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. 2023 Jul:180:1-9.
doi: 10.1016/j.yjmcc.2023.04.005. Epub 2023 Apr 18.

The selective RyR2 inhibitor ent-verticilide suppresses atrial fibrillation susceptibility caused by Pitx2 deficiency

Kyungsoo Kim  1 , Daniel J Blackwell  1 , Samantha L Yuen  2 , Madelaine P Thorpe  3 , Jeffrey N Johnston  3 , Razvan L Cornea  2 , Björn C Knollmann  4
Affiliations

The selective RyR2 inhibitor ent-verticilide suppresses atrial fibrillation susceptibility caused by Pitx2 deficiency

Kyungsoo Kim et al. J Mol Cell Cardiol. 2023 Jul.

Abstract

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and a major cause of stroke and morbidity. The strongest genetic risk factors for AF in humans are variants on chromosome 4q25, near the paired-like homeobox transcription factor 2 gene PITX2. Although mice deficient in Pitx2 (Pitx2+/-) have increased AF susceptibility, the mechanism remains controversial. Recent evidence has implicated hyperactivation of the cardiac ryanodine receptor (RyR2) in Pitx2 deficiency, which may be associated with AF susceptibility. We investigated pacing-induced AF susceptibility and spontaneous Ca2+ release events in Pitx2 haploinsufficient (+/-) mice and isolated atrial myocytes to test the hypothesis that hyperactivity of RyR2 increases susceptibility to AF, which can be prevented by a potent and selective RyR2 channel inhibitor, ent-verticilide. Compared with littermate wild-type Pitx2+/+, the frequency of Ca2+ sparks and spontaneous Ca2+ release events increased in permeabilized and intact atrial myocytes from Pitx2+/- mice. Atrial burst pacing consistently increased the incidence and duration of AF in Pitx2+/- mice. The RyR2 inhibitor ent-verticilide significantly reduced the frequency of spontaneous Ca2+ release in intact atrial myocytes and attenuated AF susceptibility with reduced AF incidence and duration. Our data demonstrate that RyR2 hyperactivity enhances SR Ca2+ leak and AF inducibility in Pitx2+/- mice via abnormal Ca2+ handling. Therapeutic targeting of hyperactive RyR2 in AF using ent-verticilide may be a viable mechanism-based approach to treat atrial arrhythmias caused by Pitx2 deficiency.

Keywords: Atrial fibrillation; Calcium; Cardiac ryanodine receptor (RyR2); Pitx2; ent-Verticilide.

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Conflict of interest statement

Declaration of Competing Interest R.L.C. holds equity in and serves as executive officer for Photonic Pharma LLC. This relationship has been reviewed and managed by the University of Minnesota. Photonic Pharma had no role in this study. The other authors have no actual or perceived conflicts of interest with the contents of this article.

Figures

Figure 1.
Figure 1.. Ca2+ spark properties in permeabilized atrial cardiomyocytes.
(A) Ca2+ spark frequency, (B) amplitude, (C) mass, and (D) leak. Figure panels shown with full range of values and a box bounding the 25th and 75th percentiles with median. N: Pitx2+/+ (vehicle) = 168 cells from 6 mice; Pitx2+/+ (ent-vert) = 36 cells from 2 mice; Pitx2+/− (vehicle) = 127 cells from 6 mice; Pitx2+/− (ent-vert) = 70 cells from 4 mice normalized to values from +/+ mice by day of experiment. Bonferroni-adjusted P values calculated using hierarchical clustering. (E) SR load measured by caffeine-induced Ca2+ transient amplitude. Individual values with mean ± SD.
Figure 2.
Figure 2.. ent-Verticilide did not inhibit cardiac sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA2a) function.
(A) SERCA2a ATPase activity (calculated as VpCa5.4-VpCa8) in the presence of ent-verticilide at the indicated concentrations. N = 6 replicates per concentration tested. (B) Positive control: SERCA ATPase activity in the presence of thapsigargin (SERCA2a inhibitor) at the indicated concentrations. N = 3 replicates at each concentration tested. Red lines show the result of fitting the equation: Y=Bottom+(Top−Bottom)/(1+(IC50/X)^HillSlope).
Figure 3.
Figure 3.. ent-Verticilide reduced spontaneous Ca2+ release (SCR) in Pitx2+/− atrial myocytes.
Isolated intact atrial myocytes were pre-treated with vehicle or ent-verticilide for 2.5 hours and loaded with Fura-2 acetoxymethyl to measure spontaneous Ca2+ release (SCR) events. (A) Representative Ca2+ transient from intact atrial myocytes. Atrial myocytes were field-stimulated at 3 Hz for 20 s followed by 40 s recording of SCR events in 1 μM isoproterenol (ISO) containing Tyrode solution. Application of 10 mM caffeine (Caff) was used to measure SR Ca2+ content. Summary data of SCR events (B), Ca2+ transient amplitude (C), and fractional SR Ca2+ release (D). Data are mean ± SD. n=45, 44, 33 and 45 cells from 4–6 mice in each group. P values calculated using ANOVA multiple comparisons with Dunn’s or Bonferroni’s test.
Figure 4.
Figure 4.. Pixt2+/− mice exhibited increased susceptibility to atrial fibrillation (AF) by transesophageal atrial burst pacing.
(A) Representative ECG traces of littermate WT (upper) and Pitx2+/− (lower) mice after an episode of burst pacing protocol in the presence of isoproterenol (1.5 mg/kg, i.p. injection). Pitx2+/− mice exhibit inducible AF followed by spontaneous conversion to sinus rhythm. (B) Incidence of inducible AF (+/+ 50.0 % vs. +/− 95.5 %; non-sustained AF + sustained AF). Sustained AF was defined as continuous AF over 300 sec induced by any single pacing train (+/+ 21.4 % vs. +/− 81.8 %, P=0.0005). P values were obtained using Fisher’s exact test. (C) Summary of total AF burden in WT (N=14 mice) and Pitx2 deficiency (N=22 mice). Data are mean ± SD. P values were obtained using the Mann-Whitney test.
Figure 5.
Figure 5.. Anti-arrhythmic efficacy of ent-verticilide on pacing-induced AF in both Pitx2+/+ and Pitx2+/− mice.
(A, B) Incidence of inducible AF (Pitx2+/+: vehicle 54.6 % vs. ent-vert 45.5 %, Pitx2+/−: vehicle 100.0 % vs. ent-vert 65.0 %; non-sustained AF + sustained AF) by burst pacing stimulation in Pitx2+/+ and Pitx2+/− mice. Sustained AF was defined as continuous AF over 300 sec induced by any single pacing train. ent-Verticilide significantly reduced sustained AF compared to vehicle group in Pitx2+/− mice (vehicle 90.0 % vs. ent-vert 25.0 %, P<0.0001). (C, D) Summary of total AF burden in Pitx2+/+ and Pitx2+/− mice (N=11 and 20 mice, respectively). Data are mean ± SD. P values were obtained using Fisher’s exact test and Wilcoxon matched-pairs signed rank test.
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References

    1. Freeman J v, Wang Y, Akar J, Desai N, Krumholz H. National trends in atrial fibrillation hospitalization, readmission, and mortality for Medicare beneficiaries, 1999–2013. Circulation. 2017;135(13):1227–39. - PubMed
    1. Darbar D, Roden DM. Genetic mechanisms of atrial fibrillation: impact on response to treatment. Nat Rev Cardiol. 2013;10(6):317–29. - PMC - PubMed
    1. Kirchhof P, Bax J, Blomstrom-Lundquist C, Calkins H, Camm AJ, Cappato R, et al. Early and comprehensive management of atrial fibrillation: proceedings from the 2nd AFNET/EHRA consensus conference atrial fibrillation entitled ‘research perspectives in atrial fibrillation’. Europace. 2009;11(7):860–85. - PubMed
    1. Benjamin EJ, Chen PS, Bild DE, Mascette AM, Albert CM, Alonso A, et al. Prevention of atrial fibrillation: report from a national heart, lung, and blood institute workshop. Circulation. 2009;119(4):606–18. - PMC - PubMed
    1. Wijffels MCEF, Kirchhof CJHJ, Dorland R, Allessie MA. Atrial fibrillation begets atrial fibrillation: a study in awake chronically instrumented goats. Circulation. 1995;92(7):1954–68. - PubMed

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