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

Abstract

Aims: Abnormal intracellular calcium (Ca2+) 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) signalling pathway contributes to atrial remodelling. 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 (cAF). NFATc1 and NFATc2 mRNA levels were elevated in cAF patients compared with those in normal sinus rhythm (NSR). 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 wild-type 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 ryanodine receptor type 2 (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 signalling pathway in patients of Chinese and European descent. NFATc2 knockout prevents the progression of AF in the CREM mouse model.

Keywords: Atrial fibrillation; Atrial remodelling; Calcium handling; NFAT; RASEF.

Graphical Abstract

Figure 1

Upregulation of NFAT signalling pathway in atrial fibrillation (AF) patients. (A) NFATc1–c4 mRNA levels normalized to GAPDH levels in right atrial samples from chronic AF patients in Germany (NSR: n = 12, AF: n = 12) and (B) China (NSR: n = 5, AF: n = 5). (C–F) Western blots showing NFATc1–c4 in cytosolic and nuclear fractions from in right atrial samples from Chinese patients in normal sinus rhythm (NSR) or chronic AF. (G and H) quantifications of NFATc1–c4 proteins levels normalized to GAPDH (cytosolic fraction) or histone H3 (nuclear fraction), respectively (NSR: n = 4, AF: n = 4). AF, atrial fibrillation; NSR, normal sinus rhythm. Unpaired Student’s t-test was used, *P < 0.05, **P < 0.01.

Figure 2

Upregulation of NFAT in CREM mice. (A) and (B) shows NFATc1–c4 mRNA normalized to L7 in atrial samples from CREM WT and TG mice at 3 months (WT: n = 7, CREM: n = 5) and 7 months (WT: n = 7, CREM: n = 6), respectively. Western blots showing NFATc1 and NFATc2 in cytosolic and nuclear fractions of the atria from 3-month-old (C–F) (WT: n = 4, CREM: n = 4) and 7-month-old (WT: n = 4, CREM: n = 4) mice (G–J), respectively. Cytosolic levels were normalized to GAPDH, nuclear fractions to lamin B1. Unpaired Student’s t-test was used, *P < 0.05.

Figure 3

CREM:NFATc2^–/– mice are protected from age-dependent development of spontaneous AF. (A) Representative ECG telemetry recordings showing premature atrial contractions (PAC) in 3-month-old CREM mice. (B) Representative ECG telemetry recordings showing spontaneous AF in 7-month old CREM mice, whereas the other genotypes were in sinus rhythm. (C and D) Left: the number of PACs per hour in mice that exhibited atrial ectopy at the age of 3 and 5 months. Middle: the incidence of sAF (as a fraction of the number of mice) at the age of 3 and 5 months. Right: the duration of longest episodes of sAF (in minutes) at the age of 3 and 5 months. (E) Middle: the incidence of sAF at the age of 7 months (as a fraction of the number of mice). Right: the duration of longest episodes of sAF (in minutes) at the age of 7 months. Numbers below the bars indicate the number of animals studied (WT: n = 10, NFATc2^–/–: n = 10, CREM: n = 13, CREM:NFATc2^–/–: n = 12). One-way ANOVA followed by the Kruskal–Wallis test was applied. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Programmed electrical stimulation reveals absence of an AF substrate in CREM:NFATc2^–/– mice. (A) Representative lead 2 (L2) surface ECG, intracardiac atrial (A) and ventricular (V) electrograms at the end of an atrial burst pacing protocol, showing AF in the CREM mouse and sinus rhythm in the other genotypes at 5 months of age. (B) Percentage of mice with reproducible AF following programmed electric stimulation (PES) (WT: n = 10, NFATc2^–/–: n = 12, CREM: n = 12, CREM:NFATc2^–/–: n = 10), Fisher’s exact test was used. **P < 0.01. (C) Average duration of the longest AF episode induced by pacing in each group (WT: n = 10, NFATc2^–/–: n = 12, CREM: n = 12, CREM:NFATc2^–/–: n = 10). One-way ANOVA followed by the Kruskal–Wallis test was applied. *P < 0.05; **P < 0.01.

Figure 5

Reversal of atrial hypertrophy and atrial fibrosis in CREM:NFATc2^–/– mice. (A) Whole-mount photographs of hearts from 7-month-old mice. (B) The atrial weight to tibial length ratio (AW/TL) of 7-month-old mice. Numbers below graphs indicate number of mice per group. (C) Masson trichrome staining of fibrosis in atrial sections. (D) Quantification of atrial fibrosis. (E and F) The level of collagen I and III mRNA normalized to L7. Numbers in the bars indicate the number of animals studied. The generalized estimating equation approach was performed by the use of the binomial distribution to study the fibrosis outcomes. One-way ANOVA followed by the Holm–Sidak’s multiple comparisons test was applied to study the other data. *P < 0.05; **P < 0.01, ***P < 0.001.

Figure 6

Genetic inhibition of NFATc2 in CREM mice normalizes SR Ca²⁺ release. (A) Confocal line-scan images showing more spontaneous Ca²⁺ sparks in atrial myocytes from CREM mice compared with WT and CREM:NFATc2^–/– mice. (B) Bar graphs showing quantification of Ca²⁺ spark frequency (CaSpF). (C) Total sarcoplasmic reticulum Ca²⁺ content (SR load) and (D) CaSpF normalized to SR Ca²⁺ load. The number of mice and cells studies for each group is indicated in the graphs. The generalized estimating equation approach was performed by the use of the binomial distribution to study the dichotomous spontaneous SR Ca²⁺ release event. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 7

RASEF transcription is the potentially modulated by NFATc2. (A) ChiP-seq analysis of RASEF. Scaled tag density represents the abundance of the corresponding sequence combined with NFATc2. TSS is the transcription start site. The arrow indicates the direction of gene transcription. (B) qPCR shows that the expression of RASEF mRNA is upregulated in AF patients (n = 6) vs. NSR controls (n = 17). *P < 0.05 compared with NSR. AF, atrial fibrillation; NSR, normal sinus rhythm. (C) RASEF mRNA normalized to L7 in mouse atrial samples. The number of mice was six for each group.