M1 Macrophages cause cardiac arrhythmia in an in vitro hiPSC coculture model of inflammation-induced atrial fibrillation
Atrial fibrillation (AF) has an estimated prevalence of 1.5–2%, making it the most common cardiac arrhythmia. The mechanisms that cause and sustain AF are still not completely understood. An association between AF and systemic as well as local inflammatory processes has been reported, however, the exact mechanisms underlying this association have not been established. While it is understood that tissue resident macrophages can influence cardiac electrophysiology, the effects of activated pro-inflammatory macrophages have not been described yet. This study investigated the pro-arrhythmic effects of activated macrophages (M1) on human induced pluripotent stem cell (hiPSC)-derived atrial cardiomyocytes (aCM), to propose a mechanistic link between inflammation and AF. Two hiPSC lines from healthy individuals were differentiated to aCMs and M1 macrophages. Electrophysiology characteristics of M1 and aCM cocultures were analysed for beat rate irregularity, electrogram amplitude and conduction velocity. M1 cocultures resulted in a significant increase in beat rate irregularity and decrease in electrogram amplitude compared to other conditions tested, including aCMs treated with activated M1 supernatant, which did not produce an increase in irregularity. Conduction analysis further showed significantly lower conduction homogeneity in M1 cocultures. Immunosuppression through glucocorticoids significantly decreased beat irregularity in aCM+M1 cocultures compared to vehicle. RNA sequencing performed in aCMs, revealed downregulation of various ion channels (SCNA5, KCNA5, ATP1A1), as a result of the M1 coculture. Electrophysiology related transcription changes were reversed by glucocorticoid treatment. This study establishes a causal relationship between M1 activation and the development of subsequent atrial arrhythmia, documented as irregularity in spontaneous electrical activation in aCM cocultured with activated macrophages. Further, beat rate irregularity could be alleviated using anti-inflammatory steroidal glucocorticoids. These results strongly support the relevance of the proposed hiPSC-derived coculture model and point at macrophage mediated inflammation as a potential AF mechanism. Overall design: For bulk RNA sequencing hiPSC-derived atrial cardiomyocytes (aCM) from 2 healthy donor lines in coculture with isogenic hiPSC-derived M1 macrophages (m1) were investigated. 4 conditions were analyzed: aCM only, aCM+M1 conditioned medium, aCM+M1 and aCM+M1+ 10 µM Hydrocortisone. All samples were seeded on fibronectin coated (1:100) 24 well Cellstar plates (Greiner) with 500,000 aCM per well and 125,000 M1 added to cocultures. Medium (PCM, 500 ul per well) was added according to M1 maturation/activation medium change schedule (as described before), with non-activated conditions having no added GM-CSF, LPS and IFN-?. Hydrocortisone addition was performed as previously described in the manuscript. For conditioned medium samples, 125 ul supernatant from M1 monocultures (24 well plate, 500,000 cells/well in 0.5 mL PCM) was added to appropriate samples. Addition was performed 2 hours prior to cell collection. On d8, cells were detached using TrypLe-Express (Gibco) and single cell suspensions generated. M1 cells in coculture suspensions were removed through CD14 magnetic bead cell sorting (CD14 Microbeads human, MS column, MiniMACS kit, all Miltenyi) according to manufacturer's protocol. aCM cell populations were stored at -80°C and used for RNA extraction (as previously described). Bulk RNA-seq was performed on RNA samples by SingleCellDiscoveries. Count data was analyzed using R-studio (R-studio, PBC , Boston, USA) and Deseq2. P values were analyzed using Wald test, with a P value <0.05 regarded as significant. Genes were annotated for ontologies using clusterProfiler and DOSE.