A carvedilol analogue, VK‐II‐86, prevents hypokalaemia‐induced ventricular arrhythmia through novel multi‐channel effects

QT prolongation and intracellular Ca2+ loading with diastolic Ca2+ release via ryanodine receptors (RyR2) are the predominant mechanisms underlying hypokalaemia‐induced ventricular arrhythmia. We investigated the antiarrhythmic actions of two RyR2 inhibitors: dantrolene and VK‐II‐86, a carvedilol analogue lacking antagonist activity at β‐adrenoceptors, in hypokalaemia.

Hypokalaemia is an independent risk factor for mortality and can cause life threatening ventricular arrhythmia. In a study of hospitalized patients with a serum potassium concentration [K + ] below 3 mM, mortality was 20.4%, 10 times higher than in patients without hypokalaemia (Paltiel et al., 2001). A retrospective analysis of patients presenting with sustained ventricular tachycardia (VT) or ventricular fibrillation (VF) showed that hypokalaemia was present in 35.7% of these patients, with 13.6% presenting with a serum [K + ] < 3.0 mM.
Gastrointestinal illness and recent changes to diuretic therapy were significantly correlated with the risk of VT/VF, probably because these are frequent causes of hypokalaemia (Laslett et al., 2020).
Close monitoring of serum potassium can be logistically difficult and hypokalaemia is frequently mismanaged (Paltiel et al., 2001).
Moreover, there is evidence to suggest that potassium supplements alone may be insufficient to reverse the increased mortality associated with hypokalaemia (Siscovick et al., 1994). In a retrospective observational study of heart failure patients, potassium supplements did not significantly alter mortality, but were associated with a significant increase in cardiovascular hospitalizations and heart failure progression (Ekundayo et al., 2010). There is therefore an unmet need for a safe treatment for the prevention of hypokalaemia-induced arrhythmia.
Hypokalaemia-induced arrhythmia has been attributed to reduction in key repolarizing potassium channel currents in the heart such as I K1 , I to (Firek & Giles, 1995;Killeen et al., 2007) and I Kr (Guo et al., 2009;Numaguchi et al., 2000;Sanguinetti & Jurkiewicz, 1992;Scamps & Carmeliet, 1989). This results in slowing of action potential (AP) repolarization and the formation of early afterdepolarizations (EADs). These EADs are reflected on the surface ECG as closely coupled triggered beats in the context of a prolonged QTc interval.
However, the role of intracellular Ca 2+ loading in the pathogenesis of hypokalaemia-induced arrhythmia (Tazmini et al., 2020) has been highlighted in recent studies. Hypokalaemia has been shown to inhibit Na + /K + ATPase (Aronsen et al., 2015). This causes the accumulation of intracellular Na + ions, which inhibits the Na + /Ca 2+ exchanger (NCX) from extruding Ca 2+ ions from cardiomyocytes. and rat hearts, supplemented by in silico data, that the most important arrhythmogenic mechanism in hypokalaemia is activation of CaMKII, rather than reduced repolarization reserve (Pezhouman et al., 2015).
CaMKII not only phosphorylates I Ca , phospholamban and RyR2, facilitating SR loading and diastolic Ca 2+ release, but also increases late Na + current (I Na_L ), thus further contributing to AP prolongation and intracellular Na + accumulation. CaMKII thereby initiates a positive feedback loop of progressive intracellular Na + and Ca 2+ loading.
Pezhouman et al demonstrated up-regulation of CaMKII in hypokalaemia and showed that inhibition of CaMKII, I Na_L , or I Ca prevented triggered activity in hypokalaemic mouse and rabbit hearts, even in the presence of dofetilide, an I Kr blocker used to reduce repolarization reserve (Pezhouman et al., 2015).
What is already known?
• Hypokalaemia causes ventricular arrhythmias through QT prolongation and diastolic Ca 2+ release from cardiac ryanodine receptors.
• Dantrolene and VK-II-86, a carvedilol analogue, have previously been shown to inhibit ryanodine receptors.
What does this study add?
• Ryanodine receptor inhibition with dantrolene and VK-II-86 prevents ventricular arrhythmia in the setting of hypokalaemia.
• VK-II-86 has greater efficacy through targeting ion channels important in intracellular Ca 2+ loading and repolarization.
What is the clinical significance?
• VK-II-86 is a potential antiarrhythmic in hypokalaemia, other causes of Ca 2+ overload and delayed repolarization.
• VK-II-86 has additional appeal as it is a carvedilol analogue without antagonist activity at β-adrenoceptors.
CaMKII is ubiquitously expressed in humans, making its inhibition problematic. There is no selective I Na-L inhibitor approved for anti-arrhythmic use in humans and I Ca blockers are often contraindicated due to negative ionotropy (Girouard et al., 2017). The aim of this study was to assess another drug target to ameliorate the Ca 2+ handling problems contributing to arrhythmogenesis in the setting of hypokalaemia. The two candidates we have evaluated are inhibitors of RyR2, dantrolene and the carvedilol analogue VK-II-86. Dantrolene's ability to prevent Ca 2+ -mediated arrhythmias through RyR2 inhibition has been demonstrated in rabbits with heart failure (Maxwell et al., 2012) and in mice carrying catecholaminergic polymorphic ventricular tachycardia (CPVT) mutations (Suetomi et al., 2011;Uchinoumi et al., 2010). VK-II-86 has also been shown to be anti-arrhythmic in CPVT mice, (Zhou et al., 2011) and rats overdosed with digoxin (Gonano et al., 2018 there is evidence to suggest that due to a short AP duration, EADs in mice may be caused by reactivation of non-equilibrium I Na , (Edwards et al., 2014) rather than Ca 2+ overload and release through RyR2, as in larger species (Edwards et al., 2014;Tazmini et al., 2020

| Synthesis of VK-II-86, a carvedilol analogue
VK-II-86 was synthesized from commercially available precursors using the methods described in previous studies (Wu et al., 2009;Xu et al., 2018). The exact methodology we used is described here with reference to Scheme 1: m-CPBA (5.17 g, 0.03 mol) was added portion-wise to 4-bromobut-1-ene (1) (2 ml, 0.02 mol) in CH 2 Cl 2 (20 ml) at 0 C. The reaction was stirred at room temperature for 24 h. The reaction mixture was neutralized with 0.1 N sodium hydroxide, extracted with CH 2 Cl 2 and dried over Na 2 SO 4 . The solvents were removed to give 2-(2-bromoethyl)oxirane (2) as a colourless oil in 90% yield, which was used without further purification in the next step (Wu et al., 2009 (Killeen et al., 2007). Speed of induction is important to eliminate any confounding effects of ischaemia-induced arrhythmia.
The [K + ] used in external patch clamp solutions was modified from 4 mM in control experiments to 3 mM to model hypokalaemia.
We avoided using 2 mM [K + ] in isolated cardiomyocytes because this level of hypokalaemia resulted in a high level of contracture and cell death.

| Single cell isolation of murine and canine cardiomyocytes
Mouse hearts were attached to a Langendorff perfusion system as described above. Canines were anaesthetised with isoflurane; their hearts were excised following retrograde aortic flush of heparin (1000 U L À1 ) and stored briefly in ice-cold cardioplegic solution. A right ventricular (RV) wedge was excised from the canine heart and an acute marginal branch of the right coronary artery cannulated. The RV wedge was then attached to the Langendorff perfusion system. The murine heart or canine ventricular wedge were perfused at 37 C with isolation solution for 5 min at 5 ml min À1 for the murine heart and 15 min at 10 ml min À1 for the canine wedge. Collagenase (Type 2; 0.6 mg ml À1 ; Worthington, Lakewood, NJ, USA,) and protease (0.075 mg ml À1 , type XIV, Sigma) were added to the perfusate and the tissue digested for 3-4 min in the murine and 25-35 min in the canine preparations, respectively. The heart was then perfused with a taurine buffer solution for 10 min in the mouse preparation and 20 min for the canine preparation. The murine heart tissue was dismounted from the cannula, the atria discarded, and the ventricles

| Study design and statistical analysis
Randomization by means of a random number generator producing an odd or even number was used to determine which mice were exposed to the various groups used for whole-heart Langendorff experiments.
Enzymically isolated cells were exposed to all independent variables for each experiment, therefore randomization was not necessary for single cell experiments. As the experiments were performed and analysed as part of the learning process for the first author's PhD, the study was not blinded.
There was a relative shortage of canine hearts available for this study because cardiomyocytes were isolated from spare ventricular wedges from hearts primarily being used for another NIH funded study, and the institution was experiencing a general shortage in supply. Canine patch-clamp experiments therefore analysed data from cardiac myocytes isolated from less than five dogs, with cardiac myocytes treated as independent units (n). Although not statistically tested, these data from canine hearts are still thought to be useful due to the distribution of the data and the similarity of results to the comparative murine cardiomyocytes, which were isolated from at least five hearts. The one exception to this were the experiments measuring I Na_L , since these experiments are technically very demanding, requiring seal formation with virtually no leak for accurate I Na_L measurement, and this was only achieved in three murine and two canine hearts, respectively. The technical difficulties of patch clamping enzymically isolated cardiomyocytes, especially those exposed to low potassium meant that numbers of were fitted to the I to traces from each cell and tau2 compared between groups using the Mann-Whitney U rank sum test ( Figure S1). A P < 0.05 value was considered statistically significant.
Where ANOVA tests were used, post hoc group comparisons were performed only when tests for normality and equal variance were passed. Data are expressed as mean ± SEM. Statistical analysis was performed using Sigmaplot 13.0 (Systat Software, RRID: SCR_003210).

| Materials
VK-II-86 was prepared as described above. Dantrolene (Ryanodex ® ) was supplied by Eagle Pharmaceuticals (Woodcliff Lake, NJ, USA) and ranolazine was supplied by Sigma.

| Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, and are permanently archived in the Concise Guide to PHARMACOLOGY 2021/22 (Alexander, Christopoulos, et al., 2021;Alexander, Fabbro, et al., 2021;.

| In murine ventricular cardiomyocytes, VK-II-86 did not significantly alter I to during hypokalaemic conditions
The canine AP data in Figure 4 showing that APD 30 is not signifi-   Figure S1). VK-II-86 did significantly accelerate the rate of inactivation of I to in both normal and low [K + ] ( Figure S1), reducing total I to charge and a faster recovery from activation, but the translation of these results to human heart rates is tenuous.
3.9 | In murine cardiomyocytes, VK-II-86 prevents the increase in oxidative stress that occurs during exposure to low [K + ] Data in Figure 9 show pooled mean fluorescent densities of murine ventricular cardiomyocytes isolated from 5 mouse hearts. Cells from each heart were loaded with a fluorogenic oxidative stress

| DISCUSSION
Our results demonstrate for the first time that RyR2 inhibition is an effective method of reducing the frequency and severity of ventricular arrhythmias in the context of hypokalaemia. This could have significant benefits to morbidity and mortality for patients susceptible to hypokalaemia, particularly those with heart failure. However, a multi-channel approach is more effective than selective RyR2 inhibition.
Pretreatment with dantrolene and VK-II-86, both previously shown to inhibit RyR2 (Gonano et al., 2018;Maxwell et al., 2012;Suetomi et al., 2011;Uchinoumi et al., 2010;Zhou et al., 2011), significantly reduced the incidence of ventricular arrhythmias in our whole-heart murine Langendorff model of hypokalaemia. VK-II-86 was the more effective antiarrhythmic compound, as it was able to prevent all triggered activity, sustained and non-sustained ventricular arrhythmias.  syndrome, a rare condition with a pro-arrhythmic phenotype. A reduction of I K1 has also been found in pro-arrhythmic acquired disease such as advanced heart failure (Beuckelmann et al., 1993). An in silico model of a guinea pig ventricular myocyte demonstrated that reduced I K1 results in a depolarized RMP, favouring Ca 2+ leak into the cell. The electrogenic sodium-calcium exchanger (NCX), in its efforts to export the excess Ca 2+ could depolarize the cell, contributing to the development of EADs and triggered arrhythmia (Silva & Rudy, 2003). We found a significant depolarization of the RMP in the ventricular AP for both murine and canine ventricular tissues and cells. This contrasts with previous studies reporting that hypokalaemia results in hyperpolarization of RMP (Aronsen et al., 2015;Bouchard et al., 2004;Tazmini et al., 2020). These studies all investigated the effects of acute hypokalaemia on RMP, but there are no published studies of the effects of chronic hypokalaemia on RMP. In our study, the whole-heart and isolated ventricular myocytes were exposed to  (Xia et al., 2005) and short QT syndrome type 3 (Priori et al., 2005) Up-regulated I K1 has been found in acquired AF (Dobrev et al., 2002) and has been shown to stabilize rotors in both AF and ventricular fibrillation Samie et al., 2001). Therefore, it is advantageous that, in the present study, VK-II-86 did not significantly up-regulate the inward or outward components of I K1 , in normal [K + ].
Canine and murine cardiomyocytes both showed an increase in I Na-L when incubated in low [K + ]. This finding is in agreement with those of Pezhouman et al. (2015), who demonstrated that low [K + ], through inhibition of Na + /K + ATPase and subsequent intracellular Ca 2+ loading, induced CaMKII activation and led to a positive feedback loop by up-regulating I Na_L and I Ca . The finding of increased I Na-L in hypokalaemia also has implications as to why hypokalaemia is proarrhythmic in patients with the Long QT syndrome. Pezhouman et al found that pretreatment with the I Na-L inhibitor GS-967 and the I Ca inhibitor nifedipine prevented the development of EADs and ventricular arrhythmia. It is therefore encouraging that VK-II-86 prevented the increase in I Ca and I Na_L in ventricular myocytes but did not significantly reduce these currents in cells held in normal [K + ], which could be harmful due to negative inotropy and QT alteration. Further investigation into the interaction of VK-II-86 with Na + channels is warranted as a recent publication found that hypokalaemia-induced AF in atrial cells lacking T-tubules is predominantly due to reactivation of non-equilibrium I Na , rather than intracellular Ca 2+ loading (Tazmini et al., 2020).
Unlike its parent carvedilol, VK-II-86 increases rather than inhibits I Kr . (Kawakami et al., 2006) Although I Kr peak tail current was significantly increased by VK-II-86 in both hypokalaemic and normokalaemic conditions, the drug did not shorten the ventricular AP of canine cardiomyocytes in 4 mM [K + ]. The small but statistically significant VK-II-86-mediated increase in I Kr in 4 mM [K + ], but not the other ion channel currents studied, indicates that there may be a direct interaction between VK-II-86 and K v 11.1, but its influence on I K1 , I Na_L and I Ca channels only in the context of hypokalaemia are more likely to be via an indirect mechanism of action. This study demonstrated that a significant increase in oxidative stress occurs in murine cardiomyocytes exposed to low [K + ] and 1 μM VK-II-86 prevented this increase. As I K1 , I Na_L and I Ca channels are all altered by oxidative stress (Trum et al., 2020;Viatchenko-Karpinski et al., 2014;Wagner et al., 2011;Xie et al., 2009), it is plausible that the ability of VK-II-86 to reduce oxidative stress is a key mechanism behind its antiarrhythmic efficacy in hypokalaemia. I Kr , I to , and RyR2 are also altered by oxidative stress (Bérubé et al., 2001;Hamilton et al., 2020;Terentyev et al., 2008;Zhao et al., 2012). VK-II-86 and its parent carvedilol contain a tricyclic carbazole moiety, which can act as an antioxidant through free radical scavenging and its ability to sequester iron in ferric ion-induced oxidation reactions. (Dandona et al., 2007).
VK-II-86 is an analogue of the β-adrenoceptor antagonist carvedilol. Carvedilol inhibits diastolic Ca 2+ leak from RyR2, although the concentration required for this action (0.3-1 μM) is much higher than the concentration required for antagonism of β-adrenoceptors ($1 nM). (Zhou et al., 2011) Patients taking high doses of carvedilol to inhibit RyR2, are often unable to tolerate the β-adrenoceptor blockade of a 1-μM plasma concentration. The advantage of VK-II-86 is that when it was tested in vivo on transgenic mice carrying a CPVT variant, there was no sinus rate slowing (Zhou et al., 2011) and it, therefore, may be better tolerated by patients.
The limitations of this study are primarily that the anti-arrhythmic effect of dantrolene and VK-II-86 at the whole heart level was tested in a murine model, which may exhibit different arrhythmogenic mechanisms for EAD formation, from those in larger mammals, as suggested by Edwards et al., (2014) However, laboratory mice are one of the few species that have been fully genotyped, so these experiments have the advantage that there are unlikely to be any genetic The effects of VK-II-86 on I Kr (K v 11.1) was assessed in HEK-293 cells, a heterologous expression system. There is evidence to suggest that cellular context can have a significant effect on the biophysical properties and regulation of heterologously expressed channels (Petersen & Nerbonne, 1999). However, this system also has the advantage of analysing the effects of VK-II-86 on the human K v 11.1 isoform responsible for I Kr. .
In conclusion, the results presented here provide proof of concept for a novel anti-arrhythmic drug, VK-II-86, capable of preventing hypokalaemia-induced arrhythmogenesis because of its multi-ion channel effects, and its ability to prevent oxidative stress during hypokalaemia. VK-II-86 targets key ion channels involved in intracellular Ca 2+ loading and repolarization including those carrying I K1 , I Kr , I Ca , I Na_L and RyR2. VK-II-86 affected these channels only under conditions of hypokalaemia, preventing their dysfunction when exposed to

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request. Some data may not be made available because of privacy or ethical restrictions.