Introduction

Due to significant advances in the therapy of acute myocardial ischemia (AMI), e.g. more aggressive approaches to coronary revascularization and secondary prevention, AMI mortality has declined drastically over the last decades1. However, patients suffering from AMI with ventricular tachycardia (VT) or -fibrillation (VF), still have a fourfold increased risk of in-hospital mortality2.

The mechanisms accounting for the development of acute VT are diverse: in the acute ischemic phase, 15–30 min after AMI, the development of so called 1B arrhythmias has been linked to increased catecholamine release and disturbances in Ca2+ signaling within cardiomyocytes of the ischemic border zone, thus leading to ventricular conduction blocks and enhanced VT vulnerability3,4. In particular, oxidation and subsequent autophosphorylation of Ca2+/calmodulin dependent kinase II (CaMKII) cause malfunctioning of ryanodine receptor type 2 (RyR2) channels and dysregulation of phospholamban (PLN)-dependent cytosolic Ca2+ clearance leading to an intracellular Ca2+ leak from the sarcoplasmic reticulum (SR)5,6. Consequently, depolarization of the membrane potential is enhanced thereby causing inactivation of Na+ channels with further slowing of ventricular conduction7.

As the identification of AMI patients at risk for VT remains difficult, a therapeutic strategy would have to be applied to a broad patient population. Therefore, dietary intervention with endogenously produced anti-arrhythmic modulators appears as an attractive therapeutic approach. This concept has been recently supported by the randomized double blind trial REDUCE-IT which demonstrates that prophylactic treatment with icosapentaenoic acid-ethyl (VASCEPA) significantly reduces cardiovascular events, the rate of cardiac arrests, and the number of sudden cardiac deaths (SCD)8.

Nitro-oleic acid (9- and 10-nitro-octadec-9-cis-enoic acid; NO2-OA) is an endogenously generated unsaturated fatty acid nitroalkene derivative that is formed by oleic acid reaction with nitric oxide and nitrite-derived nitrogen dioxide (•NO2)9,10. NO2-OA is conferred with an electrophilic β-carbon that rapidly and reversibly reacts with nucleophilic cysteine-, and to a lesser extent histidine, residues via a Michael addition11. The covalent nitro adduct is essential for the metabolic effects of NO2-OA since native oleic acid is not electrophilic and ineffective in various studies12,13,14. Nitro-fatty acids have been identified as potent endogenous anti-inflammatory mediators that affect a number of signaling mediators, transcriptional regulatory proteins and ion channels, including nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells (NF-κB), kelch-like ECH-associated protein 1 (Keap1/Nrf2), mitogen-activated protein kinases (MAPK), peroxisome proliferator-activated receptor gamma (PPARγ) and transient receptor potential channels (TRP) channels15,16,17. Preclinical studies have demonstrated therapeutic benefit in a variety of disease models such as pulmonary hypertension, nephritis and, of relevance to herein, reperfusion damage after myocardial ischemia (I/R) and a subsequent attenuation of cardiac fibrosis, reduced infarct size and preserved left ventricular function18,19,20. Since NO2-OA has passed safety clinical phase I trials21 these results have been translated into two, currently recruiting, clinical phase II trials targeting pulmonary hypertension and primary focal segmental glomerular sclerosis22,23.

Very low levels of NO2-OA could be detected in human serum- and urine24 as well as in the myocardium of rodents19. Nonetheless, inflammatory stimuli like I/R induce substantial generation of NO2-OA up to µM concentrations by enhanced formation of nitrating species19. Furthermore, increased endogenous formation of nitro-linoleic acid has been demonstrated in healthy humans after dietary supplementation with conjugated linoleic acid and nitrite or nitrate25.

If anti-arrhythmic effects of nitro-fatty acids could indeed be shown, a dietary supplementation or a pure pharmaceutical-based nitro-fatty acid therapeutic strategy in patients at risk for cardiovascular events could evolve as a strategy for prevention of sudden cardiac death.

Results

Susceptibility to VT upon right ventricular stimulation

Susceptibility to VT induction 20 min after ligation of the left anterior descending artery (LAD) was drastically reduced in animals pretreated with NO2-OA (representative ECG recordings shown in Fig. 1A). Thus, the probability of VT induction, calculated as the percentage of induced VT episodes per stimulation maneuvers (Fig. 1B), as well as the total number of inducible VT episodes (Fig. 1C) were both significantly lower in NO2-OA pretreated animals. Additionally, the total time of VT episodes was reduced after NO2-OA-pretreament (Fig. 1D) indicating that VT episodes in NO2-OA pretreated ischemic hearts were rapidly self-terminating.

Figure 1
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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Homogeneity of conduction

Epicardial mapping studies within the peri-ischemic region (positioning of the multi-electrode array (MEA) is shown in Fig. 2A) clearly revealed a more homogeneous conduction pattern in NO2-OA pretreated- compared to vehicle treated animals (representative conduction maps are shown in Fig. 2B). Of interest, only weak and therefore unevaluable electrical signals were detectable within the core ischemic region (data not shown). The mean electrical latency, measured apico-septally next to the ischemic core region26, was significantly elevated after ischemia induction, indicating a reduction of electrical conduction velocity. Although latency was numerically lower in NO2-OA pretreated hearts as compared to controls, it failed to reach statistical significance (Fig. 2C). However, the index of inhomogeneity was significantly lower upon administration of NO2-OA, indicating a reduction in ventricular conduction blocks (Fig. 2D)27.

Figure 2
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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To investigate potential side effects of NO2-OA on action potential (AP) development, we performed patch-clamp analysis of isolated adult cardiomyocytes under baseline conditions. The treatment with NO2-OA revealed no differences in the AP morphology regarding resting potential (Supplemental Fig. 1A), AP overshoot, (Supplemental Fig. 1B) or AP duration (APD, Supplemental Fig. 1C) between control and NO2-OA treated cells.

Ca2+ transient analyses under isoproterenol treatment

Given that catecholamine induced disturbances in Ca2+ homeostasis are a major contributor to arrhythmia development in acute ischemia3, we investigated cytosolic Ca2+ transients and arrhythmic events in isolated adult cardiomyocytes by ratiometric fluorescence imaging (IonOptix Corp)28. The number of arrhythmic events which appeared aside from a basic 1 Hz pacing stimulus (e.g. caused by early after depolarizations (EAD) and delayed after depolarizations (DAD)), was increased after beta-adrenergic stimulation with isoproterenol (Iso)3. Crucially, this increase was prevented by NO2-OA treatment (Fig. 3A). Further investigation of single cellular calcium transients revealed that the time to Ca2+ peak concentration upon Iso challenge (Fig. 3B) was reduced after NO2-OA treatment indicating a faster cytosolic Ca2+ influx. Furthermore, an increased peak height of the Ca2+ transient (Fig. 3C) was noted in both Iso treated groups as compared to vehicle treatment indicating elevated cytosolic Ca2+ levels. Accordingly, adrenergic stimulation led to a faster cytosolic Ca2+ clearance, as indicated by the reduced time to reach 50% or 90% of the maximum cellular Ca2+ concentration relative to baseline levels (Fig. 3D, E). Of note, no differences by additional NO2-OA treatment were detected.

Figure 3
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 animals) = 8/14/12). Graphs show Mean ± SEM. Brackets indicate Mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.

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NO2-OA treatment of isolated adult cardiomyocytes under baseline conditions furthermore did not influence the characteristics of Ca2+ transients and intracellular Ca2+ levels (Supplementary Figure S2).

Given the reduced appearance of spontaneous arrhythmic events upon NO2-OA treatment after Iso stimulation, we next investigated RyR2-dependent sarcoplasmic calcium leak as a potential pro-arrhythmic molecular mechanism.

Ca2+ leak analyses under isoproterenol treatment

Analyses of Ca2+ levels in isolated cardiomyocytes showed spontaneous Ca2+ peaks after RyR2 unblocking upon Iso stimulation (between the red and black cross in Fig. 4A), whereas almost no arrhythmic events could be observed upon additional NO2-OA treatment (Fig. 4A, B). Iso treatment resulted in an increased SR-dependent Ca2+ leak which was estimated as the difference between the Fura-2 ratio recorded at the end of the 0 Na+ /0 Ca2+ Tyrode perfusion (black cross) and at the end of the RyR2 inhibitor tetracaine treatment (red cross) (Fig. 4C). Final caffeine treatment revealed that the SR Ca2+ load was not significantly elevated after additional NO2-OA treatment (Fig. 4D).

Figure 4
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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Increased SR Ca2+ levels have been closely correlated with increased SR Ca2+ leak by RyR2-phosphorylation29,30. Of interest, caffeine treatment of short-time paced cardiomyocytes (1 Hz) demonstrated equal SR Ca2+ loads in vehicle- and NO2-OA treated cells. SR Ca2+ load was significantly elevated after Iso stimulation, an effect which was slightly, but not significantly, attenuated upon additional NO2-OA treatment (Supplemental Figure S3).

Ca2+ sensitivity

We further investigated the overall influence of NO2-OA on cytosolic Ca2+ levels and arrhythmia development upon increasing Ca2+ concentrations (representative Ca2+ transients are shown in Fig. 5A). Loading of the cells with Ca2+ concentrations ranging from 0.25 to 2 mM enhanced systolic calcium transients (Fig. 5B) as well as diastolic Ca2+ levels, the latter of which were not affected by additional NO2-OA treatment (Fig. 5C). Of importance, NO2-OA treatment completely inhibited Iso induced arrhythmic Ca2+ release (Fig. 5D).

Figure 5
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; 1 mM Ca2+: 1.56 ± 4.3 vs. 0.0 ± 0.0; 2 mM Ca2+: 2.44 ± 4.82 vs. 0.0 ± 0.0). (B–D) n(cells out of 3 animals) = 9/5 for each concentration. Graphs show Mean ± SEM. Brackets indicate Mean ± SD. * P < 0.05.

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Catecholamine-induced CaMKII activity and RyR2 phosphorylation

Given its importance in RyR2 activity and dysfunction, we next investigated the effect of NO2-OA treatment on CaMKII activity by performing a substrate binding assay using GST-HDAC4 419-670, which contains a CaMKII activity-dependent binding site31. CaMKII activity of Iso stimulated isolated cardiomyocytes was significantly reduced after NO2-OA treatment (Fig. 6A). In accordance, NO2-OA markedly diminished the Iso induced increase of pro-arrhythmic RyR2 phosphorylation on Ser281432 as revealed by immunoblotting (Fig. 6B). CaMKII-mediated Thr17-phosphorylation of PLN, which enhances sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) activity33, was increased upon Iso treatment and not further affected by NO2-OA treatment (Fig. 6C).

Figure 6
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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Discussion

Herein, we show that the electrophilic fatty acid nitroalkene NO2-OA prevents induction of VT after AMI in vivo by attenuating CaMKII dependent RyR2 leak. These results are of significant clinical relevance since sudden cardiac death related to AMI is most frequently attributed to the occurrence of sustained VT or ventricular fibrillation (VF)34. Although the absolute rates of VT and VF have declined during the era of percutaneous coronary intervention, patients suffering from VT or VF prior to revascularization still have a significantly impaired outcome and are at a higher risk for stent thrombosis. These data may underestimate the true incidence, since prehospital SCD before fibrinolysis or any other interventions were not included in this analysis35. In the present study, we modeled a clinical scenario of myocardial ischemia by LAD ligation and demonstrated that intraperitoneal injection of NO2-OA 20 min before he ischemic episode sustainably reduced ventricular arrhythmic events specifically in the acute phase after AMI.

The prospect of using NO2-OA as a therapeutic option for the treatment of acute VT after AMI is underlined by further beneficial effects of this fatty acid nitroalkenes in various cardiovascular diseases. As we and others have demonstrated, the application of NO2-OA preserves left ventricular ejection fraction and reduces infarct size in a mouse model of AMI19,36. Moreover, NO2-OA also protects from angiotensin II induced atrial fibrillation12.

Mechanistically, the occurrence of ventricular arrhythmic events during the acute phase of AMI is attributed to development of proarrhythmic substrates with subsequent formation of reentry circuits3. The concomitantly enhanced sympathetic tone leads to cellular Ca2+ overload, resulting in a disturbed Ca2+ homeostasis, accompanied by diastolic Ca2+ leak further increasing the vulnerability for VT37. On a cellular level, alterations in Ca2+ transients have been linked to the emergence of alternans and afterdepolarizations which may trigger VTs4, as well as to reduced sodium channel availability resulting in ventricular conduction slowing. This increases the wavelength of reentry circuits thereby increasing the vulnerability to VTs38,39,40.

To investigate the effects of NO2-OA on cellular Ca2+ transients and sarcoplasmic Ca2+ load, both important in arrhythmia development in AMI, we performed analyses of electrically stimulated isolated cardiomyocytes. We did not detect major changes in both Ca2+ transient characteristics and in SR Ca2+ load upon NO2-OA treatment.

We quantified PLN phosphorylation, a protein regulating cytosolic Ca2+ clearance into the SR by SERCA2a5, at its CaMKII specific phosphorylation site Thr17 under Iso stimulation. Iso increased PLN phosphorylation but no further change was detectable upon treatment with NO2-OA. This indicates that NO2-OA does not influence cytosolic Ca2+ clearance by CaMKII mediated phosphorylation of PLN on this specific site, although we could not fully exclude potential other target sites or phosphorylation mechanisms being modified by NO2-OA.

To further examine the underlying molecular effects of NO2-OA-mediated VT prevention in detail, we measured RyR2-dependent Ca2+ leak from the SR in isolated adult cardiomyocytes treated with Iso. We observed an increased number of arrhythmic events (e.g. EADs and DADs) upon Iso treatment that was markedly reduced by NO2-OA. Therefore, we hypothesized that NO2-OA beneficially influences CaMKII-dependent modulation of Ca2+-homeostasis. Indeed, CaMKII activity and subsequent RyR2 phosphorylation on the critical serine residue (Ser2814), which has been linked to CaMKII-mediated Ca2+ leak in cardiomyopathy and catecholamine treatment41, was significantly attenuated by NO2-OA in Iso-stressed cardiomyocytes (Fig. 6B). These results are in accordance with recent data demonstrating that increased RyR2 phosphorylation and thereby altered Ca2+ sensitivity may lead to local Ca2+ waves, subsequently depolarizing the membrane potential and forming DADs. These local events furthermore trigger Ca2+ waves in adjacent cells and create propagating arrhythmic events42.

Speculating on underlying biochemical mechanisms, it is noted that CaMKII activity and subsequent RyR2 phosphorylation is regulated by S-nitrosylation of specific cysteines (e.g. Cys273/ Cys290)43. Previous work shows that NO2-OA could act as a NO-donor for S-nitrosylation via a modified Nef reaction. This minor and controversial reaction, as opposed to the signaling responses induced by the post-translational alkylation of protein thiols, occurs in very low yields and only yields NO in aqueous buffered solutions when no protein is present44. Thus, it is anticipated that under pathological conditions, NO2-OA acts via a reversible nitroalkylation of functionally-significant cysteines11,19. The further investigation of critical protein targets in this model of cardioprotection will be the subject of further studies.

As this study was performed in a small animal model, a number of limitations may apply when trying to translate the present findings to human pathology. The mechanisms underlying VT in the setting of AMI are the result of multiple alterations, e.g. loss of viable myocardium with loss of cell-to-cell contact, changes in cell membrane potential, ion composition, and other factors predisposing for sudden cardiac death which are only partly mirrored by our experimental setting3.

Excessive catecholamine stimulation may be just one of multiple pathways leading to VT development during AMI modulated by NO2-OA. However, if other mechanisms, e.g., those responsive to acidosis and cell hypoxia, are altered by NO2-OA remains to be investigated.

In summary, we show for the first time that, by inhibiting CaMKII, NO2-OA modulates a critical pathway of electrical remodeling following AMI and thus has potential as a pre-emptive anti-arrhythmic strategy for patients at risk for developing AMI.

Conclusions and perspectives

Our current data reveal that NO2-OA rapidly stabilizes myocellular Ca2+ homeostasis by inhibition of CaMKII activity in the setting of AMI (Fig. 7). Crucially, herein we show for the first time that, by modulation of this pathway, NO2-OA markedly suppresses the susceptibility to ventricular arrhythmias.

Figure 7
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/).

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There are limited treatment options for prevention of acute arrhythmic tachycardias within the first 72 h after AMI, especially considering the amount of time to reach therapeutic levels for available drugs45. In this regard, the long-term dietary supplementation of NO2-OA or its precursors, might be a therapeutic option to protect AMI patients against subsequent VT / SCD.

Materials and methods

An extended description of the methods can be found within the Supplemental Material.

Animal studies

Male, 8- to 12-week old FVB/N mice (Charles River) were used for all animal studies. All animal studies were approved by the local Animal Care and Use Committees (Ministry for Environment, Agriculture, Conservation and Consumer Protection of the State of North Rhine-Westphalia: State Agency for Nature, Environment and Consumer Protection (LANUV), NRW, Germany) and follow ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines.

Left anterior descending artery (LAD) ligation

LAD ligation to induce myocardial ischemia was performed as described by Mollenhauer et al.26.

Right ventricular stimulation

A detailed stimulation protocol can be found within the Supplemental Material. In short, right ventricular stimulation was performed 20 min after induction of myocardial ischemia, while the mouse was kept under anesthesia, according to a standardized protocol that initially included a programmed ventricular stimulation with a fixed S1S1 interval to whose last S1-impulse a short-coupled additional stimulus was applied. After 10 s of recovery, automated burst stimulation was performed according to the protocol. VT were defined as a series of repetitive ventricular ectopic beats lasting for > 200 ms26.

In vivo electrophysiological mapping

In brief, directly after electrophysiological investigation the heart was exposed by thoracotomy. A 32-electrode microelectrode array (MEA, Multichannel Systems, Reutlingen, Germany) was positioned on the epicardial surface of the left ventricle apico-septally of the peri-ischemic region as described previously26. Field potentials were recorded using a 128-channel, computer-assisted recording system (Multichannel Systems). Data was evaluated according to Lammers et al.46. In short, inter-electrode conduction latencies (reciprocal conduction velocity) were calculated for all neighboring electrodes. Of each neighboring quadruplet of electrodes, the largest latency was taken and plotted as a phase map. From this phase map the mean latency of conduction was calculated. The variation coefficient of the phase map was calculated (Percentile (P): P5–95/P50) to receive the index of inhomogeneity as velocity independent factor of conduction inhomogeneity. Phase maps were calculated using custom-programmed software (Excel).

Administration of nitro-oleic acid

Nitro-oleic acid (9- and 10-nitro-octadec-9-cis-enoic acid; NO2-OA), administered at 20 nmol/g body weight, was solvated in polyethylene-glycol/ethanol (85:15, vol/vol), or 100 ml of vehicle (polyethylene-glycol/ ethanol, 85:15). These preparations were injected i.p. 20 min prior to LAD ligation. NO2-OA was synthesized as previously described47.

Isolation of adult ventricular cardiomyocytes

Ventricular myocytes were obtained from 8 to 12 week old male wild-type mice (FVB/N) as described previously28.

Assessment of Ca2+ transients

All experiments were performed at 37 °C within 6 h after cell isolation, as described previously using ratiometric Ca2+ imaging with Fura-2 dye (IonOptix)28.

For Ca2+ transient analyses the cells were incubated with either 0.1% EtOH as control, 10 µM Iso in 0.1% EtOH or 10 µM Iso and 5 µM NO2-OA in 0.1% EtOH for 10 min. Calcium transients were recorded under electrically stimulated biphasic field pulses (20 V, 4 ms) at a frequency of 1 Hz for 1 min.

SR Ca2+ leak and load were measured according to a modified protocol28,48. Fura-2 loaded ventricular cardiomyocytes were incubated with 10 µM Iso for 7 min and stimulated for 3 min at 1 Hz, 20 V, 4 ms until cellular Ca2+ transients reached a steady state followed by a burst stimulation with 4 Hz for 30 s. Directly after the last pulse the pacing was stopped and the normal Tyrode solution was substituted by a 0 Na+ /0 Ca2+ Tyrode supplemented with 10 mmol/l EGTA and 1 mmol/l of the RyR2 inhibitor tetracaine in which Na+ was replaced by Li+. This condition allowed measuring intracellular Ca2+ levels in a closed system without trans-sarcolemmal Ca2+ fluxes and prevents SR Ca2+ leak into the cytoplasm. After 40 s recording time the solution was switched back to 0 Na+ /0 Ca2+ Tyrode without tetracaine for another 40 s to unblock the ryanodine receptors and allow potential Ca2+ leak. SR Ca2+ leak was estimated as the difference between the Fura-2 ratio recorded at the end of the 0 Na+ /0 Ca2+ Tyrode perfusion with and without tetracaine. At the end of the protocol, 10 mM caffeine was applied to evaluate the total SR Ca2+ content.

Ca2+ sensitivity: Fura-2 loaded ventricular cardiomyocytes were incubated with Tyrode solution containing 0.25 mM, 0.5 mM, 1 mM or 2 mM of Ca2+ together with either 10 µM Iso in 0.1% EtOH or 10 µM Iso and 5 µM NO2-OA in 0.1% EtOH. For each calcium concentration cells were paced at 1 Hz for 30 s following a 20 s non-pacing period in which pro-arrhythmic calcium release events was counted. Ca2+ transient height was determined within the last paced Ca2+ transient before the non-paced period.

Ca2+ transient characteristics were calculated by the IonWizard (IonOptix) software.

Immunoblotting

Protein extraction and immunoblotting were performed as previously described26. Briefly, membranes were incubated with the following antibodies overnight at 4 °C: anti-GAPDH (1:1000; Santa Cruz), anti-P-PLN-Thr17, anti-PLN, anti-P-RyR2-Ser2814 (1:5000; Badrilla), anti-RyR2 (1:2000; Sigma-Aldrich). Full blots are shown in the Supplemental Material section.

CaMKII activity assay

CaMKII activity was assessed by detecting the amount of endogenous CaMKII that binds to GST-HDAC4 amino acids 419 to 670 (containing the CaMKII activity–dependent binding domain) as described previously31. Bound active CaMKII, CaMKII input, and GST-HDAC4 419-670 input were resolved by SDS-PAGE and detected by immunoblot using HDAC4 (1:1000; Santa Cruz) and CaMKII antibodies (1:1000; BD Bioscience). Protein loading was normalized to GAPDH (1:10 000; Sigma-Aldrich).

Statistical analysis

Results are expressed as mean ± standard error of the mean. Gaussian normality was tested via Shapiro–Wilk normality test. Unpaired Student’s t-test or one-way ANOVA were used for gaussian distributed data, whereas non-gaussian distributed data were analyzed using Kruskal Wallis test, each followed by appropriate post-hoc test. Results of Fig. 5B, C are normalized against their respective control within the same animal. An alpha level of P < 0.05 was considered statistically significant. All statistical calculations were carried out using GraphPad Prism 8.2.1 (GraphPad). *P < 0.05, **P < 0.01, ***P < 0.001.