Genetic inhibition of nuclear factor of activated T-cell c2 prevents atrial fibrillation in CREM transgenic mice

Abstract

Aims Abnormal intracellular calcium handling contributes to the progressive nature of atrial fibrillation (AF), the most common sustained cardiac arrhythmia. Evidence in mouse models suggests that activation of the nuclear factor of activated T-cell (NFAT) signaling pathway contributes to atrial remodeling. Our aim was to determine the role of NFATc2 in AF in humans and mouse models. Methods and results Expression levels of NFATc1-c4 isoforms were assessed by quantitative reverse transcription-polymerase chain reaction in right atrial appendages from patients with chronic AF. NFATc1 and NFATc2 mRNA levels were elevated in chronic AF (cAF) patients compared with those in sinus rhythm (SR). Western blotting revealed increased cytosolic and nuclear levels of NFATc2 in AF patients. Similar findings were obtained in CREM-IbΔC-X transgenic (CREM) mice, a model of progressive AF. Telemetry ECG recordings revealed age-dependent spontaneous AF in CREM mice, which was prevented by NFATc2 knockout in CREM: NFATc2-/- mice. Programmed electrical stimulation revealed that CREM: NFATc2-/- mice lacked an AF substrate. Morphometric analysis and histology revealed increased atrial weight and atrial fibrosis in CREM mice compared with WT controls, which was reversed in CREM: NFATc2-/- mice. Confocal microscopy showed an increased Ca2+ spark frequency despite a reduced sarcoplasmic reticulum (SR) Ca2+ load in CREM mice compared with controls, whereas these abnormalities were normalized in CREM: NFATc2-/- mice. Western blotting revealed that genetic inhibition of Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of S2814 on RyR2 in CREM: RyR2-S2814A mice suppressed NFATc2 activation observed in CREM mice, suggesting that NFATc2 is activated by excessive SR Ca2+ leak via RyR2. Finally, chromatin immunoprecipitation sequencing from AF patients identified Ras And EF-Hand Domain-Containing Protein (RASEF) as a direct target of NFATc2 mediated transcription. Conclusion Our findings reveal activation of the NFAT signaling pathway in patients of Chinese and European descent. NFATc2 knockout prevents the progression of AF in the CREM mouse model. TRANSLATIONAL PERSPECTIVE Atrial fibrillation (AF) is a progressive disease characterized by electrical and structural remodeling which promotes atrial arrhythmias. This study provides evidence for increased ‘nuclear factor of activated T-cell’ (NFAT) signaling in patients with chronic AF. Studies in the CREM transgenic model of progressive AF revealed that the NFATc2 isoform mediates atrial remodeling associated with AF substrate development. Chromatin immunoprecipitation sequencing of atrial biopsies from AF patients identified ‘Ras And EF-Hand Domain-Containing Protein’ (RASEF) as a downstream target of NFATc2-mediated transcription, suggesting that targeting these factors might be beneficial for curtailing AF progression.

Atrial Proteomic Profiling Reveals a Switch Towards Profibrotic Gene Expression Program in CREM-IbΔC-X Mice with Persistent Atrial Fibrillation

“…Specifically, we utilized the well-characterized CREM-Tg mouse model of spontaneous AF development and progression that was previously shown to mimic major aspects of AF in humans, such as AF progression, atrial remodeling and fibrosis, and intracellular Ca 2+ handling abnormalities. [11,13,25,26,[28][29][30] Our results show that 9-month-old CREM-Tg mice with persistent AF develop enlarged atria and atrial fibrosis, mimicking the phenotype seen in patients with advanced persistent AF. Since the molecular pathways involved in atrial remodeling are not well understood, we performed mass spectrometry-based unbiased proteomic profiling of atrial tissues obtained from 9-month-old CREM-Tg mice.…”

“…There were signs of left ventricular dilatation without increases in wall thickness, consistent with a mild dilated cardiomyopathy ( Supplemental Table 1 ), consistent with prior studies. [25, 26] Furthermore, the amount of atrial fibrosis was examined using picrosirius red staining of longitudinal cardiac sections ( Fig. 1E ).…”

“…CREM-Tg mice have been reported as an effective AF model, demonstrating similarities in disease progression comparable to that observed in human AF. [11,13,25,26,[28][29][30] To uncover additional areas of resemblance of molecular alterations in CREM-Tg mice in comparison with human patients, we obtained a publicly available dataset, [24] showing the atrial proteomic landscape in patients with persistent AF. We used the same significance threshold (fold change >1.2 and adjusted p value <0.1) as reported in this study and identified 480 significant DEPs in the human perAF patients compared to 98 in CREM-Tg mice (Fig.…”

Enhanced Ca 2+ -Dependent SK-Channel Gating and Membrane Trafficking in Human Atrial Fibrillation

“…Total RNA was isolated from RA whole-tissue homogenates and RA cardiomyocytes from Ctl patients and patients with cAF, and reverse-transcription or digital polymerase chain reactions were performed as described in the Supplemental Material using 30 primers based on published sequences (see Major Resources Table ).…”

In Vivo Cardiac Electrophysiology in Mice: Determination of Atrial and Ventricular Arrhythmic Substrates

“…While electrophysiology studies can be performed ex vivo in Langendorff‐perfused isolated hearts, key limitations include inadequate perfusion and oxygenation, lack of contractility and electromechanical feedback, and the absence of cardiac innervation. Ambulatory ECG recordings using implantable telemetry devices can provide meaningful insights into the occurrence of spontaneous cardiac arrhythmias in awake, freely moving mice (Keefe et al., 2023; Li et al., 2014; McCauley & Wehrens, 2010; Ni et al., 2022). However, young and healthy mice are generally resistant to the development of spontaneous arrhythmias, even when proarrhythmic genetic modifications are introduced (Campbell et al., 2020; McCauley et al., 2011; Moreira et al., 2020).…”