Cytoskeletal disarray increases arrhythmogenic vulnerability during sympathetic stimulation in a model of hypertrophic cardiomyopathy

Figures

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  Figure 1. Representative electrocardiograms (ECG) before (A–B) during (C–D) and 2 h after (E–F) i. p. injection of 2 × 20 mg/kg ISO in a wt (A, C, E) and a αMHC403/+ mouse (B, D, F). Signal-averaged complexes show distinct effects of ISO on wt (G) and αMHC403/+ mouse (H). Representative ECG recordings in an αMHC403/+ mouse before (I) and after 1 mg/kg atenolol (J) and following ISO (K) treatment. (L) Recovery of the αMHC403/+ mouse 2 h post-ISO. (M–O) R–R intervals plotted against time for ISO treated wt (M), αMHC403/+ mouse (N) and αMHC403/+ mouse treated with atenolol (Ate) 10 min prior to ISO as indicated (O). The irregular RR intervals representing arrhythmias can be seen as dots following ISO.

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  Figure 2. Representative AP recordings from wt (A) and αMHC403/+ ventricular myocytes (B), in the absence (black lines) or presence of 100 nM ISO (red). AP durations at 90% repolarization in the absence (wt n = 62, N = 21; αMHC403/+ n = 63, N = 19), or presence of isoproterenol (wt n = 10, N = 7; αMHC403/+ n = 17, N = 5) (C). Representative AP train recordings (9 Hz) on wt (D and F) and αMHC403/+ cardiac myocytes (E and G), under control conditions (D, E) or the presence of 100 nM ISO (F, G). Zoomed area shows the last 3 s of the 10 s recordings. Representative AP recordings from wt (H) and αMHC403/+ cardiac myocytes (I), in the absence (black) or presence of 10 μM forskolin (blue). Representative AP train recordings in control conditions (J) or the presence of 10 μM forskolin (K) on αMHC403/+ cardiac myocytes. Arrows indicate DADs.

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  Figure 3. Representative potassium current recordings from wt (A, C) and αMHC403/+ (B, D) ventricular myocytes under control conditions (A, B) or the presence of 100 nM ISO (C, D). Whole-cell voltage-gated outward K+ (KV) currents were evoked in response to 500 ms depolarizing voltage steps to test potentials between − 60 and + 40 mV, in 10 mV increments, from a holding potential of − 70 mV (− 70, 0 mV and positive test potentials shown). Inward rectifying K+ currents (IK1) evoked in response to hyperpolarization to − 120 mV. (E–F) Scatter plot with bar graphs show IK density (pA/pF) values for different KV current components as means ± SEM. wt n = 8–15, N = 5 αMHC403/+ n = 10–16 N = 5*p < 0.05 control versus ISO, †p < 0.05 wt versus αMHC403/+ (G) Representative immunofluorescence and corresponding Western blot images for KV4.2, Kir2.1, Kir6.2 potassium channels from wt and αMHC403/+ hearts as indicated. Relative optical density (Rel OD) values were calculated using VDAC (voltage dependent anion channel) as loading control. For further details please see section “Materials and methods”.

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  Figure 4. Representative immunofluorescence images of ventricular myocytes isolated from wt and αMHC403/+ hearts, immunolabelled with connexin 43, caveolin-3 and SAP97. Scale represents 20 μm. Representative Western blot images with relative optical density values are also shown for the same proteins, along with corresponding loading controls. *p < 0.05 wt versus αMHC403/+. For further detail see section “Materials and methods”.

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  Figure 5. Representative calcium current recordings and IV traces from wt (A) and αMHC403/+ (B) ventricular myocytes, under control conditions (black lines) or the presence of ISO (red). Scatter plot with bar graphs show the cell capacitance (C) used to calculate ICa density (pA/pF) (D); and (E) the rate of inactivation (tau). Number of cells used for the study: wt n = 26, with ISO n = 13; αMHC403/+ n = 42, with ISO n = 22. (F) Representative Western blot images of CaV1.2 protein using total heart homogenates from wt (N = 5) and αMHC403/+ hearts (N = 4) repeated in triplicate. (G) In vitro PKA phosphorylated immunoprecipitated protein samples were used to fluorescently detect total phosphoprotein as well as PKA-specific phosphorylation. (H, I) Representative Fluo-4 calcium transients acquired on current clamped ventricular myocytes isolated from wt (H) and αMHC403/+ mice (I) under control conditions (black traces, n = 23, N = 5; and n = 18, N = 5 respectively) or the presence of 100 nM ISO (red traces, (n = 13, N = 5; and n = 11, N = 5 respectively). Scatter plots with bar graphs show relative amplitude (as F/F0, J), exponential rise time (ms) (K) and exponential decay (tau) (L). (M–N)…

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  Figure 6. ISO-induced super-clustering response of CaV1.2 in ventricular myocytes. (A–B) TIRF images (top row) and super-resolution GSD localization maps (bottom row) of immunostained CaV1.2 channels in fixed adult ventricular myocytes isolated from wt (A) and αMHC403/+ mice (B). Control (left column) and ISO-stimulated (right column) myocytes are displayed side-by-side for comparison. Cluster ROIs indicated by yellow boxes in the GSD images appear magnified below the relevant image (bottom row). Mean CaV1.2 channel cluster areas ± S.E.M. (indicated by red lines and error bars) are summarized for each condition in the aligned dot-plot (C). *p < 0.05 for comparisons as indicated. n = 14–27 ventricular myocytes, from N = 4–7 mice.

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  Figure 7. Colocalization of β1ΑRs with CaV1.2 is altered in αMHC403/+ mutant myocytes. (A) and (B): TIRF images (top row), super-resolution GSD localization maps (middle row) and binarized images (bottom row) of immunostained CaV1.2 channels and β1ARs in representative fixed adult ventricular myocytes isolated from WT mice under control (A) or ISO-stimulated conditions (B). Merged two-channel image showing relative distributions of CaV1.2 and β1ARs is shown in the third column with the colocalized binarized image. (C, D) Same layout format for myocytes isolated from αMHC403/+ mice. (E) Mean β1ARs cluster areas ± S.E.M. (indicated by red lines and error bars) are summarized for each condition in the aligned dot-plot. (F) % colocalization of CaV1.2 with β1ARs and G: β1ARs and CaV1.2 are summarized. *p < 0.05 for comparisons as indicated. n = 14–27 ventricular myocytes, from N = 4–7 mice.