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RATIONALECardiac 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.

In neocortical layer-5 pyramidal neurons, the action potential (AP) is generated in the axon initial segment (AIS) when the membrane potential (Vm) reaches the threshold for activation of two diverse voltage-gated Na+ channels, that differ in spatial distribution and biophysical properties. Yet, the understanding of specific functional differences between these two channels remains elusive. Here, using ultrafast Na+, Vm and Ca2+ imaging in combination with partial block of Nav1.2 by a recent peptide, we demonstrate an exclusive role of Nav1.2 in shaping the generating AP. Precisely, Ca2+ influx via Nav1.2 activates BK Ca2+-activated K+ channels determining the amplitude and the early phase of repolarisation of the AP in the AIS. We mimicked this result with a NEURON model where the role of the different ion channels tested reproduced the experimental evidence. The exclusive role of Nav1.2 reported here is important for understanding the physiology and pathology of neuronal excitability.

The patch-clamp technique and more recently the high throughput patch-clamp technique have contributed to major advances in the characterization of ion channels. However, the whole-cell voltage-clamp technique presents certain limits that need to be considered for robust data generation. One major caveat is that increasing current amplitude profoundly impacts the accuracy of the biophysical analyses of macroscopic ion currents under study. Using mathematical kinetic models of a cardiac voltage-gated sodium channel and a cardiac voltage-gated potassium channel, we demonstrated how large current amplitude and series resistance artefacts induce an undetected alteration in the actual membrane potential and affect the characterization of voltage-dependent activation and inactivation processes. We also computed how dose–response curves are hindered by high current amplitudes. This is of high interest since stable cell lines frequently demonstrating high current amplitudes are used for safety pharmacology using the high throughput patch-clamp technique. It is therefore critical to set experimental limits for current amplitude recordings to prevent inaccuracy in the characterization of channel properties or drug activity, such limits being different from one channel type to another. Based on the predictions generated by the kinetic models, we draw simple guidelines for good practice of whole-cell voltage-clamp recordings.