Selective blockade of Cav1.2 (α1C) versus Cav1.3 (α1D) L-type calcium channels by the black mamba toxin calciseptine

L-type voltage-gated calcium channels are involved in multiple physiological functions. Currently available antagonists do not discriminate between L-type channel isoforms. Importantly, no selective blocker is available to dissect the role of L-type isoforms Cav1.2 and Cav1.3 that are concomitantly co-expressed in the heart, neuroendocrine and neuronal cells. Here we show that calciseptine, a snake toxin purified from mamba venom, selectively blocks Cav1.2 -mediated L-type calcium currents (ICaL) at concentrations leaving Cav1.3-mediated ICaL unaffected in both native cardiac myocytes and HEK-293T cells expressing recombinant Cav1.2 and Cav1.3 channels. Functionally, calciseptine potently inhibits cardiac contraction without altering the pacemaker activity in sino-atrial node cells, underscoring differential roles of Cav1.2− and Cav1.3 in cardiac contractility and automaticity. In summary, calciseptine is a selective L-type Cav1.2 Ca2+ channel blocker and should be a valuable tool to dissect the role of these L-channel isoforms.

major role in SAN pacemaker activity and impulse conduction through the AVN.In this respect, mice in which Ca v 1.3 has been genetically ablated (Ca v 1.3 −/− ) show prominent SAN bradycardia, susceptibility to atrial fibrillation and atrioventricular blocks 8,9 .In SAN pacemaker myocytes, Ca v 1.3-mediatedI CaL (I Cav1. 3 ) is characterized by a more negative activation threshold and slower inactivation kinetics than Ca v 1.2-mediatedI CaL (I Cav1.2 ) and is essential for the generation of diastolic depolarization underlying SAN automaticity 8,10 (for review see 11 ).
Despite the importance of dissecting the physiopathological roles of Ca v 1.2 and Ca v 1.3 channels in cardiac myocytes and neuronal cells, no selective pharmacological tool is currently available.Both Ca v 1.2 and Ca v 1.3 are sensitive to antagonist and agonist dihydropyridines (DHPs) and to non-DHP drugs, such as phenylalkylamine (i.e.Verapamil) and benzothiazepine-like compounds (i.e.Diltiazem) without significant selectivity 1 .
Animal venoms constitute an immense reservoir for identification of new toxins and toxin-derived tools for modulating the physiological function of ion channels.In particular, toxins showing three-fingers molecular scaffold class include several cardiotoxic peptides that abound in snake venoms.Three fingers toxin peptides potently block a variety of proteins including nicotinic receptors 12 , acetylcholinesterase 13 , acid-sensitive ion channels (ASIC) 14 , as well as cytotoxins that disrupt the lipid membrane 15 .More than 30 years ago, M. Lazdunski 's group purified a 60-amino acid three-fingers polypeptide from the venom of the black mamba (Dendroaspis polylepis) called calciseptine (Cas).Cas was shown to selectively block I CaL , to abolish all contraction in portal vein, thoracic aorta and uterine smooth muscle and to completely eliminate cardiac contractions 16 .These properties constitute clear adaptation to snake's predatory behavior to induce very rapid and robust hypotension and dramatic negative inotropic effect in the prey, also in combination with the effects of the other venom components.Interestingly, Cas specifically blocks I CaL , without affecting N-type, T-type and also the skeletal muscle (Ca v 1.1) L-type Ca 2+ channels isoforms 16 .In addition, previous data show that Cas does not affect voltage sensitive Na + and K + channels in cardiac ventricular cells, NlE115 neuroblastoma cells, RINmSF and HIT insulinomas and chicken dorsal root ganglia 16 .
In this work, we show that Cas selectively blocks Ca v 1.2 channels, while Ca v 1.3 is resistant to Cas.We show that Cas potently decreases cardiac contractility without affecting heart rate of Langendorffperfused mouse heart.In line with potent inhibition of contraction, Cas blocks I Cav1.2 in mouse ventricular myocytes.We show that while Cas partially inhibits total I CaL (I Cav1.2 +I Cav1.3 ) in SAN myocytes from wildtype mice, it totally blocks I CaL in myocytes from Ca v 1.3 −/− counterparts, where only Ca v 1.2 is functional.Selectivity of Cas blockade of Ca v 1.2 versus Ca v 1.3 is confirmed in HEK-293T expressing recombinant Ca v 1.2, Ca v 1.3 channels.Notably, the currents mediated by both Ca v 1.3 42a and Ca v 1.3 42 splice variants--characterized by short and long C-terminus, respectively 17 -are totally resistant to Cas.Finally, while DHP nifedipine robustly slows pacemaker activity of SAN myocytes, selective blockade of I Cav1.2 by Cas reduces the action potential amplitude, leaving unaffected the cell firing rate.In conclusion, our study shows that Cas selectively blocks Ca v 1.2 channels in the heart, providing an important tool in the panel of calcium channel modulators to help dissect the role of this channel isoform in heart physiology and automaticity.

Calciseptine inhibited contraction and spared automaticity of Langendorff perfused hearts
We studied the effects of Cas on cardiac contractility and automaticity in isolated hearts from mice before and after perfusion of native toxin (100 nM).Perfusion of Cas strongly reduced left ventricular (LV) contraction amplitude (51 ± 3 mmHg vs 9 ± 2 mmHg.Fig. 1a, b), without changing baseline diastolic pressure (8 ± 2 mmHg vs 8 ± 3 mmHg.Fig. 1a, b).Averaged inter-beat (RR) intervals were not affected by toxin perfusion (273 ± 17 ms vs 271 ± 17 ms, p > 0.05.Fig. 1c), indicating that Cas did not disrupt the cardiac pacemaker mechanism (automaticity).In addition, we did not observe episodes of arrhythmia upon perfusion of Cas.We then evaluated the effects of Cas on heart inotropism and lusitropism by measuring the contraction velocity (dP/dt max) and the relaxation velocity (dP/dt min), respectively.Perfusion of Cas (100 nM), significantly reduced dP/dt max (1.7 ± 0.2 mmHg/ms vs 0.4 ± 0.1 mmHg/ms) and dP/dt min (−1.2 ± 0.1 mmHg/ms vs −0.1 ± 0.1 mmHg/ms), indicating negative inotropic and lusitropic effects of the toxin (Fig. 1d).Furthermore, relaxation time constant (Ƭ) and time-to-peak interval (or time to maximal pressure), parameters related to contraction duration, were not affected by perfusion of Cas (10 ± 1 ms vs 13 ± 2 ms, and 54 ± 1 ms vs 50 ± 3 ms, respectively.Supplementary Fig. 1).Taken together these data demonstrated differential effect of Cas on heart contractility and automaticity, two distinct functions that are reliant on L-type voltage dependent Ca 2+ channels subtypes expression and regulation.
Calciseptine potently and selectively blocked L-type Ca 2+ current (I Cav1.2 ) in isolated ventricular myocytes and I Cav1.2 in HEK-293T cells expressing Ca v 1.2 We evaluated the effects of Cas on isolated adult mouse ventricular myocytes in which I CaL is mediated by Ca v 1.2 (I Cav1.2 ) 8 .For voltages ranging from −25 mV to +40 mV, I Cav1.2 in rod-shape ventricular myocytes was dose-dependently reduced by perfusion of Cas (Fig. 2).Application of nifedipine (3 µM), blocked residual Ca 2+ current in myocytes exposed to Cas 100 nM and Cas 300 nM, but failed to have an effect when cells were previously perfused with Cas 1 µM (Supplementary Fig. 2).
Whole cell recording of I CaL on recombinant Ca v 1.3 42a and Ca v 1.3 42 channels 18 and Ca v 1.2 channels expressed in HEK-293T cells before and after perfusion is shown in Fig. 3. Using a one-step voltage clamp protocol (holding potential −60 mV, voltage test 0 mV) we showed that Cas exhibited an inhibitory effect on I CaL in Ca v 1.2 transfected cells in a dose dependent manner.100 nM Cas inhibited the 37% of I Cav1.2 (Supplementary Fig. 3a), while 300 nM and 1 µM Cas produced respectively an inhibition of 70% and 80% of I Cav1.2 (Fig. 3a).Dose-effect relationship showed that Cas inhibits I Cav1.2 with IC 50 value of 92 ± 18 nM (Supplementary Fig. 3b).We then investigated Cas effects on recombinant Ca v 1.3 channels.We did not record a statistically significant reduction of recombinant I Cav1.3-42a and I Cav1.3-42expressed in HEK-293T cells by Cas at all concentrations tested (up to 1 µM) (Fig. 3a).Averaged current-to-voltage (I-V) relationships recorded upon perfusion of 300 nM of Cas showed inhibition of I Cav1.2 by ~60% at membrane voltages ranging from −20 mV to + 30 mV (Fig. 3b, Supplementary Fig. 4).Application of Cas significantly shifted the activation curve of I Cav1.2 .The V 0.5 for activation was −3 ± 2 mV in control conditions and 3 ± 2 mV during Cas application, respectively (n = 8, Wilcoxon test, p = 0.0078).We observed no significant effect of Cas on both I Cav1.3-42a and I Cav1.3-42 at all voltages tested (Fig. 3b, Supplementary Fig. 4).These data clearly indicate that Cas is a potent inhibitor of Ca v 1.2 channels irrespective of the Ca v 1.3 channel variant.We next investigated Cas selectivity on recombinant Ca v 3.1 T-type, Ca v 2.2 N-type and Ca v 2.1 P/Q type calcium channels expressed in HEK-293T cells.Moreover, knowing the propensity for several calcium channel blockers to affect human ether-a-go-go-related gene (hERG) 19 outward current, we also evaluated the effect of Cas on recombinant hERG current expressed in Xenopus oocytes.Cas had no effect on the aforementioned recombinant currents investigated (Supplementary Fig. 5 and Supplementary Fig. 6).

Calciseptine selectively inhibited I Cav1.2 in isolated mouse SAN myocytes
We studied the effects of Cas perfusion on total I CaL in isolated SAN pacemaker cells from wild-type and mutant Ca v 1.3 −/− mice (Fig. 4).We   20 (Fig. 4a, Supplementary Fig. 7).To further characterize the effect of the toxin on I CaL , we used SAN myocytes isolated from Ca v 1.3 −/− mice, in which the only residual I CaL is I Cav1.  .Cas (1 µM) totally blocked I CaL in isolated Ca v 1.3 −/− SAN myocytes (Fig. 4b, Supplementary Fig. 8).We then measured, in Ca v 1.3 −/− SAN myocytes, peak I CaL evoked by a voltage clamp step protocol at 0 mV from two different holding potentials, −60 mV and −40 mV respectively, in absence and after Cas 300 nM perfusion.We did not observe any statistically significant difference in the percentage of I CaL block (68 ± 3% vs 67 ± 4% of total Ca v 1.2-mediated current for holding potential of −40 mV and −60 mV, respectively.Supplementary Fig. 9a) suggesting no voltage dependence of Cas block on I Cav1.2 .In addition, we evaluated the frequency dependence of Cas block by modifying the frequency of the voltage clamp protocol (step to 0 mV from a holding potential of −60 mV).We did not observe any statistically significant difference in the degree of inhibition by 300 nM Cas in the three conditions tested (62 ± 6% at 0.2 Hz, 63 ± 6% at 1 Hz and 58 ± 8% at 5 Hz.Supplementary Fig. 9b).Taken together, these data show that Cas strongly discriminates between I Cav1.2 and I Cav1.3 .The toxin selectively inhibits the Ca v 1.2 channels isoform, without showing dependency from holding potential or frequency of stimulation, indicating that inhibtion of I Cav1.2 by Cas was not voltage-or use-dependent.

Calciseptine reduces the action potential amplitude and spares automaticity in SAN pacemaker myocytes
To examine the effect of the toxin on SAN cell automaticity, we recorded spontaneous action potentials (APs) in SAN myocytes from Wild-Type mice using the perforated patch-clamp technique (Fig. 5a; Fig. 5g).Consistently, 300 nM and 1 µM Cas did not affect AP firing rate (Fig. 5b, h, Table 1), but reduced AP amplitude (Fig. 5c, i, Table 1).Concomitant perfusion of Cas 300 nM and nifedipine 3 µM (Fig. 5b, Table 1) or of Cas 1 µM and nifedipine 3 µM (Fig. 5h, Table 1) induced a significantly decrease in the beating rate in SAN myocytes with respect either to control or to sole condition of Cas perfusion.Concomitant application of Cas and nifedipine 3 µM did not reduce AP amplitude further (Fig. 5c, i, Table 1).Slopes of linear (SLDD) and exponential (SEDD) part of diastolic depolarization did not change either following perfusion of Cas at 300 nM (Fig. 5d, e, Table 1) or at 1 µM (Fig. 5j, k, Table 1).Both SLDD and SEDD showed statistically significant decrease only when nifedipine 3 µM was added to the perfusion solutions (Fig. 5d, e and Fig. 5j, k, Table 1).Furthermore, we evaluated action potential duration in SAN myocytes exposed to Cas and Cas plus nifedipine 3 µM.While 300 nM Cas did not affect AP duration, we observed a reduction in action potential duration at 30%, 50% and 70% of repolarization only after concomitant application of Cas 300 nM and nifedipine 3 µM (Fig. 5f).By contrast, 1 µM Cas had statistically significant effect on AP duration at 30% and 50% of AP repolarization.Again, concomitant perfusion of Cas 1 µM and nifedipine 3 µM reduced AP duration at 30%, 50% and 70% of repolarization (Fig. 5l).We did not observe statistically significant differences in the maximum diastolic potential, the threshold potential and in action potential upstroke velocity of action potentials recorded in myocytes under control condition and after perfusion of Cas at either 300 nM or at 1 µM (Supplementary Fig. 10).

Discussion
Our study shows that the black mamba venom toxin Cas selectively inhibits Ca v 1.2 channels underlying I Cav1.2, without affecting I Cav1.3 in the heart.Cas displays selectivity between the two cardiac of L-type Ca 2+ channel subunits, enabling pharmacologic distinction between the functional roles of these L-type Ca 2+ channel isoforms.
L-type Ca 2+ channels are required for multiple functions in the heart, including regulation of early organogenesis, structural development, cardiac physiology and automaticity 21,22 .A variety of arrhythmias including inherited QT syndrome 23 have been associated with alterations in L-type channels 4 .Consequently, Ca v 1 class channels members have been, are and will be prime targets for medical therapy 21,[24][25][26] .Pharmacological inhibition of Ca v 1.2 channels accounts for most of therapeutically relevant antihypertensive and antiischemic effects, predominantly by reducing peripheral vascular resistance and negative inotropy.Dihydropyridines (DHPs) are among the most used Ca v 1 channel antagonists 27 with a long history of success in the treatment of several cardiovascular pathologies 28,29 .However, one potential limitation of DHPs in therapeutic perspective is their lack of selectivity for different isoforms of the Ca v 1 family.To date, over 100 DHPs were screened in an attempt to identify a selective antagonist able to distinguish among Ca v 1.2 and Ca v 1.3 30 .However, no candidate molecule issued from this screen study showed selectivity for  one or the other isoform.This same study indicated highly similar DHP binding site at both Ca v 1.2 and Ca v 1.3 channels, suggesting that other classes of compounds needed to be identified to achieve selectivity 30 .Other potent synthetic blockers with completely different structure as compared to DHPs have been identified for their action on L-type Ca 2+ channels [31][32][33] , but to the best of our knowledge, they have not been tested for their specificity for the different isoforms of Ca v 1 channels.Animal toxins have played an essential role in the physiological exploration of the bioelectric mechanisms of the heart and the brain.Over the last five decades, toxins have allowed investigation and characterization of the functional roles of several classes of ion channels; e.g.tetrodotoxin for the voltage-sensitive Na + channel 34 , apamin for Ca 2+ -sensitive K + channels 35 , omega conotoxins for N-type Ca 2+ channels 36 , psalmotoxin for channels 37 .Since the discovery of these leading toxins, tremendous information has accumulated on the molecular identification of L-type Ca 2+ channel isoforms in the and cardiovascular tissues.Thus, new pharmacological tools dissect the roles played by different Ca 2+ channels that discriminate between isoforms belonging to the L-type Ca v 1 gene family are needed.is of particular importance in the heart in which two L-type isoforms Ca v 1.2 and Ca v 1.3 could not be distinguished pharmacologically so far.
In this work, we investigated if Cas can discriminate between cardiac Ca v 1.2 and Ca v 1.3, and if potential selectivity could be used to study the respective physiological functions of Ca v 1.2 and Ca v 1.3 in determination of heart rate and contractility.
Our results show that Cas has selective negative inotropic effect on isolated perfused heart without affecting heart rate or inducing arrhythmia in isolated hearts.A previous study reported sinus bradycardia with unaltered contractile properties in isolated heart from Ca v 1.3 −/− mice 38 suggesting that while Ca v 1.2 is the only L-type Ca 2+ channel isoform involved in heart contractility, Ca v 1.3 plays a major role in heart automaticity and conduction.In addition, the evidence that, contrary to what was observed in Ca v 1.3 −/− mice, perfusion of Cas does not induce arhythmias, sinus bradycardia or heart block is in line with the hypothesis that Cas does not affect the I Cav1.3 .Consequently, these results indicate that pure negative inotropic effect of Cas is due to selective block of Ca v 1.2 vs Ca v 1.3 L-type Ca 2+ channels.We provide further evidence for selective Ca v 1.2 inhibition by Cas in SAN myocytes isolated from wild-type and Ca v 1.3 −/− mice.Indeed, voltage clamp recordings in Wild-Type SAN myocytes showed that Cas account for around 45% reduction of total I CaL .Considering that previous data reported that in SAN cells the I Cav1.2 /I Cav1.3 current density ratio is between 1:2 8 to 1:1 10 , it is reasonable to postulate that the residual I CaL current recorded in SAN wild-type cells correspond to I Cav1.3 .This point is supported by our recordings of I CaL in isolated Ca v 1.3 −/− SAN myocytes after Cas perfusion.control conditions I CaL peak current density in Ca v 1.3 −/− SAN cells was quantitively similar to the amount of current by Cas in Wild-Type SAN myocytes, further indicating that Cas acted selectively on I Cav1.2 .No residual I CaL was recorded in Ca v 1.3 −/− SAN myocytes after Cas perfusion, confirming the role of Cas as pure Ca v 1.2 L-type Ca 2+ current blocker.Results obtained on recombinant L-type Ca v 1.3 currents mediated by short and long variants, T-type Ca v 3.1, N-type Ca v 2.2, P/Q-type Ca v 2.1 expressed in HEK-293T and on L-type Ca v 1.2 channels in isolated ventricular myocytes further strengthen this conclusion.Since Ca v 1.2 plays a major role in shaping the AP in cardiac myocytes 39 we studied the impact of Cas application on the different parameters in spontaneous SAN AP.Cas affects both AP amplitude and duration, without affecting the slope of diastolic depolarization phase which is known to be a Ca v 1.3dependent feature 40 , stressing again the efficiency and the selectivity of the toxin.Our results are consistent with previous studies on the functional role of Ca v 1.2 in the heart using global or conditional genetic ablation of Ca v 1.2 channels.Indeed, while these studies demonstrated obligatory role of this isoform in cardiac contraction 41,42 and regulation of blood pressure 43 , they also reported lack of effect on heart rate.
The study by Teramoto and coll 44 on Cas effect on unitary Ba 2+ currents in the guinea-pig portal vein cells, suggested that the action site of Cas was outside the membrane.DHPs are prototypical organic blockers of L-type calcium channels widely used in therapeutical applications.A previous study reported that Cas at a rather high concentration could inhibit binding of DHPs in brain synaptosomes suggesting that the toxin is located in the proximity of DHP binding site 45 .
To investigate if the binding site of Cas was overlapping with that of DHPs we used isolated ventricular myocytes from mice in which Ca v 1.2 channels have been rendered insensitive to DHPs by knock-in of a point mutation in the drug binding site (Ca v 1.2 DHP−/− ) 46 .I Cav1.2 recorded in Ca v 1.2 DHP−/− myocytes showed similar sensitivity to Cas than that  those isolated from Wild-Type myocytes, suggesting that DHPs and CaS do not share the same binding site on Ca v 1.2 channels (Supplementary Fig. 11a).To futher explore the binding properties of Cas on Ca v 1.2, we also used an electrophysiological approach aimed to indicate if a toxin inhibiting a neuronal Ca 2+ channel was acting as a gating modifier 47 .We investigated the Cas-induced block Ca v 1.2 expressed in HEK-293T cells at a moderate depolarizing potential (0 mV) and at a very positive potential leading to maximal current activa-Our data show that Cas has almost no effect on outward-going I Cav1.2 , recorded at +150 mV-likely carried by Cs + a gating modifier behavior.By contrast, using the same protocol, 3 µM Nifedipine partially inhibited the I Cav1.2 elicited at +150 mV, again showing that Cas inhibits Ca v 1.2 channels with a different mechanism than DHPs (Supplementary Fig. 11b).
A very homologous snake venom peptide (FS 2 ) from black mamba was reported to have a similar action in muscle relaxation and hypotensive properties than Cas 48 .A common short peptide between two Prolines at position 42 and 47 (PTAMWP) was predicted as the binding site for Cas and FS 2 by Kini and co-workers 49 .These authors reported that this peptide called L-calchin acts like Cas and FS2 in reducing peak systolic pressure in isolated rat hearts and reducing calcium channel amplitude in rabbit cardiac myocytes but with lower potency (IC 50 ≈ 5 µM).Similar to Cas, block by L-calchin had no effect on heart rate in ex vivo rat isolated hearts.We thus synthetized L-calchin and conducted preliminary experiments on dissociated mouse ventricular myocytes (Supplementary Fig. 12).Our results showed ≈30% reduction of I Cav1.2 , in line with these previous data.Taken together, our data and comparison with previous studies on this class of peptides indicate that Cas and Cas-derived peptides are a promising starting point to develop selective inhibitors of Ca v 1 channel subtypes.
L-type Ca 2+ channels are widely expressed in the brain and, in many neuronal types, Ca v 1.2 and Ca v 1.3 are co-expressed (see ref. 50  for review) 50 .Both distinct and complementary functions for Ca v 1.2 and Ca v 1.3 have been proposed in the central nervous system.For example, Ca v 1.2 is involved in hippocampal firing 51 , spatial memory and LTP 52 , as well as in the myelinisation process 53 .It is very likely that Cas, with its high specificity for the Ca v 1.2 channel, and its high flexibility of use due to the fact that it can be stereotoxally injected in specific sites in the brain will constitute a very useful tool for future explorations of the role of Ca v 1.2 in the nervous system.Beside playing a major role in cardiac pacemaker activity, Ca v 1.3 appear to be an important player in coupling spontaneous neuronal firing with mitochondrial metabolism 54 .It has been proposed that Ca v 1.3 channels are involved in firing-dependent oxidative stress of dopaminergic neurons of substantia nigra, suggesting that these channels could constitute a potential target for Parkinson disease 55 .This has sparkled studies in the search for selective Ca v 1.3 antagonists (for review see 1,56 ).Recently, a molecule named compound 8 was described as selective for Ca v 1.3 57 with affinity ~24 µM.However, the selectivity of compound 8 is controversial 58,59 .In particular, Huang et al. 2014 58 reported that selectivity of compound 8 for Ca v 1.3 versus Ca v 1.2 is modest and dependent on the nature of the associated β subunit and possibly dependent from the Ca v 1.3 splice variants.It will thus be interesting to study selectivity of Cas in neurons coexpressing Ca v 1.2 and Ca v 1.3.

Care and use of animals
The study is in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996) and European directives (2010/63/EU).Experimental procedures were approved by the Ethical Panel of the University of Montpellier and the French Ministry of Agriculture (protocol no: 2017010310594939).Animals were housed in the IGF animal facility with free access to food and water and were exposed to 12 h light/dark cycles in a thermostatically controlled room (21-22 °C) with 40−60% humidity.

Whole heart contractility
Hearts were isolated from adult male mice anaesthetized with a first intraperitoneal injection of a mixture comprising ketamine (100 mg/kg) and xylazine (10 mg/kg), and a second one of sodium pentobarbital (45 mg/kg; Ceva Santé Animale, France).An intraperitoneal injection of heparin (7500IU/kg; Sanofi Aventis, France) was administered in order to avoid thrombus formation.A thoracotomy was performed and beating hearts were quickly removed and excised into ice-cold modified Krebs-Henseleit buffer containing (mM): NaCl 116; KCl 5; MgSO 4 1.1; NaH 2 P0 4 0.35; NaHCO 3 27; CaC1 2 1.8 and glucose 10.Buffer was gassed with 95% O 2 and 5% CO 2 , giving a pH of 7.4 at 37 °C.Before use, the buffer was initially passed through a 0.2 µm filter (Sarstedt, USA).The aorta was cannulated under magnification and hearts were quickly mounted and perfused on the Langendorff perfusion system (Isolated heart system, EMKA Technologies, France).The coronary circulation was perfused at a constant pressure (70 mmHg) over a 1 to 4 ml/min flow range with modified Krebs-Henseleit buffer.During the stabilization phase, the heart was continuously perfused with modified Krebs-Henseleit and the electrocardiogram (ECG) was recorded through Ag/AgCl electrodes positioned on the right atrium and near the apex.Heart temperature was of 37 °C as continuously assessed by a probe positioned on the base of the heart and recorded by using a TH-5 monitoring thermometer (Phymep, USA).LV pressure was continuously measured by a home-made balloon inserted into the LV cavity by the left atrium and connected to the EMKA amplifier.After 20 min of stabilization (baseline), 100 nM Cas was perfused into the heart for 7 min.Then, the modified Krebs-Henseleit buffer was perfused for 1 h allowing to wash the effects of the Cas toxin.During the whole ex vivo protocol, the left ventricular pressure signals were continuously digitized at 0.1 Hz and recorded using an IOX data acquisition system (EMKA Technologies, France).Data were analyzed using ecgAUTOv3.3.3.12 (EMKA technologies) and MATLAB-R2021b (The MathWorks) custom-made script Cell culture and transfection protocols HEK-293T (RRID:CVCL_2737) were obtained from the European Collection of Authenticated Cell Cultures (ECACC 96121229).The identity of HEK-293T has been confirmed by STR profiling and the cells have been eradicated from mycoplasma at ECACC.We routinely tested the cells for the absence of the mycoplasma contamination.Cells were cultivated at 37 °C in DMEM supplemented with GlutaMax, 10% fetal bovine serum and 1% penicillin/streptomycin (Invitrogen, Fisher Scientific, France).Transfections were performed using jet-PEI (Ozyme, France) with a DNA mix containing either the plasmids that code for Ca v 1.2-GFP, β2a and α2δ (0.8 µg of each) or either the plasmids that code for Ca v 1.3 (with and without exon 42a), or Ca v 2.1, or either Ca v 2.2 combined with β2a and α2δ (0.8 µg of each supplemented with 0.025 µg of a plasmid coding for GFP).For Ca v 3.1 expression β2a and α2δ subunits were omitted.Plasmids coding for β2a, α2δ and Ca v 1.3 were created as described previously 58 .Plasmid coding for Ca v 1.2-GFP was obtained from Dr. Catalucci 60 .After transfection cells were placed at 30 °C for 2-3 days and dissociated with Versene (Invitrogen, Fisher Scientific, France) and plated at a density of ~35 × 10 3 cells per 35 mm Petri dish for electrophysiological recordings, which were performed the following day.

Xenopus oocyte preparation, DNA injection and electrophysiology
Xenopus laevis specimen were purchased from Tefor Paris Saclay.Animal handling and experiments fully conformed to French regulations and were approved by local governmental veterinary services (authorization number E06-152-5 delivered by the Ministère de l'Agriculture, Direction des Services Vétérinaires).Animals were anesthetized by exposure for 20 min to a 0.1% solution of 3-aminobenzoic acid ethyl ester (MS-222) (Sigma) buffered at pH 7.4.Oocytes were surgically removed and dissected away in a saline solution (ND96) containing (in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 2 MgCl2, and 5 HEPES at pH 7.4 with NaOH.Stage V and VI oocytes were treated for 3 h with (1 mg/ml, type Ia, Sigma) in the presence of trypsin inhibitor (Sigma) in ND96 to discard pSI-HERG plasmid solutions were injected in nucleus (100 23 nl/oocyte) using a pressure microinjector.The oocytes were kept at 19 °C in the ND96 saline solution supplemented with penicillin (10 mg/L) and (20 mg/L).Oocytes were studied within 2 days following injection of plasmid.In a 0.3-ml perfusion chamber, a single oocyte was impaled with two standard glass microelectrodes (1-2.5 MΩ resistance) filled with 3 M KCl and maintained under voltage-clamp using a Dagan TEV 200 amplifier.Stimulation of the preparation, data acquisition and analysis were performed using pClamp software (Axon Instruments).All experiments were performed at controlled room temperature (19-20 °C) in ND96 solutions.A perfusion system allowed local drugs external application.HERG currents were evoked every 10 s by 2.5 s depolarizing voltage steps ranging from -80 to +80 mV in 20 mV increments and then by a repolarizing step to -40 mV for 1.5 s.BeKm1 peptide was purchased from Smartox (Saint Martin d'Hères, France).

Isolation of adult ventricular cardiomyocytes
Excised hearts from C57Bl/6 J Wild-Type and mice carrying DHP insensitive Ca v 1.2 channels (Ca v 1.2 DHP−/− ) mice were quickly mounted on a Langendorff apparatus.Heart was perfused with a Ca 2+ -free Tyrode solution for 4 min followed by an enzymatic solution containing Lib-eraseTM (0.2 mg/ml) and Trypsin (0.14 mg/ml) for 3.5 min.Hearts were removed from the apparatus and stored in a "Stop solution" (10 mM BDM, 5.5 mM glucose, 12.5 μM CaCl2 and 5% Fetal Calf Serum) to block enzymatic digestion.Atria were quickly removed and the ventricles were chopped into small pieces (~1 mm 3 ) using forceps and dissociated by gentle pipetting using a flame-forged Pasteur pipette.Cell suspension was then filtered using a medical gauze and myocytes gravity settle for 20 min.To restore Ca 2+ concentration to physiological level, a Ca 2+ reintroduction buffer containing (mM): NaCl, 140; KCl, 5.4; MgCl2, 1; CaCl2, 1.8; Hepes,5 and glucose, 5.5 (pH adjusted to 7.4 with NaOH) was added progressively.Then cardiomyocytes were plated on dishes for patch clamp recording.

Toxin Calciseptine
In all experiments of cellular electrophysiology, we employed synthetized analog of Cas, commercially available at Latoxan (ID: L8109).The native peptide was purified from the venom of Dendroaspis polylepis purchased from Latoxan, as previoulsly described 16 .The pure peptide was obtained after gel filtration, cation exchange and C18 reversed phase chromatography.Its measured molecular mass (7036.26Da average) was in accordance with the mass calculated from sequence data (7036.22Da).

Statistical analysis
Statistical analysis was performed using Prism 10.0 (GraphPad Software).Data are represented as mean ± the standard error of the mean.p < 0.05 was considered statistically significant.Normality test was performed on each data set and appropriate statistical tests have been applied.Statistical tests used in each experiment are specified throughout the figure legends.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Fig. 1 | Ventricular contraction and heart rate in Cas perfused heart.a Time course of isolated perfused heart (n = 5) contraction amplitude recording before and after Cas 100 nM application.b Sample traces of pressure recordings (left) and histograms of left ventricular (LV) contraction amplitude and basal diastolic LV pressure before and after toxin perfusion (n = 5).c Representative ECG traces and

Fig. 3 |
Fig. 3 | Cas effect on L-type Ca 2+ current density in HEK-293T cells expressing Ca v 1.2, Ca v 1.3 42a or Ca v 1.3 42 channels.a Histograms represent the percentage of reduction of L-type Ca 2+ peak current density after 300 nM and 1 µM Cas perfusion in HEK-293T cells transfected with Ca v 1.2 (circles, n = 14/N = 4 and n = 7/N = 4, respectively), Ca v 1.3 42a (squares, n = 13/N = 4 and n = 8/N = 4, respectively) or Ca v 1.3 42 (triangles, n = 6/N = 3 and n = 7/N = 3, respectively) channels.Peak current density was recorded starting from a holding potential of −60 mV using an activating voltage step at 0 mV.Examples of L-type Ca 2+ current traces for each condition and for each clone tested are reported.Statistics: one-way ANOVA test followed by Tukey's multiple comparisons test.b Current-to-voltage (I-V) relationship of L-type Ca 2+ current recorded from holding potential of −60 mV in HEK-293T Ca v 1.2 (n = 11/N = 4), Ca v 1.3 42a (n = 9/N = 4) and Ca v 1.3 42 (n = 6/N = 3) transfected cells before (empty circles) and after (red filled circles) 300 nM Cas application.At peak: for Ca v 1.2 before vs 300 nM Cas, p = 0.0037; for Ca v 3.1 42a before vs 300 nM Cas, p = 0.8148; for Ca v 1.3 42 before vs 300 nM Cas, p = 0.5696.Statistics: paired two-sided Student t-test.Data are presented as mean values ± SEM. n = number of cells; N = number of independent transfections.

Fig. 4 |
Fig. 4 | 1 µM Cas effect on I CaL in isolated SAN cells from Wild-Type and Ca v 1.3 −/− mutant mice.a Current-to-voltage (I-V) relationship and sample traces of L-type Ca 2+ current recorded in isolated Wild-Type SAN cells (n = 8/N = 2) from a holding potential of −55 mV in control condition (black filled circles), after 1 µM Cas perfusion (blue circles) and after concomitant exposure to Cas 1 µM and Nife 3 µM (empty circles).At peak: before vs 1 µM Cas, p = 0.0168; 1 µM Cas vs Nife 3 µM, p = 0.0008 Statistics: one-way ANOVA test followed by Tukey multiple comparisons test.b Same as in a but in isolated SAN myocytes form Ca v 1.3 −/− (n = 8/N = 2).At peak: before vs 1 µM Cas, p = 0.0018; 1 µM Cas vs Nife 3 µM, p = 0.1036.Statistics: one-way ANOVA test followed by Tukey multiple comparisons test.Voltage-clamp step protocol used is shown at the bottom of the sample traces.Data are presented as mean values ± SEM. n = number of cells; N = number of mice.