Hyperactive ryanodine receptors in human heart failure and ischaemic cardiomyopathy reside outside of couplons

Figures

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  Figure 1. Myocyte hypertrophy and increased fraction of non-coupled release sites with an increased spark frequency in human HF. (A) Cell size and TATS characteristics in non-HF (ncells = 27–31; Nhumans = 4) and HF (ncells = 83–91; nhumans = 5). Data are presented as mean value per subject. (B) Left: Examples of confocal line-scan recording of Ca2+ transients in non-HF and HF. Middle and right: Distribution and quantification of TF50 of the Ca2+ transient in coupled (<14.5 ms) and non-coupled (>21.5 ms) sites in non-HF (ncells = 18; Nhumans = 4) and HF (ncells = 32; Nhumans = 5). (C) Left: Example of a confocal line-scan recording using TF50 of the Ca2+ transient to assign sparks to different release sites. Middle: Spark frequency in coupled and non-coupled sites in non-HF (ncells = 22; Nhumans = 4) and Right: HF (ncells = 32; Nhumans = 6) (*P-value < 0.05; **P-value < 0.01; ***P-value < 0.001).

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  Figure 2. CaMKII inhibition normalized both RyR activity and Ca2+ transients in human HF myocytes. (A) The effect of AIP in coupled and non-coupled sites in non-HF (ncells = 19; Nhumans = 4) and HF (ncells = 23; Nhumans = 6). (B) Ca2+ transient amplitude in non-HF (ncells = 19; Nhuman = 4) and HF (ncells = 25; Nhuman = 6) at 2 Hz stimulation with examples of line-scan and reduced synchrony. The fraction of the line-scan with at least half-maximal Ca2+ release as a function of time. SR Ca2+ content in non-HF and HF at 0.5 Hz (ncells = 8; Nhumans = 4 in non-HF; ncells = 16; Nhumans = 6 in HF) and 2 Hz (ncells = 11; Nhumans = 4 in non-HF; ncells = 20; Nhumans = 6 in HF). (C) Ca2+ transient amplitude in non-HF (ncells = 19; Nhuman = 4) and HF (ncells = 25; Nhuman = 6) at 2 Hz and the effect with AIP (non-HF: ncells = 19; Nhuman = 4 and HF: ncells = 23; Nhuman = 6) (*P-value < 0.05; **P-value < 0.01; ***P-value < 0.001).

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  Figure 3. CaMKII-dependent and ROS-dependent signalling examined at coupled vs. non-coupled sites after MI in pig ventricular myocytes. (A) Left: The effect of AIP in coupled sites in MI (ncells = 16; Npigs = 7) and SHAM (ncells = 15; Npigs = 4). Right: The effect of gp91 ds-tat peptide in coupled sites in MI (ncells = 14; Npigs = 4) and SHAM (ncells = 13; Npigs = 4). (B) Left: The effect of AIP in non-coupled sites in MI (ncells = 16; Npigs = 7) and SHAM (ncells = 15; Npigs = 4). Right: The effect of NAC in non-coupled sites in MI (ncells = 10; Npigs = 5) and SHAM (ncells = 10; Npigs = 3) (*P-value < 0.05; **P-value < 0.01).

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  Figure 4. Ca2+ and ROS in the dyadic cleft after MI in pig myocytes. (A) a. Example of a confocal image of a pig ventricular myocyte stained for RyR (blue), CaMKII (green), and NCX (red). The co-localization of CaMKII in coupled and non-coupled RyRs is shown using the Manders’ coefficient in SHAM (ncells = 26; Npigs = 6) and MI (ncells = 32; Npigs = 7). b. Example of tissue samples in SHAM and MI stained for gp91 (red), NCX (green) and overlay (yellow). Scale bar equals 5 µm. (B) Example and mean values of ICaL inactivation in SHAM (ncells = 19; Npigs = 3) and MI (ncells = 24; Npigs = 5). (C) The effect of L-NAME (ncells = 14; Npigs = 4) and L-VNIO (MI: ncells = 15; Npigs = 4; SHAM: ncells = 12; Npigs = 3) in coupled sites in MI and SHAM (*P-value < 0.05; **P-value < 0.01; ***P-value < 0.001).

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  Figure 5. Mito-ROS microdomain affects non-coupled sites after MI in pig myocytes. (A) The effect of mitoTEMPO on spark frequency in coupled and non-coupled sites in MI (ncells = 14; Npigs = 4) and SHAM (ncells = 13; Npigs = 5). (B) a. Example traces of NAD(P)H and FAD signals, measured over time as the mean fluorescence intensity, in SHAM and MI at 2 Hz. b. For each cell, the FAD/NAD(P)H ratio measured after pacing at 2 Hz was normalized to the rest value. The graph represents the frequency distribution with the fitted Gaussian curves in SHAM and MI (*P-value < 0.05; ***P-value < 0.001).

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  Figure 6. Ca2+ wave frequency increases and Ca2+ wave origin shifts towards non-coupled release sites after MI in pig myocytes. (A). Examples of Ca2+ waves in SHAM and MI are shown. (B) Wave frequency (SHAM: ncells = 59; Npigs = 13, MI: ncells = 62; Npigs = 16), wave-induced Ca2+ transient amplitude (SHAM: nwaves = 30 in ncells = 25 in Npigs = 9; MI: nwaves = 49 in ncells = 38 in Npigs = 11) and wave-induced INCX (SHAM: nwaves = 59 in ncells = 32 in Npigs = 9; MI: nwaves = 91 in ncells = 38 in Npigs = 11) for SHAM and MI. (C) DAD incidence, DAD amplitude and spontaneous APs in SHAM (ncells = 28; Npigs = 6) and MI (ncells = 34; Npigs = 7). (D) Left: Waves are assigned to coupled or non-coupled release sites by using the wave origin (pixel-based) and TF50 of the Ca2+ transient. Middle: The distribution of wave origins and Right: wave frequency in SHAM (ncells = 39; Npigs = 9) and MI (ncells = 62; Npigs = 16). (E) a. Example and quantification of colliding and single waves in SHAM and MI. b. Ca2+ wave peak INCX and integrated INCX in coupled and non-coupled sites in SHAM and MI. Colliding waves are indicated in green. Only colliding waves are included when originating from only coupled or…

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  Figure 7. Non-coupled release sites are more sensitive to RyR blockers and inhibition of RyR modulators after MI in pig myocytes. (A) Effects of flecainide (SHAM: ncells = 27; Npigs = 5; MI: ncells = 26; Npigs = 6), S107 (SHAM: ncells = 19; Npigs = 3; MI: ncells = 27; Npigs = 4), AIP (SHAM: ncells = 22; Npigs = 3; MI: ncells = 25; Npigs = 9) and mitoTEMPO (MI: ncells = 23; Npigs = 5) on (A) wave frequency and (B) global Ca2+ transient in SHAM and MI (*P-value < 0.05; **P-value < 0.01).