Nitro-fatty acids suppress ischemic ventricular arrhythmias by preserving calcium homeostasis

Figures

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  Figure 1. Surface and intracardiac electrocardiograms (ECG) of NO2-OA versus vehicle treated mice. (A) Representative images of ECG before induction of myocardial ischemia (Baseline; left panel), 20 min after ischemia induction without right ventricular stimulation (AMI; middle panel) and after right ventricular stimulation (STIM; right panel) with appearance of ventricular tachycardias (VT) in NO2-OA- or vehicle treated mice. Scale bar = 200 ms. (B) Probability of VT in sham versus LAD-ligated mice (AMI) with and without NO2-OA treatment (AMI vehicle: 11.2 ± 3.1% vs. AMI NO2-OA: 3.6 ± 2.3; n = 8/7). (C) Number of VT episodes (AMI vehicle: 5.4 ± 1.5 vs. AMI NO2-OA: 1.7 ± 1.1; n = 8/7) and (D) Total time of VT episodes in sham versus AMI mice with and without NO2-OA treatment (AMI vehicle: 3.2 ± 2.4 s vs. AMI NO2-OA: 0.6 ± 0.4 s; n = 8/7). Graphs show Mean ± SEM. Brackets indicate Mean ± SD. *** P < 0.001.

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  Figure 2. Epicardial mapping analyses within the peri-ischemic region by microelectrode array. (A) Scheme of left ventricular positioning of the multi-electrode array (MEA) indicating ischemic and peri-ischemic regions and cardiac electrical conduction (blue arrows). (B) Representative maps of spontaneous conduction in the peri-ischemic region with vehicle treatment (control) versus treatment with NO2-OA. Left side of the maps indicate electrodes oriented nearest to myocardial septum as shown in A. (C) Mean inter-electrode latency of electrical conduction (sham vehicle: 1.4 ± 0.5 s/m vs. AMI vehicle: 4.6 ± 3.3 s/m vs. AMI NO2-OA: 4.1 ± 2.9 s/m; n = 8/7/7) and (D) inhomogeneity index in sham animals versus 20 min of ischemia with and without NO2-OA treatment (sham vehicle: 1.4 ± 0.3 vs. AMI vehicle: 2.2 ± 0.3 vs. AMI NO2-OA: 1.6 ± 0.4; n = 8/7/7). Interelectrode distance = 300 µm. Graphs show Mean ± SEM. Brackets indicate Mean ± SD. * P < 0.05; ** P < 0.01. Figures were produced using Servier Medical Art (https://www.servier.com/).

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  Figure 3. Rhythm analyses and Ca2+ transient measurements of isolated adult cardiomyocytes under beta-adrenergic stimulation. (A) Number of arrhythmic events and representative recordings of calcium transients of isolated adult cardiomyocytes with and without Iso and Iso + NO2-OA treatment (vehicle: 0.0 ± 0.0 vs. Iso: 10.9 ± 18.6 vs. Iso + NO2-OA: 1.4 ± 3.9; n(cells out of 3 animals) = 9/12/11). Asterisks indicate time point of external stimulation. (B) Time to maximum Ca2+ peak as indicator for Ca2+ influx velocity (vehicle: 33.2 ± 9.3 ms vs. Iso: 26.1 ± 13.1 ms vs. Iso + NO2-OA: 26.3 ± 8.4 ms; n(cells out of 3 animals) = 9/15/13). (C) Relative Ca2+ peak height as indicator for the maximal amount of Ca2+ in control- versus Iso- and Iso + NO2-OA treated cells (vehicle: 0.53 ± 0.12 vs. Iso: 0.93 ± 0.25 vs. Iso + NO2-OA: 0.91 ± 0.29 ms; n(cells out of 3 animals) = 9/15/13). (D) Time to 50% Ca2+ decay (vehicle: 173.7 ± 35.9 ms vs. Iso: 106.4 ± 22.8 ms vs. Iso + NO2-OA: 89.9 ± 17.8 ms; n(cells out of 3 animals) = 9/14/13) and € 90% Ca2+ decay as indicators for cytosolic Ca2+ clearance (vehicle: 448.0 ± 12.5 ms vs. Iso: 291.1 ± 12.8 ms vs. Iso + NO2-OA: 308.0 ± 10.3 ms; n (cells out of 3…

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  Figure 4. Iso dependent Ca2+ leak analyses in isolated adult cardiomyocytes. (A) Representative Ca2+ transient recordings of isolated adult isoproterenol (Iso)- and Iso + NO2-OA treated cardiomyocytes upon field stimulation (1 Hz and 4 Hz) followed by assessment of Ca2+ leak under non-stimulated conditions after removal of the RyR2 inhibitor tetracaine. Arrows indicate spontaneous arrhythmic events (red and black crosses visualize time points of Fura-2 ratios used for Ca2+ leak calculation). (B) Total number of arrhythmic events after removal of tetracaine (between red and black crosses) of Iso or Iso + NO2-OA subjected isolated cardiomyocytes (Iso: 11.2 ± 11.42 vs. Iso + NO2-OA: 0.2 ± 0.67; n = 8/8). (C) Sarcoplasmic (SR) Ca2+ leak quantified as Δ Fura-2 ratio (Iso: 0.021 ± 0.019 Δ Fura-2 ratio vs. Iso + NO2-OA: -0.006 ± 0.013 Δ Fura-2 ratio). (D) Total SR Ca2+ load in Iso versus Iso + NO2-OA treated cardiomyocytes (Iso: 0.53 ± 0.29 vs. Iso + NO2-OA: 0.56 ± 0.33). (B–D) n(cells out of 3 animals) = 8/8. Graphs show Mean ± SEM. Brackets indicate Mean ± SD. ** P < 0.01; *** P < 0.001.

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  Figure 5. Ca2+ sensitivity analyses in isolated adult cardiomyocytes. (A) Representative Ca2+ transient recordings of isolated adult isoproterenol (Iso)- and Iso + NO2-OA treated cardiomyocytes upon increasing extracellular Ca2+ concentrations (0.25 mM–2 mM) stimulated at 1 Hz for 30 s following a non-stimulated period of 20 s in which arrhythmic Ca2+ events were recorded (asterisks). (B) Relative Ca2+ transient peak height in control- versus Iso- and Iso + NO2-OA treated cells upon different Ca2+ concentrations (Iso vs. Iso + NO2-OA [∆Fura-2]; 0.25 mM Ca2+: 0.71 ± 0.28 vs. 0.6 ± 0.23; 0.5 mM Ca2+: 0.73 ± 0.19 vs. 0.71 ± 0.0.28; 1 mM Ca2+: 1.01 ± 0.16 vs. 1.05 ± 0.16; 2 mM Ca2+: 1.24 ± 0.14 vs. 1.08 ± 0.23). (C) Relative diastolic Ca2+ levels measured between the 1 Hz pacing periods (Iso vs. Iso + NO2-OA [∆Fura-2]; 0.25 mM Ca2+: 1.32 ± 0.16 vs. 1.38 ± 0.13; 0.5 mM Ca2+: 1.35 ± 0.16 vs. 1.40 ± 0.11; 1 mM Ca2+: 1.39 ± 0.16 vs. 1.42 ± 0.11; 2 mM Ca2+: 1.45 ± 0.18 vs. 1.45 ± 0.12). (D) Mean number of arrhythmic Ca2+ release events within the non-stimulated time period (asterisks, Iso vs. Iso + NO2-OA [mean number]; 0.25 mM Ca2+: 0.5 ± 1.07 vs. 0.0 ± 0.0; 0.5 mM Ca2+: 0.22 ± 0.67 vs. 0.0 ± 0.0;…

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  Figure 6. CaMKII activity and RyR2 phosphorylation upon beta-adrenergic stimulation in isolated adult cardiomyocytes. (A) Immunoblot analyses after GST pulldown showing active (GST-HDAC4 419-670-bound) CaMKII in relation to total CaMKII (vehicle: 100% ± 36.1 vs. Iso: 116.5% ± 57.9 vs. Iso + NO2-OA: 66% ± 34.4). (B) Critical RyR2 phosphorylation under Iso stimulation and simultaneous NO2-OA treatment as indicated by immunoblotting (vehicle: 100% vs. Iso: 140% ± 22.9 vs. NO2-OA: 129% ± 19.8). (C) Phosphorylation of phospholamban (Thr17-p-PLN) in vehicle- vs. Iso- vs. Iso + NO2-OA treated cardiomyocytes (vehicle: 100% vs. Iso: 107.2% ± 0.1 vs. NO2-OA: 107.0% ± 2.9). n (cells out of 3 animals) = 9/9/9. Graphs show Mean ± SEM. Brackets indicate Mean ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001. The cropped blots are presented, and their full-length blots are included in the Supplemental Figures S4–S6.

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  Figure 7. NO2-OA prevents acute ischemia induced ventricular tachycardias. (A) Increased adrenergic signaling leads to the activation of CaMKII with subsequently dysregulated RyR2-function and impaired Ca2+ homeostasis due to a diastolic Ca2+ leak. (B) Treatment with NO2-OA restores Ca2+ homeostasis and prevents ventricular tachycardia (VT) development. Figures were produced using Servier Medical Art (https://www.servier.com/).