Downregulation of L-type Ca2+ current in patients with atrial fibrillation results from altered channel gating and disruption of membrane microdomains

Figures

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  Figure 1. Whole-cell and single-channel ICa,L current measured in RA and LA myocytes from patients with SR and AF. Current–voltage relationship of the whole-cell ICa,L (A), steady-state curves of voltage-dependent ICa,L activation (B), mean basal current density measured at +10 mV (C), and mean V50 activation voltages (D) measured in cardiomyocytes isolated from RA-SR (N/n = 4/9), RA-AF (N/n = 2/6), LA-SR (N/n = 4/4) and LA-AF (N/n = 4/7) samples. ∗P < .05, ∗∗P < .01 for SR vs AF by 1-way ANOVA with Tukey’s post-hoc test. E: Representative SICM surface topography scans are shown for RA-SR, RA-AF, and LA-AF myocytes; T-tubule and crest regions from which channel activity were recorded (T-LTCC and C-LTCC, respectively), are indicated. On the right, representative single channel traces recorded at −6.7 mV are shown to illustrate an enhanced LTCC activity (ie, more often and longer channel openings) in AF myocytes. F: A single-channel LTCC current distribution (% of occurrence) in human RA and in LA myocytes obtained from patients with SR and AF. ∗P < .05 by Fisher exact test. G: Open probability PO measured at −6.7 mV in RA and LA myocytes in SR and AF (RA-SR: T-LTCC = 12 and C-LTCC = 5…

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  Figure 2. Upregulation of PKA and CaMKII activity in AF. Western blot of human atrial tissue lysates probed for (A) auto-phosphorylated at the Thr286/287 site CaMKII, and (B) PKA-dependent phosphorylated site Ser16 of phospholamban. Statistical significance was assessed by unpaired Student’s t test, P < .001, n = 5–6 per diagnosis, 3 technical replicates per blot. AF = atrial fibrillation; CaMKII = Ca2+-calmodulin kinase II; LA = left atrium; LTCC = L-type Ca2+ channels; PKA = protein kinase A; RA = right atrium; SR = sinus rhythm.

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  Figure 3. Effect of isoproterenol (Iso, 1 mM) stimulation on single C-LTCCs in LA-AF myocytes and on the whole-cell ICa,L in RA and LA myocytes from patients with SR and AF. A: Single C-LTCC current distribution (% of occurrence) recorded in the crest microdomain. B: Open probability (PO) measured at −6.7 mV without (no treatment, n = 5 channels) and with 1 μM Iso (n = 7 channels), NS. C: Representative single channel traces at −6.7 mV showing LTCC activity recorded in the crest without and with Iso. D: Representative whole-cell peak ICa,L traces in the presence and absence of Iso. E: Relative increase in the peak ICa,L current density in the presence of Iso compared with basal current from RA-SR (n = 12), RA-AF (n = 8), LA-SR (n = 13) and LA-AF (n = 5) myocytes. Significance was assessed by 1-way ANOVA with Tukey’s post hoc test. AF = atrial fibrillation; ANOVA = analysis of variance; LA = left atrium; LTCC = L-type Ca2+ channels; RA = right atrium; SR = sinus rhythm.

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  Figure 4. Transverse T-tubules, surface topography and caveolae integrity in human RA and LA myocytes from patients with SR and AF. T-tubules network (A) and cardiomyocyte surface topography scans (B) of atrial myocytes from the patients with SR and AF. T-tubules, crests, and non-structured areas are indicated by arrows. Below, average data for T-tubule density (as % of cell volume) and Z-groove index (calculated as a ratio of z-grooves per 10 μm x 10 μm scan) are shown. ∗∗P < .01, ∗∗∗P < .001 for SR vs AF groups. P-values were determined by 2-way ANOVA with Bonferroni correction for T-tubule density and by Student’s t test for Z-groove index. C: Representative caveolin-3 (Cav-3) immunoblots from SR and AF atrial tissue homogenates and GAPDH-normalized Cav-3 levels from RA (n = 10/9 SR/AF) and LA (n = 3/4 SR/AF) tissues, 3 technical replicates per blot. ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001, respectively, for AF vs SR by Student t test. AF = atrial fibrillation; ANOVA = analysis of variance; LA = left atrium; RA = right atrium; SR = sinus rhythm.

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  Figure 5. Computational modeling of experimentally observed ICa,L downregulation in right (RA) and left (LA) atria in AF. (A) The human atrial AP and Ca2+ model includes subspaces for the cytosol, sarcoplasmic reticulum, tubule junction, and sarcolemma. The gating equations (dC/T-LTCC, fC/T-LTCC) and channel conductances (GC/T-LTCC) were modified to account for microdomain differences. Additional parameters that modify the current because of changes in T-tubule density (TTdensity_scf), microdomain occurrence (Ca_sl_scf, Ca_junc_scf), and ratio of T-tubule to crest channels (Fjunc_Ca) are described in the main text. The baseline current equations (ĪC/T-LTCC) were unmodified from Grandi et al18B through E: Experimental vs model I–V curves (B and C) and steady-state curves of voltage-dependent channel activation (D and E) in RA and LA myocytes from patients with SR and AF. Points are experimental data; lines are model results. AF = atrial fibrillation; LA = left atrium; LTCC = L-type Ca2+ channels; RA = right atrium; SR = sinus rhythm.

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  Figure 6. Relative impact of individual ultrastructural and biophysical changes on ICa,L remodeling in right (RA) and left (LA) atrial cardiomyocytes during AF. Whole-cell ICa,L computational modeling incorporating structural and biophysical channel modulation in AF. A: Contribution of individual changes and their cumulative effect on the maximum current amplitude in AF at membrane potential Vm = 0 is shown. B: Contribution of T-LTCC vs C-LTCC to the ICa,L peak current in SR and AF right and left atrial myocytes. AF = atrial fibrillation; LTCC = L-type Ca2+ channels; SR = sinus rhythm.

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  Figure 7. Overexpression of caveolin-3 upregulates membrane level of Cav1.2 and partially restores ICa,L in LA-AF myocytes. A: Immunofluorescent staining of caveolin-3 (Cav3, red) and CaV1.2 (green) in control (LA-AF, 48-hour culture, but not transfected) and caveolin-3 overexpression (LA-AF with Cav3-OE) left atrial human myocytes from patients with AF. B and C: Relative expression of caveolin-3 (B) and CaV1.2 (C) is shown for LA-AF (25 cells/3 patients and 43 cells/3 patients for 48-hour cultured control and Cav3-OE groups, respectively) and LA-SR (10 cells/1 patient and 19 cells/1 patient for 48-hour cultured control and Cav3-OE groups, respectively) groups. Statistics by Mann-Whitney after Shapiro-Wilk normality test. D: Individual Pearson coefficients are shown for LA-AF cardiomyocytes transfected with caveolin-3 (Cav3-OE, n = 10 cells from n = 3 samples) and non-transfected, but cultured (control, n = 10 cells from n = 2 samples). ∗P < .05 by unpaired Student’s t test. E: Current–voltage relationship of ICa,L and average peak ICa,L current in cultured, but not transfected (control, n = 6 cells) and transfected with Cav3 (Cav3-OE, n = 3 cells) LA-AF myocytes. ∗P < .05 by unpaired…