), whereas a trend toward increase was observed for other ECM genes, such as COL8A2 (1.5 (fold change), p=0.1). CONCLUSION: Incubation of NVRM with MPO results in depolarization of cardiomyocytes and an increase in the number of fibroblasts and ECM gene expression. Thereby, MPO increases conduction heterogeneity and susceptibility to re-entry. [Figure: see text] [Figure: see text]