Altered atrial cytosolic calcium handling contributes to the development of postoperative atrial fibrillation

Figures

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  Figure 1. Preoperative speckle-tracking echocardiography to quantify LA strain in patients who do not (Ctrl) and who do develop poAF. (A) Representative apical four-chamber echocardiogram of a poAF patient. The multi-coloured area defines the region of interest along the inner contour of the LA wall. (B) Segment nomenclature of the LA wall. (C) Representative strain curves of Ctrl (left) and poAF (right) patients. Coloured lines show strain of the different segments shown in (B). Global mean strain, denoted by the black line, is used for the measurement of total LASr, LAScd, and LASct as indicated by arrows. (D) Mean ± standard deviation LASr (left), LAScd (middle), and LASct (right). *P < 0.05, **P < 0.01, vs. Ctrl. n = patients. Comparison using the unpaired Student’s t-test. Ctrl, control; LA, left atrial; LAScd, left atrial conduit strain; LASct, left atrial contraction strain; LASr, left atrial reservoir strain; poAF, postoperative atrial fibrillation.

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  Figure 2. ICa,L-triggered CaT in atrial myocytes from patients who do not (Ctrl) and who do develop poAF. (A) Voltage-clamp protocol (0.5 Hz, upper), representative simultaneous recordings of ICa,L (middle), and triggered CaT (lower) in Ctrl (left) and poAF (right) myocytes. (B) Mean ± standard error of the mean (SEM) peak ICa,L (left) and integrated ICa, L (right). (C) Mean ± SEM diastolic and systolic [Ca2+]i (left) and resulting CaT amplitude (right). (D) Mean ± SEM time constant of decay (τ) of ICa,L-triggered CaT. *P < 0.05, ***P < 0.001 vs. Ctrl. n/N = number of myocytes/patients. Comparison using the unpaired t-test with Welch’s correction (C right and D). CaT, Ca2+ transients; Ctrl, control; poAF, postoperative atrial fibrillation.

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  Figure 3. cCaT and the corresponding transient inward currents (INCX) to assess SR Ca2+ content in atrial myocytes from patients who do not (Ctrl) and who do develop poAF. (A) Voltage-clamp protocol (upper), representative CaT and cCaT (middle), and corresponding membrane currents (IM, lower). (B) SR Ca2+ load, quantified with cCaT amplitude (left), or integrated membrane current (right). (C) Time constants of cCaT decay (indicating Ca2+ extrusion via NCX). (D) INCX as a function of [Ca2+]i. (E) Ca2+-dependence of INCX, based on the slope of linear fit to the INCX/[Ca2+]i relationship during the decay of the cCaT. (F) Representative western blots (upper, greyscale) and quantification of NCX1 expression (lower) in atrial tissue samples. Data normalized against total protein (red) in the same gel area. **P < 0.01, ***P < 0.001 vs. Ctrl. n/N = number of myocytes/patients (B, C, and E). n = number of tissue samples (F). Comparison using the unpaired Student’s t-test and t-test with Welch’s correction (B, left). CaT, Ca2+ transients; cCaT, caffeine-induced Ca2+ transients; Ctrl, control; poAF, postoperative atrial fibrillation.

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  Figure 4. SR Ca2+ leak and ryanodine receptor channel (RyR2) function in atrial myocytes from patients who do not (Ctrl) and who do develop poAF. (A) Voltage-clamp protocol (upper) and [Ca2+]i (lower) in a representative Ctrl experiment, showing the method for SR Ca2+ leak and SR Ca2+ content quantification in human atrial myocytes using the tetracaine protocol developed by Shannon et al.20 (B) Mean ± standard error of the mean (SEM) total SR Ca2+ leak in Ctrl and poAF patients (upper) and SR Ca2+ load quantified using caffeine-triggered Ca2+ transient amplitude (lower). (C) Representative western blots (upper) showing the expression of total RyR2 (green) against total protein in the same gel area (red). Lower panel shows quantification of total RyR2 expression normalized to total protein (mean ± SEM). (D) Representative western blots (upper) showing RyR2 phosphorylation at Ser2808 and Ser2814 (green) against total expression of RyR2 (red). Lower panel shows quantification of Ser2808/Ser2814 RyR2 phosphorylation levels normalized against total RyR2 (mean ± SEM). n/N = number of myocytes/patients (B). n = number of tissue samples (C and D). Comparison using the Mann–Whitney U test. Ctrl,…

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  Figure 5. SR Ca2+ ATPase (SERCA2a) and PLB expression, phosphorylation and function in atrial myocytes from patients who do not (Ctrl) and who do develop poAF. (A) Representative caffeine experiment, indicating the decay rate constant of the systolic (ICa,L-triggered) Ca2+ transient (ksyst) and the decay rate constant of the caffeine-induced Ca2+ transient (kcaff). (B) Respective rate constants ksyst (left), kcaff (middle), and kSERCA (right, calculated as the difference between ksyst and kcaff) in Ctrl and poAF (mean ± SEM). (C) Representative western blots (upper) showing the expression of SERCA2a (green) against total protein in the same gel area (red). Lower panel shows quantification of SERCA2a expression normalized against total protein (mean ± SEM). (D) Representative western blots (upper) showing the expression of total PLB, PLB phosphorylation at Ser16 and Thr17, respectively, against SERCA2a. Lower panel shows quantification of PLB, PLB-Ser16 and PLB-Thr17 normalized to SERCA2a. *P < 0.05, **P < 0.01 vs. Ctrl. n/N = number of myocytes/patients (B). n = number of tissue samples (C and D). Comparison using the unpaired Student’s t-test. Ctrl, control; PLB, phospholamban; poAF,…

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  Figure 6. Combined measurements of action potentials (APs) and Ca2+ transients (CaTs) in atrial myocytes from patients who do not (Ctrl) and who do develop poAF. (A) Representative traces of simultaneous AP (upper) and CaT (lower) recorded at various frequencies from a patient proceeding to develop poAF. (B) Mean ± standard error of the mean (SEM) of resting membrane potential (RMP) at increasing pacing frequencies. (C) Mean ± SEM of APD90 at increasing pacing frequencies (left) and diastolic intervals (right, AP restitution). (D) Mean ± SEM frequency-dependent effects on diastolic calcium (left) and CaT amplitude (right) in myocytes from Ctrl and poAF patients. (B–D) A single decay curve was fitted when no significant difference between groups was detected with an extra sum-of-squares F test. Two curves imply a global significant difference between both groups. ***P < 0.001. n–n/N = range of myocytes/patients. APD, action potential duration; Ctrl, control; poAF, postoperative atrial fibrillation..

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  Figure 7. Occurrence of alternans in atrial myocytes from patients who do not (Ctrl) and who do develop poAF. (A) Representative traces of concordant alternans in AP (amplitude and repolarization alternans; upper) and CaT (amplitude and diastolic alternans; lower) at 4 Hz measured from a patient proceeding to develop poAF. (B) The first occurrence of frequency-dependent alternans as a Kaplan–Meier plot in AP (upper panel) and CaT (lower panel). (C) Alternans threshold frequency. Data are mean ± standard error of the mean. Kaplan–Meier curves compared with the Gehan–Breslow–Wilcoxon test. **P < 0.01 vs. Ctrl. n/N = number of myocytes/patients. Alternans threshold frequency compared using the Mann–Whitney U test. AP, action potentials; CaT, Ca2+ transients; Ctrl, control; poAF, postoperative atrial fibrillation.