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Modulation of NOX2 causes Obesity- Mediated Atrial Fibrillation

ABSTRACT: Obesity is linked to an increased risk of atrial fibrillation (AF) via increased oxidative stress. While NADPH oxidase II (NOX2), a major source of oxidative stress and reactive oxygen species (ROS) in the heart predisposes to AF, the underlying mechanisms remain unclear. Here, we studied NOX2-mediated ROS production in obesity-mediated AF using Nox2-knock-out (KO) mice and mature human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCMs). Diet-induced obesity (DIO) mice and hiPSC-aCMs treated with palmitic acid (PA) were infused with a NOX blocker (apocynin) and a NOX2-specific inhibitor, respectively. We showed that NOX2 inhibition normalized atrial action potential duration and abrogated obesity-mediated ion channel remodeling with reduced AF burden. Unbiased transcriptomics analysis revealed that NOX2 mediates atrial remodeling in obesity-mediated AF in DIO mice, PA-treated hiPSC-aCMs, and human atrial tissue from obese individuals by upregulation of paired-like homeodomain transcription factor 2 (PITX2). Furthermore, hiPSC-aCMs treated with hydrogen peroxide, a NOX2 surrogate, displayed increased PITX2 expression, establishing a mechanistic link between increased NOX2-mediated ROS production and modulation of PITX2. Our findings offer insights into possible mechanisms through which obesity triggers AF and support NOX2 inhibition as a potential novel prophylactic or adjunctive therapy for patients with obesity-mediated AF.

ORGANISM(S): Homo sapiens

PROVIDER: GSE271748 | GEO | 2024/10/09

REPOSITORIES: GEO

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Project description:Introduction: Atrial fibrillation (AF) is the most common arrhythmia and affects around 1% of the population with increasing incidence and substantial morbidity. Functional effects of genetic variants associated with AF should help to identify targets involved in AF initiation and/or subsequent remodeling. Gene variants next to the gene of the transcription factor PITX2 (paired-like homeodomain transcription factor 2) showed the strongest association with AF. A significant number of AF patients showed reduced PITX2 levels in the left atrium. Mouse models have been used to study functional aspects of PITX2 gene deletion (“knock out”), but there are substantial differences in atrial electrophysiology between mouse and human . HiPSC-CM should be able to bridge the gap, provided the cells reproduce typical characteristics of human atrium. Methods: PITX2 knock out samples from a control cell line of human induced pluripotent stem cells (hiPSC) were obtained. Differentiation and generation of Atrial-like engineered heart tissue (aEHT) was performed. RNA-sequencing (RNA-Seq), Quantitative Real-time PCR (RT-qPCR), Protein analysis by Western Blot, Contraction and force analysis, action potential measurements, Calcium current measurements and differential gene expression analysis were run on the samples. Results: Effective knock out of PITX2 was confirmed by mRNA sequencing and WB. It was shown that PITX2 deficiency reduces contraction force. PITX2 deficiency induces action potential triangulation with more negative maximum diastolic potential. Activation of muscarinic receptors shortens APD in both PITX2 knockout and controls but hyperpolarization is lost in PITX2 knock out. It was shown that Ca2+ current density is smaller in PITX2 knock out than controls. We found that more negative MDP in PITX2 knock out is not associated with higher IK1. Conclusion: We improved atrial differentiation, coming very close to human atrial electrophysiology. We demonstrate that PITX2 deficiency induces key characteristics known from persistent AF.

Project description: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.

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Modulation of NOX2 causes Obesity- Mediated Atrial Fibrillation

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