Impaired Intracellular Calcium Buffering Contributes to the Arrhythmogenic Substrate in Atrial Myocytes From Patients With Atrial Fibrillation

Abstract

Background: Alterations in the buffering of intracellular Ca²⁺, for which myofilament proteins play a key role, have been shown to promote cardiac arrhythmia. It is interesting that although studies report atrial myofibrillar degradation in patients with persistent atrial fibrillation (persAF), the intracellular Ca²⁺ buffering profile in persAF remains obscure. Therefore, we aimed to investigate the intracellular buffering of Ca²⁺ and its potential arrhythmogenic role in persAF.

Methods: Transmembrane Ca²⁺ fluxes (patch-clamp) and intracellular Ca²⁺ signaling (fluo-3-acetoxymethyl ester) were recorded simultaneously in myocytes from right atrial biopsies of sinus rhythm (Ctrl) and patients with persAF, alongside human atrial subtype induced pluripotent stem cell-derived cardiac myocytes (iPSC-CMs). Protein levels were quantified by immunoblotting of human atrial tissue and induced pluripotent stem cell-derived cardiac myocytes. Mouse whole heart and atrial electrophysiology were measured on a Langendorff system.

Results: Cytosolic Ca²⁺ buffering was decreased in atrial myocytes of patients with persAF because of a depleted amount of Ca²⁺ buffers. In agreement, protein levels of selected Ca²⁺ binding myofilament proteins, including cTnC (cardiac troponin C), a major cytosolic Ca²⁺ buffer, were significantly lower in patients with persAF. Small interfering RNA (siRNA)-mediated knockdown of cTnC (si-cTNC) in atrial iPSC-CM phenocopied the reduced cytosolic Ca²⁺ buffering observed in persAF. Si-cTnC treated atrial iPSC-CM exhibited a higher predisposition to spontaneous Ca²⁺ release events and developed action potential alternans at low stimulation frequencies. Last, indirect reduction of cytosolic Ca²⁺ buffering using blebbistatin in an ex vivo mouse whole heart model increased vulnerability to tachypacing-induced atrial arrhythmia, validating the direct mechanistic link between impaired cytosolic Ca²⁺ buffering and atrial arrhythmogenesis.

Conclusions: Our findings suggest that loss of myofilament proteins, particularly reduced cTnC protein levels, causes diminished cytosolic Ca²⁺ buffering in persAF, thereby potentiating the occurrence of spontaneous Ca²⁺ release events and atrial fibrillation susceptibility. Strategies targeting intracellular buffering may represent a promising therapeutic lead in persAF management.

Keywords: atrial fibrillation; atrial remodeling; calcium signaling; cardiac arrhythmias; electrophysiology; ion channels.

Figure 1.

Characterization of atrial myocytes isolated from patients without (Ctrl) and with persAF. A, Example 3-dimensional reconstruction of confocal z-stack images of atrial myocytes from Ctrl and persAF stained with di-4-ANEPPS. B, Mean±SEM cell dimensions and volume of control and persAF myocytes. C, I_Ca,L-triggered CaT in control and persAF atrial myocytes; representative simultaneous recordings of I_Ca,L (upper, inset, voltage-clamp protocol, 0.5 Hz) and triggered CaT (Fluo-3, lower). D, Mean±SEM peak I_Ca,L (left) and integrated I_Ca,L (right). E, Mean±SEM diastolic and systolic [Ca²⁺]_i (left) and resulting CaT amplitude (middle), and time constant (τ) of decay (right). *P<0.05, **P<0.01, ***P<0.001 vs control. n/N=number of myocytes/patients. Normality of data was determined by Shapiro-Wilk test, whereas comparison was made using the Student t test with Welch correction and Mann-Whitney U test for normally and nonnormally distributed data, respectively. CaT indicates Ca²⁺ transient; Ctrl, control; and persAF, persistent atrial fibrillation.

Figure 2.

Caffeine-induced CaT and corresponding transient inward current (I_NCX) to assess SR Ca²⁺ content and buffering properties of atrial myocytes isolated from patients without (control) and with persAF. A, Representative caffeine-induced CaT (upper), associated I_NCX (middle) and integral of inward current, corrected for cell volume to give a measure of total Ca²⁺ (lower). B, Mean±SEM amplitude and time constant (τ) of decay of caffeine-induced CaT. C, Mean±SEM calculated total Ca²⁺. D, Representative buffer curves showing the relationship between cytosolic free Ca²⁺ and total Ca²⁺, fitted with a hyperbolic function. E, Mean±SEM maximum buffering capacity (B_max, left) and dissociation constant (K_d, right), determined from buffer curves. F, Mean (line) ±SEM (shaded) of calculated individual total buffer power curves as a function of free [Ca²⁺]_i. **P<0.01, ***P<0.001 vs control. n/N=number of myocytes/patients. Normality of data was determined by Shapiro-Wilk test and comparison was made using the Mann Whitney U test. Ctrl indicates control; and persAF, persistent atrial fibrillation.

Figure 3.

Myofilament protein expression and contractile response to cytosolic Ca²⁺ in control and persAF. A, Immunoblots (upper left) and quantification (upper right) of cMyBP-C (cardiac myosin binding protein-C), its phosphorylated state (P-cMyBP-C), and cTnC (cardiac troponin-C) in atrial samples from controls and patients with persAF, normalized to CSQ (calsequestrin), except for P-cMyBP-C, which was normalized to total cMyBP-C. Immunoblots (lower left) and quantification (lower right) of cTnI (cardiac troponin I) and its phosphorylated state (P-cTnI) in atrial samples from controls and patients with persAF, normalized to CSQ and total cTnI, respectively. B, Absolute (left) and normalized (right) force-pCa relationship of skinned muscle fibers of controls and patients with persAF with mean±SEM of maximum force (F_max) and calcium sensitivity (pCa₅₀). *P<0.05 vs control. n=number of patients. Normality of data was determined by Shapiro-Wilk test, whereas comparison was made using the Student t test with Welch correction. Ctrl indicates control; and persAF, persistent atrial fibrillation.

Figure 4.

Quantification of decay of total Ca²⁺ in atrial myocytes isolated from patients without (control) and with persAF. A, Representative rate of decay of total Ca²⁺ (-d[Ca²⁺]_T/dt) plotted during systolic CaT against free [Ca²⁺]_i (left) and slope of -d[Ca²⁺]_T/dt plotted against [Ca²⁺]_i (right). B, Representative rate of decay of total Ca²⁺ during caffeine-induced Ca²⁺ transient (-d[Ca²⁺]_T/dt) plotted against the corresponding free [Ca²⁺]_i (left) and slope of -d[Ca²⁺]_T/dt during caffeine plotted against corresponding [Ca²⁺]_i (right). C, Difference between slopes in A and B, indicating unaltered [Ca²⁺]_i dependence of SERCA-mediated Ca²⁺ removal. *P<0.05 vs control. n/N=number of myocytes/patients. Normality of data was determined by Shapiro-Wilk test, whereas comparison was made using the Student t test with Welch correction and Mann-Whitney U test for normally and nonnormally distributed data, respectively. Ctrl indicates control; and persAF, persistent atrial fibrillation.

Figure 5.

Ca2+ handling and Ca²⁺ buffering properties in atrial iPSC-CMs with normal (control) and reduced (si-cTnC) cTnC levels. A, Representative simultaneous recordings of I_Ca,L (upper, inset, voltage-clamp protocol, 1 Hz) and triggered CaT (lower). B, Mean±SEM peak I_Ca,L (left) and integrated I_Ca,L (right) in control (siRNA ns) and si-cTnC (siRNA cTnC) iPSC-CMs. C, Mean±SEM diastolic and systolic [Ca²⁺]_i (left) and resulting CaT amplitude (middle), and time constant (τ) of decay (right). D, Representative caffeine-induced CaT (upper), associated I_NCX (middle) and integral of inward current, corrected for cell volume to give a measure of total Ca²⁺ (lower). E, Mean±SEM amplitude (left) and time constant (τ) of decay (right) of caffeine-induced CaT. F, Mean±SEM calculated total Ca²⁺. G, Buffer curves showing the relationship between cytosolic free Ca²⁺ and total Ca²⁺, fitted with a hyperbolic function. H, Mean±SEM maximum buffering capacity (B_max, left) and dissociation constant (K_d, right), determined from buffer curves. I, Mean±SEM of calculated individual total buffer power curves as a function of free [Ca²⁺]_i. *P<0.05, **P<0.01 vs control. n=number of myocytes (2–4 differentiations). Normality of data was determined by Shapiro-Wilk test, whereas comparison was made using the Student t test and Mann-Whitney U test for normally and nonnormally distributed data, respectively. Ctrl indicates control; cTnC, cardiac troponin C; CaT, Ca²⁺ transient; iPSC-CM, induced pluripotent stem cell–derived cardiac myocyte; ns, nonsilencing; and siRNA, small interfering RNA.

Figure 6.

Incidence of Ca²⁺ sparks and action potential (AP) alternans in atrial iPSC-CMs with normal (control) and reduced (si-cTnC) cTnC levels. A, Representative confocal line scans showing SR Ca²⁺ release in the form of Ca²⁺ sparks in control (siRNA ns) and si-cTnC (siRNA cTnC) iPSC-CMs. B, Mean±SEM Ca²⁺ spark frequency (CaSpF, left) and amplitude (right). C, Representative normalized traces of AP at 0.5 Hz (upper) and 2 Hz (lower) in control (left) and si-cTnC iPSC-CMs. D, AP duration at 90% repolarization (APD₉₀) at increasing diastolic intervals (AP restitution), fitted with a 1-phase association nonlinear function to determine maximum curve slope. E, Kaplan-Meier plot indicating the percentage of iPSC-CMs without alternans in relation to the respective pacing frequency. F, Mean±SEM alternans threshold frequency. Number of myocytes without AP alternans are shown in boxes above. ***P<0.001, *P<0.05 vs control. n=number of myocytes (2 or 3 differentiations). Comparison was made using the unpaired Student t test, the Mann-Whitney U test, and the Gehan-Breslow-Wilcoxon test (E). Ctrl indicates control; cTnC, cardiac troponin C; iPSC-CM, induced pluripotent stem cell–derived cardiac myocyte; ns, nonsilencing; and siRNA, small interfering RNA.

Figure 7.

Effect of Ca²⁺ sensitization on Ca²⁺ sparks in atrial iPSC-CMs with reduced (si-cTnC) cTnC levels. A, Representative confocal line scans of atrial iPSC-CMs with normal (control, siRNA ns) and reduced (si-cTnC, siRNA cTnC) cTnC levels, pretreated with EMD57033 (EMD, 5 µmol/L). B, Mean±SEM Ca²⁺ spark frequency (CaSpF, left) and amplitude (right). *P<0.05, **P<0.01 vs control and si-cTnC. n=number of myocytes (2 differentiations). Comparison was made using the Kruskal-Wallis test followed by the Dunn post hoc test. Ctrl indicates control; cTnC, cardiac troponin C; iPSC-CM, induced pluripotent stem cell–derived cardiac myocyte; ns, nonsilencing; and siRNA, small interfering RNA.

Figure 8.

Langendorff experiments in mouse heart. A, Representative atrial electrogram traces showing effect of burst pacing (100 Hz) in the absence (control, left) and presence (right) of blebbistatin (5 µmol/L) (2 mmol/L K⁺ in both). B, Grouped bar chart showing mean±SEM inducibility of atrial arrhythmic activity for each potassium level, differentiated by the presence or absence of blebbistatin. The chart highlights significant main effects of blebbistatin, F(1, 72)=20.06, P<0.0001, and potassium, F(3, 72)=14.91, P<0.0001. C, Mean±SEM arrhythmic episode duration for each potassium level, in the presence or absence of blebbistatin. The graph highlights the significant effect of potassium, F(3, 36)=3.52, P<0.05. D, Kaplan-Meier plot showing the percentage of hearts without arrhythmic activity. *P<0.05, ***P<0.001 vs control. n=number of hearts. Comparison using 2-way ANOVA followed by a Fisher Least Significant Difference post hoc test (B and C) and the Gehan-Breslow-Wilcoxon test (D). Ctrl indicates control.