Spontaneous, pro-arrhythmic calcium signals disrupt electrical pacing in mouse pulmonary vein sleeve cells

Abstract

The pulmonary vein, which returns oxygenated blood to the left atrium, is ensheathed by a population of unique, myocyte-like cells called pulmonary vein sleeve cells (PVCs). These cells autonomously generate action potentials that propagate into the left atrial chamber and cause arrhythmias resulting in atrial fibrillation; the most common, often sustained, form of cardiac arrhythmia. In mice, PVCs extend along the pulmonary vein into the lungs, and are accessible in a lung slice preparation. We exploited this model to study how aberrant Ca(2+) signaling alters the ability of PVC networks to follow electrical pacing. Cellular responses were investigated using real-time 2-photon imaging of lung slices loaded with a Ca(2+)-sensitive fluorescent indicator (Ca(2+) measurements) and phase contrast microscopy (contraction measurements). PVCs displayed global Ca(2+) signals and coordinated contraction in response to electrical field stimulation (EFS). The effects of EFS relied on both Ca(2+) influx and Ca(2+) release, and could be inhibited by nifedipine, ryanodine or caffeine. Moreover, PVCs had a high propensity to show spontaneous Ca(2+) signals that arose via stochastic activation of ryanodine receptors (RyRs). The ability of electrical pacing to entrain Ca(2+) signals and contractile responses was dramatically influenced by inherent spontaneous Ca(2+) activity. In PVCs with relatively low spontaneous Ca(2+) activity (<1 Hz), entrainment with electrical pacing was good. However, in PVCs with higher frequencies of spontaneous Ca(2+) activity (>1.5 Hz), electrical pacing was less effective; PVCs became unpaced, only partially-paced or displayed alternans. Because spontaneous Ca(2+) activity varied between cells, neighboring PVCs often had different responses to electrical pacing. Our data indicate that the ability of PVCs to respond to electrical stimulation depends on their intrinsic Ca(2+) cycling properties. Heterogeneous spontaneous Ca(2+) activity arising from stochastic RyR opening can disengage them from sinus rhythm and lead to autonomous, pro-arrhythmic activity.

Figure 1. PVCs can be paced by electrical field stimulation (EFS), but are prone to showing spontaneous activity.

(A) PVCs displayed rapid increases of cytosolic Ca²⁺ concentration in response to EFS. Between EFS pulses, some spontaneous Ca²⁺ activity was observed (indicated by asterisks). After termination of EFS, the cells continued to show spontaneous Ca²⁺ transients. (B) 100 µM nifedipine inhibited the ability of PVCs to respond to EFS pulses, causing the spontaneous Ca²⁺ transients to become prominent. (C) Removal of extracellular Ca²⁺ stopped responses to EFS, but spontaneous Ca²⁺ transients were still evident [See fig. 3 for an explanation how spontaneous and paced activity was distinguished]. (D) Quantification of the spontaneous Ca²⁺ transients on the day of the preparation (Day 0) and the three following days, illustrating that the spontaneous activity is not significantly different between day 0 and 2 (n = 7–36 cells, 2–4 slices). (E) Normalized intensity of Oregon Green BAPTA-1 in PVCs on the day of the preparation (Day 0) and the three following days (n = 2–19 cells, 1–10 slices).

Figure 2. Characterization of PVCs and their spontaneous Ca²⁺ signaling in mouse lung slices.

(A) Cross-section of a lung slice showing a lone pulmonary vein, and the relative position of an airway and its associated pulmonary artery within the alveolar tissue. (B_i–C_ii) Phase-contrast images of PVCs revealed the characteristic striated pattern (arrows) associated with sarcomeres of cardiac myocytes that (B_ii) overlap with the striated expression pattern of RyR2 and (C_ii) RyR3 detected by immunofluorescence. (D_i) Light microscopic image and (D_ii) membrane staining of PVCs with di-8-ANNEPS predominantly highlighted peripheral membranes with little evidence for internal transverse-tubules (n = 38 cells, 5 slices). (E_i) Differential interference contrast image of a pulmonary vein cross section that displayed (E_ii) small, non-coordinated spontaneous contractions and (E_iii) larger, coordinated contractions in response to EFS (1 Hz, black tick marks; line-scan analysis along line indicated in E_i). (F_i–v) Spontaneous Ca²⁺ increases that either remain localized and only spread over a limited area (white arrows) or that travel as a Ca²⁺ wave through individual cells (red arrow). The Ca²⁺ waves often originated in the same location (e.g. red asterisks) and spread in the same direction with each wave. Relative fluorescence (Ca²⁺) increases are indicated by the pseudocolor bar (F/F₀). The time interval between images is indicated in each panel. (F_vi) Grey-scale fluorescence image of the PVCs.

Figure 3. Effect of Electrical Field Stimulation (EFS) on PVC Ca²⁺ signals.

(A) Line-scan analysis of spontaneous Ca²⁺ signals and responses to 1 Hz EFS in two neighboring cells. The cells are outlined in the 2-photon fluorescence image of PVCs in a lung slice shown in (B). In (A), the cell border is depicted by the dashed line. The spontaneous Ca²⁺ activity in Cell 1 shows a bidirectional wave (origin indicated by an asterisks, arrowheads indicate direction). The timing of the Ca²⁺ increases in both cells is independent of each other, but did occasionally coincide. EFS caused whole-cell Ca²⁺ increases, which are shown as vertical straight lines. EFS timing and pulse numbers are indicated by the top bar. The ability of Cell 1 or 2 to respond to EFS is indicated in the table; ‘+’ indicates full response, while ‘±’ indicates an incomplete response. (C) Summary, showing that the latency from an EFS-induced Ca²⁺ signal to the next spontaneous Ca²⁺ transient is significantly longer for PVCs with infrequent spontaneous Ca²⁺ activity (left). In contrast, the latency from a spontaneous Ca²⁺ transient to the next EFS-induced Ca²⁺ signal did not depend on the frequency of the spontaneous activity (right) (n >166 events in 10 cells, 3 slices).

Figure 4. Heterogeneity in the pulsed responses of neighboring PVCs to increasing frequencies of EFS.

At each EFS frequency (0 to 3 Hz), the responses of 2 contiguous PVCs are illustrated with a line-scan analysis (left, A_i to C_i) and a fluorescence (Ca²⁺) trace for Cell 1 (A_ii to C_ii) and Cell 2 (A_iii to C_iii). Within the line-scan image, the cell border between the PVCs is indicated by the dashed line. The timing of the EFS pulses is indicated by the top bar. (A) PVCs displayed spontaneous Ca²⁺ activity in the absence of EFS. (B) With 2 Hz EFS, Cell 1 displayed full pacing (FP) while Cell 2 showed partial pacing (PP). (C) With 3 Hz EFS, Cell 1 displayed alternans while Cell 2 was fully paced. (D_i–iv) Summary, showing the predominate forms of response induced by different EFS frequencies (n = 25 cells, 4 slices). (E) Correlation of spontaneous Ca²⁺ transient frequency and the EFS stimulation frequency required for full pacing (n = 25 cells, 4 slices).

Figure 5. Response of PVCs to the removal and re-addition of external Ca²⁺.

(A) A fluorescence (Ca²⁺) trace showing the reduction of spontaneous Ca²⁺ transients during a period of extracellular Ca²⁺ removal, and increased spontaneous Ca²⁺ transient activity after Ca²⁺ re-addition. (B) A representative example of (B_i) the spontaneous Ca²⁺ activity before Ca²⁺ removal, (B_ii) the restoration of the Ca²⁺ transients during Ca²⁺ re-addition and (B_iii) the prolonged Ca²⁺ waves after Ca²⁺ re-addition. (C_i–iv) Quantification of the Ca²⁺ responses of PVCs to Ca²⁺ removal and re-addition. (C_i) The frequency of the spontaneous Ca²⁺ transients is reduced by Ca²⁺ removal. (C_ii) The amplitude (ΔF/F₀) of the spontaneous Ca²⁺ transients and (C_iii) the cytosolic Ca²⁺ concentration transients are both reduced by Ca²⁺ removal and increased after Ca²⁺ re-addition. (C_iv) The duration of Ca²⁺ transients increases at Ca²⁺ re-addition (n = 35 cells, 12 slices). (D) A fluorescence (Ca²⁺) trace showing the effect of external Ca²⁺ removal while 1 Hz EFS was applied. EFS-induced pacing of the cell was lost during Ca²⁺ removal and was not re-established when extracellular Ca²⁺ was restored. (E) Summary of the (E_i) Ca²⁺ responses to EFS before the Ca²⁺ removal, (E_ii) Ca²⁺ signals following Ca²⁺ re-addition and (E_iii) the prolonged Ca²⁺ waves established after Ca²⁺ re-addition. (F) shows (F_i–_iv) Line-scan analysis of phase-contrast images measuring pulmonary vein contraction (cf. Fig. 1). (F_i) No contraction without EFS. (F_ii) Coordinated contractions with 1 Hz EFS. (F_iii) In the absence of external Ca²⁺, EFS did not evoke contractions. (F_iv) Strong uncoordinated contractions after the re-addition of Ca²⁺.

Figure 6. Caffeine stimulates Ca²⁺ signals in PVCs.

(A_i) A Ca²⁺ trace and (B_i) line-scan analysis of spontaneous Ca²⁺ signals, illustrating that 1 mM caffeine activated a reversible increase in the spontaneous Ca²⁺ activity, which was reversible (A_ii and B_ii). (C_i) A Ca²⁺ trace and (D_i) line-scan analysis of Ca²⁺ signals paced by 1 Hz EFS illustrating that 1 mM caffeine increased the spontaneous Ca²⁺ activity and caused a loss of pacing, which was reversible (C_ii and D_ii). (E) Summary of the response to 1 mM caffeine showing (E_i) increased frequency of the spontaneous Ca²⁺ signals and increased basal Ca²⁺ levels in (E_ii) the absence of EFS (n = 46 cells, 9 slices) or (E_iii) the presence of EFS (n = 35 cells, 8 slices).

Figure 7. KCl stimulates Ca²⁺ signals in PVCs.

(A_i, A_ii) Ca²⁺ traces and (B_i, B_ii) line-scan analysis (selected details) showing that KCl increased spontaneous Ca²⁺ signals in PVCs. (C) Ca²⁺ trace and (D_i, D_ii) selected line-scan analysis showing the that KCl reversibly caused a loss of pacing and increased spontaneous Ca²⁺ signals in PVCs during 1 Hz EFS. (E_i –_iv) Line-scan analysis of phase-contrast images of a pulmonary vein measuring contraction (cf. Fig. 1). (E_i) No contractions without EFS. (E_ii) 1 Hz EFS induced contractions. (E_iii) KCl changed the coordinated contractions to uncoordinated fibrillations. (E_iv) EFS responses were restored after KCl wash-out. (F_i–iii) Quantification of the PVC responses to KCl addition. Summaries showing that KCl (F_i) increased the frequency of the spontaneous Ca²⁺ signals and (F_ii) the basal Ca²⁺ concentration in PVCs without EFS (n = 17 cells, 4 slices). KCl also (F_iii) increased the basal Ca²⁺ concentration in PVCs paced with 1 Hz EFS (n = 24 cells, 4 slices).