Suppression of spontaneous ca elevations prevents atrial fibrillation in calsequestrin 2-null hearts

Abstract

Background: Atrial fibrillation (AF) risk has been associated with leaky ryanodine receptor 2 (RyR2) Ca release channels. Patients with mutations in RyR2 or in the sarcoplasmic reticulum Ca-binding protein calsequestrin 2 (Casq2) display an increased risk for AF. Here, we examine the underlying mechanisms of AF associated with loss of Casq2 and test mechanism-based drug therapy.

Methods and results: Compared with wild-type Casq2+/+ mice, atrial burst pacing consistently induced atrial flutter or AF in Casq2-/- mice and in isolated Casq2-/- hearts. Atrial optical voltage maps obtained from isolated hearts revealed multiple independent activation sites arising predominantly from the pulmonary vein region. Ca and voltage mapping demonstrated diastolic subthreshold spontaneous Ca elevations (SCaEs) and delayed afterdepolarizations whenever the pacing train failed to induce AF. The dual RyR2 and Na channel inhibitor R-propafenone (3 μmol/L) significantly reduced frequency and amplitude of SCaEs and delayed afterdepolarizations in atrial myocytes and intact atria and prevented induction of AF. In contrast, the S-enantiomer of propafenone, an equipotent Na channel blocker but much weaker RyR2 inhibitor, did not reduce SCaEs and delayed afterdepolarizations and failed to prevent AF.

Conclusions: Loss of Casq2 increases risk of AF by promoting regional SCaEs and delayed afterdepolarizations in atrial tissue, which can be prevented by RyR2 inhibition with R-propafenone. Targeting AF caused by leaky RyR2 Ca channels with R-propafenone may be a more mechanism-based approach to treating this common arrhythmia.

Keywords: atrial fibrillation; calsequestrin 2; delayed afterdepolarization; propafenone; ryanodine receptor calcium release channel; tachycardia, ventricular.

Figure 1

Casq2−/− hearts are susceptible to pacing induced AF. AF susceptibility was evaluated by atrial burst pacing (50Hz, 2s). Representative ECG records from Casq2+/+ (A) and Casq2−/− (B,C) hearts. Atrial tachyarrhythmias resembling atrial flutter (B) and AF (C) were observed in Casq2−/− hearts. For statistical analysis, episodes of AF and atrial flutter were grouped together and labelled as AF. Average rate (D) and duration (E) of AF in each group. n = 10 Casq2+/+ and 12 Casq2−/−, **P<0.01***P<0.001)

Figure 2

Representative optical voltage maps of Casq2−/− atria during sinus rhythm (A) and AF (B). Voltage maps were recorded from the posterior aspect of the heart. Asterisks indicate points of earliest activation. The corresponding bright-field image is in the leftmost panels. LA left atrium, RA right atrium, PV pulmonary veins, IVC inferior vena cava. The square in the ECG record indicates time of optical maps. Bottom right panels: Composite activation maps. Note that during sinus rhythm (A), only a single focal activation originates in the posterior aspect of the right atrium close to the crista terminalis where the sinoatrial node is located. Depolarization wavefronts spread towards the right appendage and the left atrium travelling across the posterior atrial walls. During AF (B), three independent depolarizations appear quasi simultaneously (two from the PV region, one near the IVC). Activation wavefronts spread only to a limited area surrounding the respective foci, followed by electrical silence (panel 15 ms), and repetitive activation from the same foci (panel 18 ms).The AF activation map (lower right panel) shows near-simultaneous activation originating from anatomically-distinct foci. C) Anatomical origin of atrial activation during AF. Left panel: Summary of atrial activation sites during AF episodes in 7 Casq2−/− hearts. Size of the circles is proportional to number of events arising from that specific area. Right panel: Distribution of atrial activation sites by region: right atrium (RA), pulmonary vein region (PV) and left atrial appendage (LA).

Figure 3

Anatomical origin of DADs in intact Casq2−/− atria. A) Representative example of a delayed afterdepolarizations (DAD). The red optical voltage record originating from the atria shows a deflection in the post pacing pause consistent with a DAD. Only deflections greater than 10% atrial of action potential amplitude were considered as DADs. No influence from ventricular fluorescence (black signal) is present during the atrial DAD. B) Anatomical distribution of DADs in Casq2−/− atria: The majority of the DADs occurred in the pulmonary vein region.

Figure 4

Action potential duration (APD) and conduction velocity (CV) of Casq2+/+ and Casq2−/− atria. A) Representative example of action potentials recorded optically from the RA appendage in Casq2+/+ and Casq2−/− hearts. B) Average APD at 90% repolarization (ADP90) measured during constant pacing at 100 ms pacing cycle length was not statistically different. C) Optical activation maps of the right atrial appendage during pacing. CV was calculated by defining isochrones where the time of maximum upstroke velocity of the fluorescent signal was the same. D) Average CV values were not statistically different between Casq2+/+ and Casq2−/− atria. N=4–5 hearts per group.

Figure 5

Ca handling is impaired in isolated atrial myocytes (A, B) and intact atria (C–E) of Casq2−/− hearts. A and B left panels: Representative line scans of Ca sparks (A) and Ca waves (B) obtained in permeabilized atrial myocytes. Right panels: averaged data. N=35–45 cells per group, ***P<0.001. C) Examples of fluorescent Ca signals obtained from Ca maps of Casq2+/+ and Casq2−/− hearts. Casq2−/− atria frequently exhibit spontaneous Ca elevations (SCaE) after the rapid pacing train that are absent in Casq2+/+ atria (D). Similar to DADs, SCaE were observed only in some regions of the atria. E) Anatomical distribution of SCaE in intact atria.

Figure 6

R-propafenone (R-Prop) but not S-propafenone (S-Prop) reduces the frequency of spontaneous Ca waves (SCW) in permeabilized Casq2−/− atrial myocytes and suppresses pacing-induced SCW and triggered beats in intact atrial Casq2−/− myocytes. (A) Representative line scans recorded from permeabilized Casq2−/− atrial myocytes in presence of vehicle (VEH, DMSO), R-Prop (10 μM) or S-Prop. (B) Averaged data. N=5–8 cells per group, *P<0.05, ***P<0.001. (C) Representative Ca fluorescence records from Fura2-AM loaded, intact atrial myocytes after 15 min exposure to VEH, R-Prop (3μM) or S-Prop (3μM), or Triggered Beats (TBs, arrow) and SCWs (arrowhead) were induced by a 20s pacing train (3Hz). (D) Incidence of TBs and SCWs. ***P<0.01 vs. VEH, #P<0.05 vs. S-Prop; n= 9–13 per group.

Figure 7

RyR2 channel block is necessary for prevention of pacing-induced SCaE and AF in Casq2−/− atria. (A) Representative examples of fluorescent Ca signals obtained from Ca maps of intact Casq2−/− hearts. As opposed to R-propafenone (R-Prop), administration of 3 μM of S-propafenone (S-Prop) or 20 μM of lidocaine (Lido) did not significantly reduce the rate (B) and amplitude (C) of SCaE in intact atria compared to vehicle. Despite a similar level of Na channel block evidenced by comparable QRS widening (D), S-propafenone and lidocaine failed to prevent AF in Casq2−/− hearts (E). N = 5–6 Casq2−/− hearts, **P<0.01.