Enhanced sarcoplasmic reticulum Ca2+ leak and increased Na+-Ca2+ exchanger function underlie delayed afterdepolarizations in patients with chronic atrial fibrillation

Abstract

Background: Delayed afterdepolarizations (DADs) carried by Na(+)-Ca(2+)-exchange current (I(NCX)) in response to sarcoplasmic reticulum (SR) Ca(2+) leak can promote atrial fibrillation (AF). The mechanisms leading to delayed afterdepolarizations in AF patients have not been defined.

Methods and results: Protein levels (Western blot), membrane currents and action potentials (patch clamp), and [Ca(2+)](i) (Fluo-3) were measured in right atrial samples from 76 sinus rhythm (control) and 72 chronic AF (cAF) patients. Diastolic [Ca(2+)](i) and SR Ca(2+) content (integrated I(NCX) during caffeine-induced Ca(2+) transient) were unchanged, whereas diastolic SR Ca(2+) leak, estimated by blocking ryanodine receptors (RyR2) with tetracaine, was ≈50% higher in cAF versus control. Single-channel recordings from atrial RyR2 reconstituted into lipid bilayers revealed enhanced open probability in cAF samples, providing a molecular basis for increased SR Ca(2+) leak. Calmodulin expression (60%), Ca(2+)/calmodulin-dependent protein kinase-II (CaMKII) autophosphorylation at Thr287 (87%), and RyR2 phosphorylation at Ser2808 (protein kinase A/CaMKII site, 236%) and Ser2814 (CaMKII site, 77%) were increased in cAF. The selective CaMKII blocker KN-93 decreased SR Ca(2+) leak, the frequency of spontaneous Ca(2+) release events, and RyR2 open probability in cAF, whereas protein kinase A inhibition with H-89 was ineffective. Knock-in mice with constitutively phosphorylated RyR2 at Ser2814 showed a higher incidence of Ca(2+) sparks and increased susceptibility to pacing-induced AF compared with controls. The relationship between [Ca(2+)](i) and I(NCX) density revealed I(NCX) upregulation in cAF. Spontaneous Ca(2+) release events accompanied by inward I(NCX) currents and delayed afterdepolarizations/triggered activity occurred more often and the sensitivity of resting membrane voltage to elevated [Ca(2+)](i) (diastolic [Ca(2+)](i)-voltage coupling gain) was higher in cAF compared with control.

Conclusions: Enhanced SR Ca(2+) leak through CaMKII-hyperphosphorylated RyR2, in combination with larger I(NCX) for a given SR Ca(2+) release and increased diastolic [Ca(2+)](i)-voltage coupling gain, causes AF-promoting atrial delayed afterdepolarizations/triggered activity in cAF patients.

Figure 1. I_Ca,L-triggered Ca²⁺-transients (CaT) in sinus rhythm (Ctl) and cAF

A, Top: Voltage-clam p protocol (0.5 Hz). Below: Simultaneous recording of I_Ca,L (middle) and triggered CaT (Fluo-3, bottom) in Ctl (left) and cAF (right) myocytes. B, Mean±SEM Peak-I_Ca,L (left) and integrated I_Ca,L (right). C, Mean±SEM diastolic and systolic [Ca²⁺]_i (left) and resulting CaT-amplitude (right). D, Mean±SEM time-constant (τ) of decay of I_Ca,L-triggered CaT. E, Mean±SEM “coupling efficiency” of Ca²⁺-influx and SR Ca²⁺-release. **P<0.01 and ***P<0.001, respectively vs. corresponding means in Ctl. Numbers indicate myocytes/patients.

Figure 2. Caffeine-induced Ca²⁺-transients (cCaT) to assess SR Ca²⁺ content and the corresponding transient inward currents (I_NCX) in atrial myocytes from Ctl and cAF patients

A, Voltage-clamp protocol to quantify SR Ca²⁺ content (Fluo-3) and corresponding I_NCX (top), cCaT (middle) and inward NCX current during caffeine (10-mmol/L) application (bottom) following steady-state stimulation for 1 -minute at 0.5 Hz in a Ctl (left) and in a cAF (right) myocyte. B, Mean±SEM cCaT-amplitude (left) and integrated inward NCX (right). C, Mean±SEM time constant (τ) of decay of cCaT (left) and Peak-I_NCX during caffeine (10-mmol/L) application (right). D, I_NCX as a function of [Ca²⁺]_i obtained during CaT together with the mean±SEM slope-fit from linear regression obtained from decay phase. E, mRNA and protein levels of NCX1 in Ctl (sinus rhythm) and cAF atria. *P<0.05 vs. corresponding means in Ctl. Numbers indicate myocytes/patients (B, C, D) and tissue samples (E), respectively.

Figure 3. Quantification of diastolic SR Ca²⁺-leak with tetracaine in voltage-clamped atrial myocytes from sinus rhythm (Ctl) and cAF patients

A, Experimental protocol for determination of SR Ca²⁺-leak (Fluo-3). After steady-state stimulation for 1-minute at 0.5 Hz, the bath solution is rapidly switched to sodium- and calcium-free (0Na⁺, 0Ca²⁺) solution. Tetracaine (1-mmol/L) blocks RyR2, and the shift downward in resting [Ca²⁺]_i is proportional to SR Ca²⁺-leak. After at least 30-seconds and tetracaine washout, SR Ca²⁺-content is measured with 10-mmol/L caffeine. B,C, Mean±SEM tetracaine-dependent decrease in resting [Ca²⁺]_i (SR Ca²⁺-leak; B) and for SR Ca²⁺-leak normalized to SR Ca²⁺-content (C) in control myocytes and myocytes pretreated (30-minutes) with the CaMKII-inhibitor KN-93 (1-μmol/L), its inactive analogue KN-92 (1-μmol/L) and the PKA-inhibitor H-89 (1-μmol/L), respectively. *P<0.05 and **P<0.01 vs. corresponding means in Ctl. Numbers within columns indicate myocytes/patients. D,E, SR Ca²⁺-leak plotted vs. SR Ca²⁺-load. Curves are from exponential regression. *P<0.05 and ***P<0.001, respectively vs. corresponding rate constant in Ctl (F-test).

Figure 4. RyR2 single-channel recordings of sinus rhythm (Ctl) and cAF patients

A, Single-channel tracings were obtained using planar lipid-bilayer recordings of RyR2 channels from sinus rhythm (left) and cAF (right) patients, respectively. Channel openings (o) are shown as upward deflections from the closed (c) level. The measurements were performed at indicated cytosolic (cis) [Ca²⁺] levels. The open probability (Po), mean open time (To), and mean closed time (Tc) are shown below each respective tracing. B, Mean+SEM RyR2 Po in Ctl and cAF at increasing cis [Ca²⁺]. C–E, Mean±SEM RyR2 Po before and after the CaMKII-inhibitor KN-93 (10-μmol/L, C), its inactive analog KN-92 (10-μmol/L, D) and the PKA-inhibitor H-89 (10-μmol/L, E) in Ctl and cAF, respectively. *P<0.05, **P<0.01 and ***P<0.001, respectively vs. corresponding control values in cAF. Numbers indicate channels/patients.

Figure 5. Spontaneous SR Ca²⁺-release events (SCaEs) with corresponding inward I_NCX in myocytes from Ctl and cAF patients

A, Voltage-clamp protocol (top) and representative recordings of SCaEs (Fluo-3, middle) and corresponding I_NCX (bottom) from a Ctl (left) and cAF (right) myocyte, respectively following steady-state stimulation for 1-minute at 0.5 Hz. B, Susceptibility to SCaEs in Ctl and cAF. *P<0.05 vs. Ctl (Fisher’s exact test). C, Mean±SEM frequency (left), latency (middle), and amplitude (right) of SCaEs in Ctl and cAF. D, Mean±SEM amplitude of SCaE-generated I_NCX. C,D, *P<0.05 and **P<0.01 vs. corresponding means in Ctl. Numbers indicate myocytes/patients.

Figure 6. Simultaneous recordings of membrane voltage (V_m) and [Ca²⁺]_i in atrial myocytes from Ctl and cAF patients

A, Current-clamp protocol (0.5 Hz, top) together with simultaneous recordings of triggered AP (middle) and CaT (Fluo-3, bottom) in a Ctl (left) and in a cAF (right) myocyte. B, Mean±SEM resting membrane potential, AP amplitude, APD₅₀ and APD₉₀, respectively. C, Mean±SEM diastolic and systolic [Ca²⁺]_i levels (left) and resulting CaT-amplitude (right). *P<0.05 vs. corresponding means in Ctl. Numbers indicate myocytes/patients.

Figure 7. Incidence of SCaEs and corresponding DADs in current-clamped atrial myocytes from Ctl and cAF patients

A, Representative recording of [Ca²⁺]_i (Fluo-3) and corresponding membrane-voltage (V_m) oscillations (DADs/triggered APs) in a Ctl and a cAF-myocyte, respectively, following steady-state stimulation for 1-minute at 0.5 Hz. B, Enhanced susceptibility to spontaneous Ca²⁺-release events (SCaEs) and SCaE-induced DADs in cAF vs. Ctl. *P<0.05 vs. Ctl (Fisher’s exact test). C, Mean±SEM for frequency (left) and latency (right) of SCaEs. D, Mean±SEM amplitude of SCaEs (top left), magnitude of corresponding V_m-change (top right), and the calculated [Ca²⁺]_i-membrane voltage coupling gain (bottom). *P<0.05 vs. corresponding means in Ctl. Numbers indicate myocytes/patients.

Figure 8. Increased AF susceptibility and SR Ca²⁺-leak in S2814D knock-in mice

A, Representative traces of Ca²⁺-spark (Fluo-4) recordings in WT and S2814D myocytes in absence and presence of KN-93 (10-μmol/L). B, Mean±SEM Ca²⁺-spark frequency (CaSF). Numbers indicate myocytes from at least 3 mice in each group. *P<0.05 and **P<0.01 vs. WT. C, Surface L1-ECG and intracardiac atrial electrogram showing P-wave absence and irregular RR-intervals in S2814D mice following atrial-burst pacing (right), suggestive of AF. WT-mice typically reverted to sinus rhythm immediately following atrial pacing (left). D,E Bar graphs summarizing the incidence of reproducible AF in WT, S2814D-control mice and S2814D-mice treated with KN-93 (10-μmol/kg, i.p.). Numbers indicate mice.