AbstractRATIONALECardiac rhythm, conduction and synchronization of electrical activity require the coordinated action of different types of ion channels that differ according to transmural and regional specificities. Classical pharmacology affects these ion channels in a non-regionalized way which explains why treating arrhythmias, that often occur in specific foci, has often limited efficacy in addition to negative side-effects on non-targeted organs. Photopharmacology is an emergent technology that has the potential to counteract all the negative aspects of classical pharmacology by restricting drug activity in a spatio-temporal manner.OBJECTIVEWe tested the potential of photopharmacology in specifically regulating heart activity by using a caged derivative of a natural peptide inhibitor of the ERG channel, BeKm1. The peptide was uncaged and activity monitoredin vitroon a cell line expressing the hERG channel, on human cardiomyocytes derived from iPS cells, andex vivoandin vivoon zebrafish larvae and rat hearts.METHODS AND RESULTSCaged BeKm-1 is inactive and fully active upon uncaging. Uncaging of the peptide on human iPS-derived cardiomyocytes enlarges the action potential duration and triggers arrhythmias. Uncaging also triggers bradycardia and disturbs cardiac conduction within the atria in perfused rat hearts upon illumination. The potency of photopharmacology for cardiac electrical modulation was further validated in zebrafish larvae where illumination of the caged compound induces bradycardia and atrio-ventricular desynchrony. Finally, in anesthetized rats, illumination of the caged peptide in the right atria, containing the sino-atrial node, leads to bradycardia without arrhythmia.CONCLUSIONSThis report demonstrates that photopharmacology, using the caged peptide strategy, can be used for dynamically regulating cardiac electrical activityin vivoand that spatial illumination restriction can dissociate the bradycardic effect from the arrhythmic one. The technology is applicable to all kinds of cardiac ion channels and regions of interest to create arrhythmogenic models or investigate new clinical applications.

Hemorrhagic shock (HS) is the consequence of an important loss in blood volume leading to an insufficient delivery in oxygen. HS causes metabolic changes, multi-organ dysfunction and inflammation. The acute kidney injury related to HS is associated with hyperkalemia mainly through an alteration of Na/K ATPase pump. In a previous O-GlcNAcylomic study, we have identified Na/K ATPase as potentially O-GlcNAcylated protein. O-GlcNAcylation is a post-translational modification with a putative beneficial effect in shock situation. This study aims at evaluating if Na/K ATPase is O-GlcNAcylated and its potential impact during the early phase of hemorrhagic shock. Wistar rats were subjected to a HS protocol induced via exsanguination and then randomly treated or not with NButGT (10 mg/kg), a O-GlcNAcase inhibitor, to increase O-GlcNAc levels. Blood pressure and heart rate were monitored throughout the experiment. Blood was collected from the animals to perform a complete blood count and a blood gas analysis. Kidney was immediately frozen for histological and biochemical studies. Immunoprecipitation and Wheat Germ Agglutinin (WGA) based lectin affinity gel electrophoresis were used to study the O-GlcNAcylation of Na/K ATPase. NButGT increased by 2 O-GlcNAc levels in heart and kidney. This increase in O-GlcNAc restored mean arterial pressure (MAP) (P < 0.01) and bicarbonates (P < 0.01). In treated group, kalemia (Sham: 4.2 ± 0.1; HS: 4.7 ± 0.1; NButGT: 4.3 ± 0.1 mmol/L; P < 0.05) and natremia (Sham: 141.5 ± 0.4; HS: 139.8 ± 0.5; NButGT: 141.9 ± 0.4 mmol/L; P < 0.01) were restored. The study of the O-GlcNAcylation of the Na/K ATPase by immunoprecipitation and WGA gel validated its O-GlcNAcylation. Histological analysis of kidney revealed that HS reduced NA/K ATPase expression during HS and treatment by NButGT restored it (Sham: 15.1 ± 1.1; HS: 10.3 ± 0.6; NButGT: 13.78 ± 1.1; % area; P < 0.05). Our results demonstrate that increase O-GlcNAcylation during hemorrhagic shock restored MAP and reduced markers of metabolic acidosis. In our study, animal treatment with NButGT to increase O-GlcNAc levels, restored natremia and kalemia. This effect is associated with an increase in Na/K ATPase expression. To our knowledge, our study validates the O-GlcNAcylation of the Na/K ATPase. With regard to our results, now it will be relevant to study the effect of O-GlcNAcylation on the activity and localisation.

RATIONALE Cardiac rhythm, conduction and synchronization of electrical activity require the coordinated action of different types of ion channels that differ according to transmural and regional specificities. Classical pharmacology affects these ion channels in a non-regionalized way which explains why treating arrhythmias, that often occur in specific foci, has often limited efficacy in addition to negative side-effects on non-targeted organs. Photopharmacology is an emergent technology that has the potential to counteract all the negative aspects of classical pharmacology by restricting drug activity in a spatio-temporal manner. OBJECTIVE We tested the potential of photopharmacology in specifically regulating heart activity by using a caged derivative of a natural peptide inhibitor of the ERG channel, BeKm1. The peptide was uncaged and activity monitored in vitro on a cell line expressing the hERG channel, on human cardiomyocytes derived from iPS cells, and ex vivo and in vivo on zebrafish larvae and rat hearts. METHODS AND RESULTS Caged BeKm-1 is inactive and fully active upon uncaging. Uncaging of the peptide on human iPS-derived cardiomyocytes enlarges the action potential duration and triggers arrhythmias. Uncaging also triggers bradycardia and disturbs cardiac conduction within the atria in perfused rat hearts upon illumination. The potency of photopharmacology for cardiac electrical modulation was further validated in zebrafish larvae where illumination of the caged compound induces bradycardia and atrio-ventricular desynchrony. Finally, in anesthetized rats, illumination of the caged peptide in the right atria, containing the sino-atrial node, leads to bradycardia without arrhythmia. CONCLUSIONS This report demonstrates that photopharmacology, using the caged peptide strategy, can be used for dynamically regulating cardiac electrical activity in vivo and that spatial illumination restriction can dissociate the bradycardic effect from the arrhythmic one. The technology is applicable to all kinds of cardiac ion channels and regions of interest to create arrhythmogenic models or investigate new clinical applications.

IKr current, a major component of cardiac repolarization, is mediated by human Ether-à-go-go-Related Gene (hERG, Kv11.1) potassium channels. The blockage of these channels by pharmacological compounds is associated to drug-induced long QT syndrome (LQTS), which is a life-threatening disorder characterized by ventricular arrhythmias and defects in cardiac repolarization that can be illustrated using cardiomyocytes derived from human-induced pluripotent stem cells (hiPS-CMs). This study was meant to assess the modification in hiPS-CMs excitability and contractile properties by BeKm-1, a natural scorpion venom peptide that selectively interacts with the extracellular face of hERG, by opposition to reference compounds that act onto the intracellular face. Using an automated patch-clamp system, we compared the affinity of BeKm-1 for hERG channels with some reference compounds. We fully assessed its effects on the electrophysiological, calcium handling, and beating properties of hiPS-CMs. By delaying cardiomyocyte repolarization, the peptide induces early afterdepolarizations and reduces spontaneous action potentials, calcium transients, and contraction frequencies, therefore recapitulating several of the critical phenotype features associated with arrhythmic risk in drug-induced LQTS. BeKm-1 exemplifies an interesting reference compound in the integrated hiPS-CMs cell model for all drugs that may block the hERG channel from the outer face. Being a peptide that is easily modifiable, it will serve as an ideal molecular platform for the design of new hERG modulators displaying additional functionalities.