Cellular and molecular mechanisms of atrial arrhythmogenesis in patients with paroxysmal atrial fibrillation

Abstract

Background: Electrical, structural, and Ca2+ -handling remodeling contribute to the perpetuation/progression of atrial fibrillation (AF). Recent evidence has suggested a role for spontaneous sarcoplasmic reticulum Ca2+ -release events in long-standing persistent AF, but the occurrence and mechanisms of sarcoplasmic reticulum Ca2+ -release events in paroxysmal AF (pAF) are unknown.

Method and results: Right-atrial appendages from control sinus rhythm patients or patients with pAF (last episode a median of 10-20 days preoperatively) were analyzed with simultaneous measurements of [Ca2+]i (fluo-3-acetoxymethyl ester) and membrane currents/action potentials (patch-clamp) in isolated atrial cardiomyocytes, and Western blot. Action potential duration, L-type Ca2+ current, and Na+ /Ca2+ -exchange current were unaltered in pAF, indicating the absence of AF-induced electrical remodeling. In contrast, there were increases in SR Ca2+ leak and incidence of delayed after-depolarizations in pAF. Ca2+ -transient amplitude and sarcoplasmic reticulum Ca2+ load (caffeine-induced Ca2+ -transient amplitude, integrated Na+/Ca2+ -exchange current) were larger in pAF. Ca2+ -transient decay was faster in pAF, but the decay of caffeine-induced Ca2+ transients was unaltered, suggesting increased SERCA2a function. In agreement, phosphorylation (inactivation) of the SERCA2a-inhibitor protein phospholamban was increased in pAF. Ryanodine receptor fractional phosphorylation was unaltered in pAF, whereas ryanodine receptor expression and single-channel open probability were increased. A novel computational model of the human atrial cardiomyocyte indicated that both ryanodine receptor dysregulation and enhanced SERCA2a activity promote increased sarcoplasmic reticulum Ca2+ leak and sarcoplasmic reticulum Ca2+ -release events, causing delayed after-depolarizations/triggered activity in pAF.

Conclusions: Increased diastolic sarcoplasmic reticulum Ca2+ leak and related delayed after-depolarizations/triggered activity promote cellular arrhythmogenesis in pAF patients. Biochemical, functional, and modeling studies point to a combination of increased sarcoplasmic reticulum Ca2+ load related to phospholamban hyperphosphorylation and ryanodine receptor dysregulation as underlying mechanisms.

Keywords: atrial fibrillation; calcium; computational biology; electrophysiology; sarcoplasmic reticulum.

Figure 1

Atrial-cardiomyocyte action-potential (AP) characteristics in sinus-rhythm (Ctl) or paroxysmal atrial fibrillation (pAF) patients. A. Atrial APs recorded at 0.5 Hz. B. AP-duration at 20%, 50%, and 90% repolarization (APD₂₀, APD₅₀, and APD₉₀). C. Resting membrane potential (RMP). D. AP-amplitude. n/N=numbers of myocytes/patients. Comparisons using multi-level mixed-effects models.

Figure 2

L-type Ca²⁺-current (I_Ca,L) and I_Ca,L-triggered Ca²⁺-transient properties. A. Voltage-clamp protocol (V_m, applied at 0.5 Hz) and simultaneous recordings of I_Ca,L (I_M) and I_Ca,L-triggered Ca²⁺-transients (top to bottom) in a Ctl (left) or pAF (right) myocyte. B. Peak I_Ca,L-amplitude. C. Diastolic and systolic (I_Ca,L-triggered) Ca²⁺-levels, and corresponding Ca²⁺-transient (Δ[Ca²⁺]_i). D. Time-constant of Ca²⁺-transient decay. *P<0.05 vs. Ctl. n/N=numbers of myocytes/patients. Comparisons using multi-level mixed-effects models.

Figure 3

Spontaneous SR Ca²⁺-release events (SCaEs). A. Intracellular [Ca²⁺] (top) and membrane potential (bottom) in Ctl (left) and pAF (right) cardiomyocytes. SCaEs and delayed afterdepolarizations (DADs) were assessed during a one-minute follow-up period after cessation of 0.5 Hz-stimulation in 2.0-mmol/L extracellular-[Ca²⁺]. B. Prevalence of DADs exceeding 20 mV (left) and SCaEs (right). C. Intrinsic frequency of SCaEs (left), SCaE-amplitudes (middle) and corresponding membrane-potential changes (right). *P<0.05, **P<0.01 vs. Ctl. n/N=numbers of myocytes/patients. Comparisons using multi-level mixed-effects models.

Figure 4

SR Ca²⁺-content and Na⁺-Ca²⁺-exchange (NCX) current (I_NCX) in atrial myocytes from Ctl and pAF-cardiomyocytes. A. Voltage-clamp protocol (top), and resulting I_Ca,L- and caffeine-triggered Ca²⁺-transients (middle), or membrane-currents (I_m, bottom). B. SR Ca²⁺-load, quantified with caffeine-triggered Ca²⁺-transient amplitude (left), or integrated membrane-current (right). C. Time-constants of caffeine-triggered Ca²⁺-transient decay (reflecting Ca²⁺-extrusion via NCX). D. I_NCX as a function of [Ca²⁺]. E. Ca²⁺-dependence of NCX, based on slope of linear fit to the I_NCX/[Ca²⁺]_i relationship during the decay of the caffeine-triggered Ca²⁺-transient. F. Representative Western blots (top) and mean NCX1 protein-expression (bottom) in atrial tissue-samples. Calsequestrin (CSQ) was loading-control. **P<0.01 vs. Ctl. n/N=numbers of myocytes/patients (B,C,E). N=number of tissue-samples (F). Comparisons using multi-level mixed-effects models (B,C,E) or one-way ANOVA (F).

Figure 5

SR Ca²⁺-ATPase (Serca2a) and phospholamban (PLB) expression, phosphorylation and function in Ctl and pAF-patients. A. Representative Western blots (top) for total Serca2a and total PLB protein-expression, as well as Ser16-PLB phosphorylation, Thr17-PLB phosphorylation, and calsequestrin (CSQ) expression. Bottom panel shows quantification of total Serca2a and PLB expression, and relative Ser16/Thr17 PLB phosphorylation-levels. Group data are normalized to CSQ. B. Representative example of a caffeine experiment, highlighting the decay rate of the systolic (I_Ca,L-triggered) Ca²⁺-transient (k_syst) and the decay rate of the caffeine-induced Ca²⁺-transient (k_caff). C. Respective rate-constants k_syst (left), k_caff (middle) and k_Serca (right, obtained as the difference between k_syst and k_caff) in Ctl and pAF-patients. Numbers indicate tissue samples per group (panel A) or myocytes/patients (panel C). *P<0.05 vs. Ctl. Comparisons using one-way ANOVA (A) or multi-level mixed-effects models (C).

Figure 6

SR Ca²⁺-leak and ryanodine-receptor channel (RyR2) function in Ctl and pAF-patients. A. Voltage-clamp protocol (top) and [Ca²⁺]_i (bottom) in a representative Ctl experiment, illustrating the method for SR Ca²⁺-leak and SR Ca²⁺-content quantification in human atrial myocytes using the tetracaine protocol developed by Shannon et al. B. Total SR Ca²⁺-leak in Ctl and pAF-patients. C. SR Ca²⁺-load quantified using caffeine-triggered Ca²⁺-transient amplitude. D. Representative Western blots showing total RyR2, Ser2808- or Ser2814-phosphorylated RyR2 in tissue-homogenates (left) or SR-fractions (right). Bottom panels show total RyR2 expression and Ser2808 and Ser2814 phosphorylation levels (relative to total RyR2 expression) in corresponding Ctl and pAF-samples. E. Representative RyR2 single-channel recordings in lipid-bilayers showing channel openings (upward deflections) at 150 nmol/L cytosolic (cis) [Ca²⁺] in RyR2 isolated from Ctl or pAF-samples (left). RyR2 open-probability (P_o) in Ctl or pAF-samples (right). Data are shown relative to Ctl. Numbers indicate myocytes/patients (in panels B,C), tissue samples (panel D), or channels/patients (panel E). * P<0.05 versus Ctl. Comparisons using multi-level mixed-effects models (B,C,E) or one-way ANOVA (D).

Figure 7

Computational modeling of human atrial Ca²⁺-handling. A. Schematic of a single longitudinal segment, highlighting the 18 transverse Ca²⁺-domains, subcellular Ca²⁺-diffusion, subcellular compartments (Cyt: cytosol, SR: sarcoplasmic-reticulum, subsarc.: subsarcolemmal space), and Ca²⁺-transport mechanisms (Serca2a: SR Ca²⁺-ATPase; RyR2: ryanodine-receptor channel type-2; I_Ca,L: L-type Ca²⁺-current; I_NCX: Na⁺-Ca²⁺-exchange current; I_pmCa: plasmalemmal Ca²⁺-ATPase). Ca²⁺-buffers and other ion-currents are also incorporated into the model but omitted here for clarity. B. Experimental voltage-clamp protocol (top), along with simulated whole-cell I_Ca,L-triggered Ca²⁺-transient (CaT) and whole-cell caffeine-triggered Ca²⁺-transient (cCaT; bottom). Inset: transverse line-scan representation of [Ca²⁺]_i at center of virtual cell, showing Ca²⁺-wave propagation from subsarcolemma to cell-center; color-scale below. C. Comparison between key Ca²⁺-handling properties in the model (Mdl, black bars) compared to experimental data in Ctl myocytes (Exp, white bars, from Figures 2,4,6). Top left: peak I_Ca,L-amplitude, Top middle: CaT-amplitude, Top right: time-constants of CaT-decay, Bottom left: SR Ca²⁺-leak, Bottom middle: SR Ca²⁺-load (amplitude of cCaT), Bottom right: time-constants of cCaT-decay. Numbers indicate myocytes/patients (for experimental data). Experimental data are shown as mean±SEM; model simulations produce single value (no error-bar relevant).

Figure 8

Computational analysis of SR Ca²⁺-leak and spontaneous Ca²⁺-release events (SCaEs). A. Voltage-clamp protocol and transverse line-scan representation of [Ca²⁺]_i at center of virtual cell in the Ctl model, Ctl model with increased SR Ca²⁺-uptake, Ctl model with RyR2 dysregulation and pAF model combining both elements (top to bottom). The protocol shows the two final depolarization-triggered Ca²⁺-transients at steady state, followed by a quiescent period to assess SCaEs, and simulated application of tetracaine and caffeine to determine SR Ca²⁺-leak and SR Ca²⁺-load. B. Quantification of incidence (top) and average amplitude (middle) of SCaEs. Bottom panel shows amplitude of SCaE-induced membrane currents. Results are presented as relative changes versus Ctl for increased SR Ca²⁺-uptake (⬆Serca2a), RyR2-dysregulation (⬆RyR2), and pAF models. C. Similar to panel B for SR Ca²⁺-load (top) and SR Ca²⁺-leak (bottom).