Approved drugs ezetimibe and disulfiram enhance mitochondrial Ca2+ uptake and suppress cardiac arrhythmogenesis

Treatment of cardiac arrhythmia remains challenging due to severe side effects of common anti‐arrhythmic drugs. We previously demonstrated that mitochondrial Ca2+ uptake in cardiomyocytes represents a promising new candidate structure for safer drug therapy. However, druggable agonists of mitochondrial Ca2+ uptake suitable for preclinical and clinical studies are still missing.


| INTRODUCTION
While mortality and morbidity rates related to cardiovascular diseases are generally declining, arrhythmia-related incidents are still on the rise (Benjamin et al., 2018). This is in part related to limited effectiveness and major side effects of common anti-arrhythmic drugs. Antiarrhythmic drugs of Vaughan Williams class I, III and IV act by targeting plasma membrane ion channels and suppressing propagation of ectopic signals. However, due to their effects on the cardiac action potential and thus cardiac conduction speed, they are prone to proarrhythmic side effects. Therefore, novel therapeutic strategies that suppress the initiation of arrhythmogenic signals inside cardiomyocytes are currently in focus of the search for novel and safer anti-arrhythmic therapies.
We have recently demonstrated a critical role of mitochondrial Ca 2+ uptake for the regulation of cardiac rhythmicity (Shimizu et al., 2015). The cardiac contraction cycle is initiated by influx of extracellular Ca 2+ into cardiomyocytes and subsequent Ca 2+ release predominantly from the sarcoplasmic reticulum (SR) to initiate muscle contraction (Bers, 2002(Bers, , 2008. Mitochondria are located in close proximity to the SR (Rog-Zielinska et al., 2016) and can rapidly take up Ca 2+ on a beat-to-beat interval (Robert et al., 2001) through a selective mitochondrial Ca 2+ uptake pathway consisting of pore proteins in both mitochondrial membranes and several positive and negative regulators (Baughman et al., 2011;De Stefani et al., 2011;Kirichok et al., 2004;Waldeck-Weiermair et al., 2013). In cardiomyocytes, the mitochondrial Ca 2+ uptake pathway is directly tethered to the Ca 2+ release sites of the SR. Within the Ca 2+ microdomain around the Ca 2+ release sites of the SR, the local cytosolic Ca 2+ concentration reaches values high enough to activate the mitochondrial Ca 2+ uptake pathway allowing for rapid and direct shuttling of Ca 2+ from the SR into mitochondria (De la Fuente et al., 2016;De la Fuente & Sheu, 2019). Under pathological conditions, erratic Ca 2+ release of single Ca 2+ release sites in form of Ca 2+ sparks followed by Ca 2+ waves during diastole leads to spontaneous contractions and arrhythmia (Allen et al., 1984). Pharmacological activation of mitochondrial-Ca 2+ uptake locally buffers these events and thereby suppresses arrhythmogenic signals in cardiomyocytes, while systolic events remain unaltered (Shimizu et al., 2015). Indeed, treatment with agonists of mitochondrial-Ca 2+ uptake suppressed episodes of arrhythmia in a murine model for catecholaminergic polymorphic ventricular tachycardia (CPVT) (Schweitzer et al., 2017). Interestingly, despite enhancing mitochondrial Ca 2+ uptake over several days, no signs of severe adverse effects, for example, apoptosis, were observed neither in cellular nor in animal models (Schweitzer et al., 2017;Shimizu et al., 2015). Modulation of mitochondrial-Ca 2+ uptake could thus serve as novel pharmacological strategy for the treatment of human cardiac arrhythmias. This can be accomplished by agonists of either the voltagedependent anion channel 2 (VDAC2) in the outer mitochondrial membrane or the mitochondrial Ca 2+ uniporter in the inner mitochondrial membrane. Two such compounds, the VDAC2 gating modulator efsevin (Shimizu et al., 2015;Wilting et al., 2020) and the mitochondrial Ca 2+ uniporter agonist kaempferol (Montero et al., 2004), were found to be effective in reducing arrhythmia, thus providing a proof of concept that mitochondrial Ca 2+ uptake enhancers (MiCUps) represent, in this respect, a pharmacologically highly relevant class of molecules. However, both drugs are so far only used experimentally and are currently too poorly characterized to have clinical potential.

What is already known
• Cardiac arrhythmia results from imbalances in cellular Ca 2+ homeostasis.

What does this study add
• The two clinically approved drugs, ezetimibe and disulfiram activate mitochondrial Ca 2+ uptake.
• Both efficiently suppress arrhythmogenesis in translational models in a nanomolar range.

What is the clinical significance
• The results strengthen mitochondrial Ca 2+ uptake as a pharmacological target.
• The identification of clinically approved drugs allows for repurposing studies.
Furthermore, both are effective at concentrations around 10-20 μM at the in vitro target site, making them poor candidates for clinical use.
Here, we applied a three-step protocol to identify novel MiCUps. First, we screened a chemical library consisting of 727 compounds with a history of use in human clinical trials for novel mitochondrial Ca 2+ uptake enhancers using an established screening platform for mitochondrial Ca 2+ uptake modifiers . We identified three hits, the FDA-and EMA-approved drugs, ezetimibe and disulfiram, and the natural compound honokiol, which significantly increased mitochondrial-Ca 2+ uptake in permeabilized HeLa cells. To transfer these results to a cardiac system, we measured SR-mitochondria Ca 2+ transfer in a standardized cultured cardiomyocyte assay (Schweitzer et al., 2017;Wilting et al., 2020) and found that two of them, ezetimibe and disulfiram, enhanced SR-mitochondria Ca 2+ transfer at significantly lower concentrations than efsevin and kaempferol. Finally, we performed efficacy testing in translational arrhythmia models (Schweitzer et al., 2017;Shimizu et al., 2015)  Microplate Counter, PerkinElmer). Drug screens were analysed as described previously . Briefly, after smoothing the dynamics of mitochondrial-Ca 2+ -dependent luminescence obtained for each compound using a cubic spline function as described previously , both peak (maximal amplitude of the luminescence signal) and uptake rate (left slope) were automatically determined. Based on these parameters a score (S drug ) was assigned to each compound as shown in Figure 1.

| Human iPSC cardiomyocytes
Human iPSCs from a 60-year-old male donor presenting with a severe form of CPVT were generated as described previously (Moretti et al., 2010). Spontaneously beating areas were explanted after 2-

| Confocal Ca 2+ imaging
Ca 2+ waves were analysed by confocal microscopy as described previously (Schweitzer et al., 2017). In brief, cardiomyocytes were loaded with 1-μM Fluo-4, AM (Thermo Fisher), and Ca 2+ transients were elicited by electric field stimulation using a S48 square pulse Stimulator (Grass Technologies, Warwick, RI, USA) on an inverted confocal microscope (Leica TCS SP5 or Zeiss LSM 880). Line scan series were generated along the long axis of a myocyte. After reaching steady state pulsing was stopped and cells were analysed for the occurrence of spontaneous diastolic Ca 2+ waves. Every experiment contained a group with isoprenaline and only preparations that were sensitive to stimulation with isoprenaline were used for drug testing. Depending to the quality of the preparation and the yield of cells, the remaining cells were randomly attributed to experimental groups. All groups were measured in random order to avoid the influence of a decrease in the quality of cells over time and were stopped when cell quality decreased as indicated by a hyperexcitability or insensitivity to external stimuli.

| Materials
Test substances ezetimibe and disulfiram were purchased from Molekula (Germany). All other laboratory chemicals were purchased from Sigma-Aldrich (Germany) or Carl Roth GmbH (Germany) unless noted otherwise in the methods section. Laboratory devices are specified in the respective methods sections.

| Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in the IUPHAR/BPS Guide to PHARMACOL-OGY http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018).

| Screening for novel mitochondrial Ca 2+ uptake enhancers
Since pharmacological enhancement of mitochondrial-Ca 2+ uptake can prevent arrhythmogenesis in cardiomyocytes (Schweitzer et al., 2017;Shimizu et al., 2015), but agonists of mitochondrial-Ca 2+ uptake are still scarce and experimental, we set out screen for novel, potent MiCUps for preclinical and clinical testing. As a first step, we took advantage of a previously established and validated mitochondrial-Ca 2+ uptake assay. In a previous study, we have devel-  Figure 1c). This revealed dose-dependent enhancement for honokiol, ezetimibe and disulfiram starting at 3-6 μM but not for cefatrizine, which was thus considered a false positive hit from the primary screen. We next confirmed that hits from our screen are indeed enhancers of mitochondrial Ca 2+ uptake rather than blockers of mitochondrial Ca 2+ extrusion, which would likewise lead to enhanced mitochondrial Ca 2+ accumulation. Therefore, we measured mitochondrial Na + /Ca 2+ exchanger-mediated mitochondrial Ca 2+ extrusion in permeabilized HeLa cells: unlike the established NCLX blocker CGP-37157, neither of our three candidate drugs affected mitochondrial Na + /Ca 2+ exchanger activity ( Figure 2).
3.2 | Ezetimibe and disulfiram enhance SRmitochondria Ca 2+ transfer in cardiomyocytes Significant differences were described for mitochondrial Ca 2+ uptake between non-excitable cells like HeLa cells and cardiomyocytes (Chen et al., 2011;Mammucari et al., 2018). In particular, in cardiomyocytes a specialized mechanism of SR-mitochondria Ca 2+  (Schweitzer et al., 2017;Wilting et al., 2020) to generate doseresponse relationships and was previously successfully used to investigate the role of VDAC2 in promoting SR-mitochondria Ca 2+ transfer (Min et al., 2012;Wilting et al., 2020). Rapid mitochondrial uptake of Ca 2+ released from the SR by a caffeine pulse was significantly enhanced by ezetimibe and disulfiram, while no significant effect of honokiol could be observed (Figure 3a,b). Strikingly, analysis of dose-response curves revealed that the two active substances, ezetimibe and disulfiram, enhanced mitochondrial-Ca 2+ uptake at markedly lower concentrations compared to the established MiCUps, the mitochondrial Ca 2+ uniporter activator kaempferol (Montero et al., 2004) and the VDAC2 modifier efsevin (Wilting et al., 2020).
To exclude a putative false-positive result due to a potential effect of ezetimibe and/or disulfiram on SR Ca 2+ release, we next measured F I G U R E 3 Direct transfer of Ca 2+ from the sarcoplasmic reticulum into mitochondria in cardiomyocytes. (a) Representative recordings of mitochondrial (mt)-Ca 2+ uptake (black line) in permeabilized HL-1 cardiomyocytes. Superfusion with 10-mM caffeine induced uptake of Ca 2+ released from the SR into mitochondria, which was enhanced by 1-μM ezetimibe and 1-μM disulfiram. Ruthenium red as a blocker of RyR, VDAC and mitochondrial Ca 2+ uniporter was used to block SR-mitochondria Ca 2+ transfer as a negative control. cytosolic Ca 2+ levels in intact ezetimibe-and disulfiram-treated HL-1 cardiomyocytes using fura-2 and analysed basal Ca 2+ levels and Ca 2+ release after superfusion with 10-mM caffeine (Figure 3c). We observed no significant change for either baseline fura-2 fluorescence ratio (R 340nm/380nm ) or release of Ca 2+ from the SR (ΔR) ( Figure 3c). In conclusion, the above data indicates that both, ezetimibe and disulfiram, specifically enhance mitochondrial-Ca 2+ uptake in HL-1 cardiomyocytes without affecting baseline cytosolic Ca 2+ levels or SR Ca 2+ release and have significantly lower EC 50 values then the established MiCUps kaempferol and efsevin.

| Ezetimibe and disulfiram suppress arrhythmia in a zebrafish model of Ca 2+ -overload induced arrhythmia
Consequently, we set out to evaluate the potency of the newly identified MiCUps identified in our screening approach to suppress arrhythmia in translational models. Prior to application of MiCUps to these models, we confirmed that both substances, ezetimibe and disulfiram, effectively enhance mitochondrial Ca 2+ uptake also in intact HeLa cells at concentrations comparable to those effective in permeabilized cells ( Figure S1). While we found enhanced mitochondrial-Ca 2+ (tre tc318 ), lacking a cardiac isoform of the Na + /Ca 2+ exchanger, display Ca 2+ overload-induced cardiac arrhythmia (Langenbacher et al., 2005;Shimizu et al., 2015). In contrast to wild-type embryos, which consistently show rhythmic cardiac contractions at day 1 of development.
Homozygous tre embryos display a hypercontracted heart that shows only chaotic contractions within the myocardium (Langenbacher et al., 2005). This phenotype can be rescued by treatment with efsevin (Shimizu et al., 2015). We therefore tested the newly identified MiCUps ezetimibe and disulfiram for their potential to restore rhythmic cardiac contractions in this model (Figure 4a). Addition of 0.05-μM ezetimibe restored rhythmic cardiac contractions in roughly one quarter of tre embryos and approximately 60% of tre embryos at concentrations above 1 μM (Figure 4b), while only a tenth of control embryos treated with vehicle showed synchronized contractions. In agreement with the dose-response curves in HL-1 cardiomyocytes slightly higher concentrations of disulfiram were needed to obtain a similar phenotype rescue in tre embryos, where 0.1 μM disulfiram did not induce a significant effect but again approximately one half to 60% of all tre embryos were rescued at concentrations above 0.5 μM.
Although not all embryos treated with ezetimibe or disulfiram showed rhythmic contractions, this rescue efficiency is comparable to the rescue efficiency initially described with 10 μM efsevin (Shimizu et al., 2015). Interestingly however, zebrafish embryos treated with disulfiram showed signs of intoxication such as a lack of pigmentation

| Ezetimibe and disulfiram suppress arrhythmogenesis in cardiomyocytes of a murine tachycardia model
We next tested both compounds for their anti-arrhythmic potential in freshly isolated cardiomyocytes of a murine model for catecholaminergic polymorphic ventricular tachycardia (CPVT) (Cerrone et al., 2005). Consistent with the phenotype of CPVT, cardiomyocytes from RyR2 R4496C/WT mice develop spontaneous Ca 2+ waves during diastole when stimulated with catecholamines ( Figure 5a) (Schweitzer et al., 2017;Sedej et al., 2010 through mutated RyR2s and represent the origin of ectopic cardiac excitations that cause arrhythmia (Allen et al., 1984). Both MiCUps dose-dependently reduced the number of isoprenaline (ISO)-induced Ca 2+ waves to levels comparable to unstimulated control cells ( Figure 5b). Interestingly, and comparable to our zebrafish data, disulfiram displayed toxic effects at higher concentrations (10 μM) reflected by an elevation of basal cytosolic Ca 2+ levels. Cells treated with 10-μM disulfiram showed very high fluo-4 fluorescence (51.5 ± 4.8FU compared to 15.3 ± 0.5FU under isoprenaline alone) and extensive spontaneous activity, which prevented further analysis ( Figure S2).
To confirm that this striking effect in CPVT cardiomyocytes is solely attributable to the enhanced mitochondrial-Ca 2+ uptake induced by the two MiCUps, we crossbred RyR2 R4496C mice with MCU À/À mice, which lack the central pore forming subunit of the mitochondrial Ca 2+ uniporter complex (Pan et al., 2013). In freshly isolated RyR2 R4496C/WT /MCU À/À cardiomyocytes, we again observed an induction of diastolic Ca 2+ waves upon isoprenaline treatment compared to untreated control cells. Strikingly, application of the highest effective doses, 10 μM ezetimibe and 1 μM disulfiram, failed to reduce Ca 2+ waves in both cases (Figure 5c), indicating that mitochondrial Ca 2+ uptake is the effective target of ezetimibe and disulfiram.
Apart from diastolic Ca 2+ waves, systolic Ca 2+ activity was suggested as a potential trigger for arrhythmia in CPVT (Němec et al., 2010;Němec et al., 2016). We therefore also analysed systolic Ca 2+ transients of our recordings for the occurrence of secondary systolic Ca 2+ -elevations (SSCEs) and found that a significantly higher portion of transients recorded from cells treated with isoprenaline showed secondary systolic Ca 2+ -elevations then transients recorded however, higher concentrations of disulfiram induced higher spontaneous Ca 2+ activity, which might again be attributed to the enhanced baseline Ca 2+ observed under disulfiram ( Figure S2).

| Ezetimibe and disulfiram suppress arrhythmogenesis in human iPSC-derived CPVT cardiomyocytes
Finally, we tested both substances for their ability to suppress arrhythmogenic Ca 2+ waves in human cells to estimate the translatability of our data and the potential of ezetimibe and disulfiram to serve as candidates for a human therapy ( Figure 6). To this aim, we used iPSC-derived cardiomyocytes from a CPVT patient. Comparable to murine CPVT cardiomyocytes and in line with the CPVT phenotype, these cells did not display spontaneous diastolic Ca 2+ waves under unstimulated control conditions while addition of isoprenaline (ISO) induced prominent Ca 2+ waves (Figure 6a,b). Strikingly, addition of ezetimibe and disulfiram reduced Ca 2+ waves to control conditions. 4 | DISCUSSION

| Mitochondrial Ca 2+ uptake enhancer screen
We have previously demonstrated that the VDAC2 agonist efsevin as well as the mitochondrial Ca 2+ uniporter agonist kaempferol potently suppress arrhythmia in murine and human models of CPVT (Schweitzer et al., 2017;Shimizu et al., 2015). However, in regard of a clinical application of these substances, it is of note that both enhance SR-mitochondria Ca 2+ transfer with an EC 50 of around 5 μM, a concentration that is likely to favour off-target effects. Furthermore, data concerning efsevin's bioavailability, pharmacodynamics, stability and toxicity are largely missing. Though kaempferol was used in clinical studies before, it was only used at a low dose as a nutritional supplement and was shown to bind multiple targets including the NF-κB (Kadioglu et al., 2015), the fibroblast growth factor (Lee et al., 2018) and other signalling pathways (Kim et al., 2015;Wu et al., 2017;Yao et al., 2014 (Arduino & Perocchi, 2018;Di Marco et al., 2020;Kon et al., 2017;Nathan et al., 2017;Woods & Wilson, 2020), agonists still remain scarce.
We therefore set up a three-step protocol to identify novel MiCUps with anti-arrhythmic effects: primary screening was performed in a previously validated HeLa-cell based assay that allows screening of large compound libraries in a system that is cost efficient and automatable. Further target validation was performed in HL-1 cells allowing for testing of multiple candidate substances at multiple concentrations on cardiac myocytes. Finally, only candidates passing steps one and two were further evaluated for their anti-arrhythmic potential in translational animal models thereby reducing animal usage.
Using this approach, we screened the NCC consisting of 727 compounds with a history of use in human clinical trials to identify novel specific enhancers of mitochondrial-Ca 2+ uptake. Three compounds, honokiol, disulfiram and ezetimibe, were selected as hits based on their stimulatory effect on mt-Ca 2+ uptake without blocking mitochondrial Ca 2+ extrusion in primary drug screens as well as dosedependent measurements in permeabilized HeLa cells. We then further tested these compounds for their ability to selectively enhance the transfer of Ca 2+ from the SR into mitochondria in cardiomyocytes as the mechanism proposed to be the molecular prerequisite for the anti-arrhythmic effect of MiCUps (Schweitzer et al., 2017;Shimizu et al., 2015;Wilting et al., 2020). Interestingly, honokiol, which displayed the most pronounced effects in HeLa cells, was inactive in this system, while ezetimibe and disulfiram consistently enhanced mitochondrial-Ca 2+ uptake. This might be attributable to differences in the mitochondrial Ca 2+ uptake pathway between non-excitable and excitable cells and highlights the importance of re-evaluation of hits from our primary screening platform in cardiac cells. After passing both assays, ezetimibe and disulfiram were then further tested for their anti-arrhythmogenic potential in translational models and both were shown to indeed suppress arrhythmogenesis.
Taken together, we successfully identified two novel, potent MiCUps by applying a combination of a HeLa-based chemical screening followed by cardiomyocyte-specific target validation and final testing in arrhythmia models. With this protocol, we identified ezetimibe and disulfiram as novel candidates for the use in further preclinical and eventually clinical tests on the efficacy of MiCUps for the treatment of cardiac arrhythmia, but also as valuable compounds for further basic research on the mitochondrial Ca 2+ uptake pathway and preclinical research for other indications. In this respect enhancing mitochondrial Ca 2+ uptake was recently suggested to be beneficial to facilitate cerebral blood flow after traumatic brain injury (Murugan et al., 2016) and to promote metabolism/secretion coupling in type 2 diabetes (Bermont et al., 2020).

| Cardiac selectivity of novel mitochondrial Ca 2 + uptake enhancers
In this and previous studies, we successfully applied MiCUps on different models from cell cultures to in vivo systems, but never observed gross effects, for example, apoptosis, on other tissues than the heart (Schweitzer et al., 2017;Shimizu et al., 2015).  well tolerated, it is however not first-line therapy, due to a lower efficiency compared to the commonly used statins. Ezetimibe is membrane permeable (Alhayali et al., 2018) and orally bioavailable, and plasma concentrations reach a maximum approximately 2 h after intake. It is enterohepatically metabolized (Kosoglou et al., 2005). However, approximately 80%-90% of ezetimibe is rapidly metabolized into ezetimibe-glucoronide (Kosoglou et al., 2005). Though both forms were shown to be active inhibitors of NPC1L1, we found that ezetimibe glucoronide is less effective to reduce Ca 2+ waves in RyR2 R4496C/WT cardiomyocytes ( Figure S3). Thus, although plasma concentrations of approximately 300 nm for both forms were described after a 10 mg per 10 days oral intake (Ezzet et al., 2001), which would be well in the effective range in our studies, further experiments in preclinical models need to evaluate effective in vivo doses.
Disulfiram is an inhibitor of acetaldehyde dehydrogenase and is used for the treatment of alcohol abuse, but serious side effects limit the use of the drug. Also in our experiments, disulfiram induced severe malformations in zebrafish and showed signs of cellular toxicity in intact HeLa cells and cardiomyocytes at higher concentrations. It is of note however that disulfiram is administered to zebrafish embryos at a very sensitive step of development while it is envisioned to serve as an anti-arrhythmic drug for adult subjects. Furthermore, the disulfiram concentration needed for efficient rescue in zebrafish was significantly higher compared to concentrations applied in cellular models which might be explained by differences in the differentiation state of cardiomyocytes in the distinct models or a limited uptake into zebrafish embryos through the embryonic skin. However, also in cardiomyocytes, we observed enhanced Ca 2+ activity during diastole upon treatment with higher doses disulfiram. This might at least in part be explained by a direct destabilizing effect of disulfiram on the RyR2 during diastole, which would be in agreement with a recently identified modulatory effect of disulfiram on the skeletal muscle isoform RyR1 (Rebbeck et al., 2017). Furthermore, disulfiram was shown to influence the permeability of the inner mitochondrial membrane and to release Ca 2+ from mitochondria at higher concentrations (Balakirev & Zimmer, 2001;Chávez et al., 1989) which is in line with our results from intact cells.
Although these results speak against the use of disulfiram as an antiarrhythmic agent in clinical use, on the other hand and consistent with our findings, previous preclinical studies already demonstrated an antiarrhythmic potential of disulfiram in different animal models (Fossa et al., 1982;Fossa & Carlson, 1983). These effects might now be explained by activation of mitochondrial-Ca 2+ uptake. It is of note however that these reports also observed a negative inotropic effect of disulfiram at higher concentrations which might be explained by the unspecific binding to RyR2 or other unspecific effects as the ones outlined above. Taken together, further investigations on drug efficacy and safety are definitely needed for the use of disulfiram.
Summarizing our results, we have identified two novel mitochondrial Ca 2+ uptake enhancers that are already in clinical use. However, before clinical tests for the treatment of human arrhythmia can be performed with these substances, follow-up studies comprising in vivo studies with different dosing and administration regimes are needed to determine effective doses and time points of administration to evaluate the potential of MiCUps to be used as preventive therapy to reduce the risk for arrhythmias or to stop an acute episode of arrhythmia, respectively.