Tachycardia-induced silencing of subcellular Ca2+ signaling in atrial myocytes

Abstract

Atrial fibrillation (AF) is characterized by sustained high atrial activation rates and arrhythmogenic cellular Ca2+ signaling instability; however, it is not clear how a high atrial rate and Ca2+ instability may be related. Here, we characterized subcellular Ca2+ signaling after 5 days of high atrial rates in a rabbit model. While some changes were similar to those in persistent AF, we identified a distinct pattern of stabilized subcellular Ca2+ signaling. Ca2+ sparks, arrhythmogenic Ca2+ waves, sarcoplasmic reticulum (SR) Ca2+ leak, and SR Ca2+ content were largely unaltered. Based on computational analysis, these findings were consistent with a higher Ca2+ leak due to PKA-dependent phosphorylation of SR Ca2+ channels (RyR2s), fewer RyR2s, and smaller RyR2 clusters in the SR. We determined that less Ca2+ release per [Ca2+]i transient, increased Ca2+ buffering strength, shortened action potentials, and reduced L-type Ca2+ current contribute to a stunning reduction of intracellular Na+ concentration following rapid atrial pacing. In both patients with AF and in our rabbit model, this silencing led to failed propagation of the [Ca2+]i signal to the myocyte center. We conclude that sustained high atrial rates alone silence Ca2+ signaling and do not produce Ca2+ signaling instability, consistent with an adaptive molecular and cellular response to atrial tachycardia.

Figure 10. Ca²⁺ signaling silencing in human AF.

(A) Representative transverse confocal linescan and derived local CaTs from a CTL cell (patient in sinus rhythm) during steady-state field stimulation (0.5 Hz) showing similar amplitudes of ss and cc CaTs. (B) As in A for an AF cell showing the significantly blunted cc CaT. (C) Reduced cc but preserved ss CaT amplitude in human AF cells (CTL: n = 39 cells, 9 patients; AF: n = 23 cells, 10 patients). **P < 0.01.

Figure 9. Ca²⁺ sparks and Ca²⁺ waves.

(A) Ca²⁺ spark frequency, amplitude, mass, and Ca²⁺ spark–mediated Ca²⁺ leak in a CTL cell in ss (CTL: n = 250, 21 cells, 5 animals; RAP: n = 210, 19 cells, 5 animals) and cc (CTL: n = 80, 21 cells, 5 animals; RAP: n = 90, 19 cells, 5 animals) domains. (B) As in A for RAP cells. ss (n = 210, 19 cells, 5 animals) and cc (n = 90, 19 cells, 5 animals) regions. (C) Ca²⁺ wave inducibility (CTL: n = 33 cells, 6 animals; RAP: n = 34 cells, 6 animals). (D) Ca²⁺ wave frequency at baseline and after stimulation with 1 μmol/l isoproterenol in the same cell (CTL: n = 25 cells, 5 animals; RAP: n = 22 cells, 5 animals).

Figure 8. RyR2 in atrial myocytes.

(A) Confocal immunofluorescence image of RyR2 clusters in a CTL cell. (B) As in A, but with an RAP cell. Both panels show transverse spacing of RyR2 clusters. Enlarged panels show the region of interest (ROI) that was used to obtain the transverse RyR2 cluster distance. Below are the intensity profiles along each ROI. ^§, denotes the peaks of intensity, and their distance is measured as the spacing between the RyR2 clusters. Representative histograms of transverse RyR2 cluster spacing from 1 animal (10 cells). (C) Percentiles and heterogeneity index (p95-p5/p50) for CTL and RAP cells (CTL: 408 ROI, n = 51 cells, n = 8; RAP: 420 ROIs, n = 60 cells, n = 8). (D) Western blot analyses showing changes in total RyR2 protein expression and phosphorylation in RAP cells (CTL: n = 18; RAP: n = 15). n = number of animals. *P < 0.05.

Figure 7. Fast [Ca²⁺] buffering components.

(A) Pro-Q Diamond staining (ProQ) to evaluate phosphorylation of myofilament proteins and SYPRO Ruby (SYPRO) to analyze total protein expression. cMyBP-C, cardiac myosin–binding protein C; TnT and TnI, cardiac TnT and TnI; MLC2, myosin light chain 2. (CTL: n = 17; RAP: n = 14.) (B) TnI protein expression. (C) Reduced TnI phosphorylation at Ser23/24 (CTL: n = 17; RAP; n = 14). (D) Increased calmodulin (CaM) and unchanged TnC protein expression levels (E) in RAP (relative to CTL and normalized to GADPH; CTL: n = 15; RAP: n = 11). *P < 0.05.

Figure 6. Intracellular Ca²⁺ buffering strength.

Confocal linescans and derived local CaTs of a CTL cell (2 Hz) at baseline (A) and after application of 1 μmol of the Ca²⁺ chelator BAPTA-AM (n = 13 cells, 4 animals) (B). Similar ss and cc CaT amplitudes in CTL cells during steady-state stimulation (2 Hz) (A) and reduced cc CaT amplitude after treatment with 1 μmol/l BAPTA-AM (B). (C) Left panel: Example of intracellular Ca²⁺ buffering strength in an RAP cell and a CTL cell plotting the reverse ∫I_NCX (total Ca²⁺) and the falling phase of the whole-cell CaT (epifluorescence, fluo-3; free Ca²⁺) that were simultaneously recorded during fast application of caffeine (10 mmol/l). Right panel: Slopes of intracellular Ca²⁺ buffering strength in CTL versus RAP cells (CTL: n = 15 cells, 5 animals; RAP: n = 13 cells, 6 animals). (D) Confocal linescans of a CTL cell at baseline (left) and after addition of the Ca²⁺ sensitizer EMD (3 μmol/l) (n = 12 cells, 4 animals). *P 0.05; **P 0.01.

Figure 5. Altered Na⁺ homeostasis in RAP.

(A and B) Representative ratiometric measurements of [Na⁺]_i in a CTL cell and an RAP cell with in situ calibration. (C) [Na]_i at rest and during stimulation. **P 0.01; ***P 0.001. (D) Δ[Na]_i in stimulated myocytes (CTL: 12 cells, 10 animals; RAP 9 cells, 7 animals). (E) Protein expression levels of NKA (NKA, CTL: n = 15; RAP: n = 11). (F) Protein expression levels of PLM and changes in PLM phosphorylation levels at Ser63 and Ser68 (CTL, PLM: n = 15, Ser63: n = 14, Ser68: n = 15; RAP, PLM: n = 7, Ser63: n = 7, Ser68: n = 6). *P 0.05.

Figure 4. Upregulation of NCX and unchanged SR Ca²⁺ content.

(A) Left panel: Original recordings of whole-cell CaTs (whole-cell epifluorescence, fluo-3) during caffeine application (10 mmol/l). Right panel: Caffeine-induced CaT amplitude (CTL: n = 22 cells, 5 animals; RAP: n = 16 cells, 5 animals). (B) SR Ca²⁺ content: Original recordings of I_NCX during caffeine application (10 mmol/l) and SR Ca²⁺ content based on integrated I_NCX (CTL: n = 18 cells, 4 animals; RAP: n = 16 cells, 5 animals). (C) Example of linear (slow) phase of I_Ncx free [Ca²⁺]_i plot obtained during simultaneous measurement of I_NCX and [Ca²⁺]_i (whole-cell epifluorescence, fluo-3) during fast application of caffeine (10 mmol/l) in voltage-clamped atrial myocytes (CTL: n = 14 cells, 4 animals; RAP: n = 13 cells, 5 animals). (D) Left panels: Transverse confocal linescans during caffeine application depicting domain-specific SR Ca²⁺ load in a CTL cell and an RAP cell. (CTL: n = 15 cells, 4 animals; RAP: n = 13 cells, 4 animals). Right panel: Ratio of cc/ss caffeine-induced CaT amplitudes as an approximation of domain-specific SR Ca²⁺ content. **P 0.01.

Figure 3. Role of I_Ca,L reduction.

(A) Confocal linescan and derived local CaTs in a CTL myocyte before (left panel) and after (right panel) treatment with the Ca²⁺ channel antagonist nitrendipine (Nitr) (0.3 μmol/l). (B) Regional CaT amplitudes at baseline and after application of nitrendipine (n = 15 cells, 4 animals). (C) Averaged (n = 20) recordings of whole-cell CaT in CTL cells, CTL cells treated with nitrendipine, and RAP cells. (D) Whole-cell CaT amplitude, time to peak, and decay time (CTL and CTL plus nitrendipine: n = 14 cells, 4 animals; RAP: n = 22 cells, 5 animals). ***P 0.001.

Figure 2. Failure of centripetal Ca²⁺ wave propagation after RAP.

(A) Transverse confocal linescan and derived local CaTs from a CTL cell showing similar amplitudes of ss and cc CaTs. (B) Transverse confocal linescan and derived local CaTs from an RAP cell showing the significantly blunted cc CaT. (C) Reduced cc but preserved ss CaT amplitude in RAP cells at 2 Hz (CTL: n = 28 cells, 6 animals; RAP: n = 26 cells, 6 animals). ***P 0.001. F, fluorescence intensity; F₀, baseline fluorescence intensity.

Figure 1. Contractile and electrical remodeling after RAP.

(A) Fractional myocyte shortening (CTL: n = 18 cells, 6 animals; RAP: n = 21 cells, 5 animals). (B) Cellular AP and APD at 2 Hz (CTL: n = 15 cells, 7 animals; RAP: n = 12 cells, 6 animals). (C) Left panel: Original recordings of I_Ca,L in a CTL and RAP cell. Right panel: Current-voltage relation (CTL: n = 19 cells, 6 animals; RAP: n = 17 cells, 5 animals).*P 0.05;**P 0.01; ***P 0.001.