Dr Claire O Malécot
email: claire.malecot@univ-tours.fr

Low voltage-activated channels in rat pulmonary vein cardiomyocytes: Coexistence of a non-selective cationic channel and of T-type Ca channels

Keywords: Basic Science Research, Ion Channels/Membrane Transport, Electrophysiology Calcium channels, non-specific cationic channel, LVA currents CM, cardiomyocytes, FLVA, fast low voltage-activated, FSLVA, fast activating pulmonary vein, SLVA, slow low voltage-activated, TTX, tetrodotoxin

Low voltage-activated channels in rat pulmonary vein cardiomyocytes: coexistence of a non-selective cationic channel and of T-type Ca channels

Claire O Malécot

INTRODUCTION

Ectopic activity arising from the pulmonary veins (PV) myocardial sleeve has been shown to play an important role in the onset of atrial fibrillation (AF) in humans [START_REF] Haïssaguerre | Spontaneous initiation of atrial fibrillation by ectopic beats originating in the pulmonary veins[END_REF]. However, although many studies were undertaken to decipher the underlying mechanism, none has succeeded so far and the question remains open [START_REF] Mahida | Science linking pulmonary veins and atrial fibrillation[END_REF]. In the rat, our laboratory has shown that application of the catecholamine norepinephrine (NE) induced an unusual automatic activity in PV myocardial sleeve, but not in the left atria (LA; [START_REF] Maupoil | Ectopic activity in the rat pulmonary vein can arise from simultaneous activation of alpha1-and beta1-adrenoceptors[END_REF]). This catecholaminergic automatic activity (CAA), which markedly differs from the classical cardiac pacemaker activity, consists of bursts of high frequency (4 to 5 Hz) slow action potentials organized in a saw-tooth pattern [START_REF] Doisne | Catecholaminergic automatic activity in the rat pulmonary vein: electrophysiological differences between cardiac muscle in the left atrium and pulmonary vein[END_REF]. In some way, this bursting activity looks similar to that of some hippocampal neurones for which T-type calcium channels have been found to noticeably contribute to their neuronal firing states (bursting and tonic; [7 for review, 8]). Like Ca v 1.3 channels, T-type Ca channels are also involved in cardiac automaticity as genetic inactivation of Ttype Ca v 3.1 calcium channel in mice results in bradycardia and slowing of atrio-ventricular conduction (for review, [START_REF] Mangoni | Functional role of L-type Cav1.3 Ca 2+ channels in cardiac pacemaker activity[END_REF][START_REF] Mesirca | Functional role of voltage gated Ca 2+ channels in heart automaticity[END_REF]).

The cellular electrophysiological characteristics of PV cardiomyocytes (CM) have been compared to those of atrial tissue in dog and several differences have been reported [START_REF] Ehrlich | Cellular electrophysiology of canine pulmonary vein cardiomyocytes: action potential and ionic current properties[END_REF][START_REF] Melnyk | Comparison of ion channel distribution and expression in cardiomyocytes of canine pulmonary veins versus left atrium[END_REF].

Concerning the T-type Ca 2+ current, dog PV and LA CM were found to have identical current-voltage relationships (7 and 8 CM studied, respectively, [START_REF] Ehrlich | Cellular electrophysiology of canine pulmonary vein cardiomyocytes: action potential and ionic current properties[END_REF]), but data on rat PV and LA CM are not available in the literature to our knowledge. Previous studies from our laboratory have shown contribution of extracellular calcium in the induction of CAA [START_REF] Bredeloux | Contribution of calcium to the catecholaminergic automatic activity of rat pulmonary veins[END_REF] as well as of TTX-sensitive sodium channels to the slope of slow depolarization between burst of CAA [START_REF] Malécot | A TTX-sensitive resting Na + permeability contributes to the catecholaminergic automatic activity in rat pulmonary vein[END_REF]. However, the trigger mechanism which should occur at relatively negative potentials [START_REF] Doisne | Catecholaminergic automatic activity in the rat pulmonary vein: electrophysiological differences between cardiac muscle in the left atrium and pulmonary vein[END_REF] remains still unknown. If low (T-type) or medium (Ca v 1.3) voltage-activated calcium channels were involved in triggering CAA, we hypothesized that rat PVCM should present a Ca 2+ current different from that found in LACM, in terms of type or amplitude. Therefore, the objective of our study was to examine the properties of low voltage activated (LVA) Ca 2+ currents in a large number of PV and LA CM. We report here that both rat PV and LA CM unexpectedly present a fast non-selective LVA cationic channel. Moreover, a large T-type Ca 2+ current which was not observed in LACM was present in about 14 % of PVCM.

METHODS

Experiments were carried out on male Wistar rats (350-480g; CER Janvier, Le Genest St Isle, France. All protocols have been approved by the local ethical committee (Comité d'Ethique en Expérimentation Animale Val de Loire n°019, Tours, France; approval reference number APAFIS#6649-2016090711251954 v2) and conform to the guidelines from Directive 2010/63/EU of the European Parliament. Additional information is given in the Supplemental Material.

Cardiomyocytes Isolation

Cardiac myocytes were enzymatically (liberase plus protease) isolated as previously described [START_REF] Malécot | A TTX-sensitive resting Na + permeability contributes to the catecholaminergic automatic activity in rat pulmonary vein[END_REF][START_REF] Pasqualin | Structural heterogeneity of the rat pulmonary vein myocardium: consequences on intracellular calcium dynamics and arrhythmogenic potential[END_REF] at 37°C from the left and right superior pulmonary veins (PV) and left atria. All procedures are summarized in the Supplemental material. After isolation, isolated CM were kept in normal Tyrode solution (in mM: 140 NaCl, 5.4 KCl, 1 MgCl 2 , 1.36 CaCl 2 , 0.33 NaH 2 PO 4 , 10 HEPES buffer and 11 glucose, pH adjusted to 7.4 with NaOH) until use. Only cardiomyocytes presenting clear cut striations were used in this study. Therefore, they did not correspond to either vascular smooth muscle or endothelial cells that one would expect to find in the pulmonary vein (i.e, [START_REF] De Almeida | The cardiac muscle in the pulmonary vein of the rat: a morphological and electrophysiological study[END_REF][START_REF] Hosoyamada | Structural organization of pulmonary veins in the rat lung, with sppecail emphasis on the musculature consisting of cardiac and smooth muscles[END_REF][START_REF] Klavins | Demonstration of striated muscle in the pulmonary veins of the rat[END_REF][START_REF] Mueller-Koecker | Of rodents and humans: a light microscopic and ultrastructural study on cardiomyocytes in pulmonary veins[END_REF].

Patch-clamp recording of the calcium current I Ca

The whole-cell recording configuration was used to study I Ca at room temperature (22-25°C; see Supplementary Material for details). The cell under study was directly superfused by gravity (small capillary -300 µm internal diameter -positioned within less than [START_REF] Hosoyamada | Structural organization of pulmonary veins in the rat lung, with sppecail emphasis on the musculature consisting of cardiac and smooth muscles[END_REF] Membrane capacitance (C m ) was calculated by integration of the capacitive currents in response to a series of 10 hyperpolarizing pulses applied from -70 mV (amplitude and duration: 10 mV, 10 ms) and then averaged. Calcium current amplitude was measured as peak inward current minus current flowing at the end of the 300 ms activating pulse, except when otherwise stated.

All data were normalised to the cell capacitance C m and are expressed as mean ± SE in pA/pF or nS/pF when quantified.

Patch-clamp recording of the fast sodium current I Na

The whole cell sodium current was recorded from a holding potential of -110 mV at room temperature in conditions where K + and Ca 2+ currents were blocked, as previously described [START_REF] Malécot | A TTX-sensitive resting Na + permeability contributes to the catecholaminergic automatic activity in rat pulmonary vein[END_REF]

Relative permeability ratios and ion activities calculations

Relative permeability ratios for Ca 2+ over Na + (P' Ca /P Na ) or K + (P' Ca /P K ) were calculated from the shifts of the apparent reversal potential of the Ca 2+ current induced by the addition of Na + or K + to the extracellular solution, using a modified Goldman, Hodgkin and Katz equation (GHK) to take into account both divalent and monovalent ions, as described by Lewis [START_REF] Lewis | Ion-concentration dependence of the reversal potential and the single channel conductance of ion channels at the frog neuromuscular junction[END_REF]. Ion activity coefficients were calculated in each condition with the Davies equation S6 (see Supplementary Material for details).

Chemicals and Reagents

Chemicals were of reagent grade and obtained from Sigma-Aldrich (Saint Quentin Fallavier, France), Tocris, (R&D Systems, Lille, France) and Alomone Labs Ltd, (Jerusalem, Israel). Reagents were prepared as 10 -2 M stock solutions in distilled water or DMSO, as needed, and kept frozen (-20°C) until just prior to dilution in saline solutions.

Statistics

Data are presented as mean values  SE, with the number of experiments indicated as n.

Statistical significance was assessed with Mann-Whitney, Wilcoxon, Student's t-test or Fisher exact test where appropriate (SigmaStat V3.5, Systat Software Inc., Point Richmond, CA, USA). A P<0.05 value was considered to be significant.

RESULTS

In pulmonary vein cardiomyocytes (PVCM) held at a negative potential in Na + and K + -free extracellular solution containing 2 or 5 mM Ca 2+ , several types of Ca 2+ current could be activated by relatively small depolarizations. They could be distinguished by their kinetics: either very fast (FLVA, Fig. 1a1) or slow activating (SLVA, Fig. 1a2) and inactivating currents, or a mixture of both fast activating and slow inactivating current (FSLVA, Fig. 1a3). As a consequence, three types of currentvoltage (IV) relationships were observed (Fig. 1b andS1). Whereas SLVA-I Ca had a classical IV curve (open circles in Figs. 1b andS1a), that of FLVA-I Ca (filled triangles) presented an unusual shape: peak current density was maximum at around -35 mV, decreased between -25 to -20 mV and finally increased for more positive potentials. This is a characteristic of the IV curve of the TTX-sensitive calcium current, I Ca(TTX) (see discussion). The third type of IV curve (filled circles), typical of the presence of a T-type Ca 2+ current, was found in only a minority of PVCM: 21 out of 148 CM in the presence of 2 mM Ca 2+ and 10 out of 99 CM in the presence of 5 mM. In left atrial (LA) CM, FLVA-I Ca could also be recorded (Fig. S1b), but the T-type Ca 2+ current, if present, was quite small (filled circles in Fig. S1b). The aim of the following experiments was to characterize these three low voltageactivated currents components in PVCM.

Characteristics of the fast LVA current in PVCM

In 2 mM extracellular Ca 2+ , the fast LVA Ca 2+ current (FLVA-I Ca ) was observed in 63 out of 148 PVCM. It was activated in a narrow range of low negative voltages between  -55 to -30 mV. Its amplitude was relatively small ( -0.6 pA/pF), representing  13 % of maximum peak Ca 2+ current recorded at +15 mV (I Camax ; Table 1 andFig S1a). In 19 CM with no apparent FLVA-I Ca in 2 mM Ca 2+ , increasing the extracellular Ca 2+ concentration to 5 mM revealed its presence in 14 of them. In another set of experiments in the direct presence of 5 mM extracellular Ca 2+ , FLVA-I Ca was found in 45 out of 80 PVCM (Fig. 1a1 andb). Thus, FLVA-I Ca was not present in all PVCM, unlike the classical Ltype current. Compared with data recorded with 2 mM Ca 2+ , in 5 mM Ca 2+ the peak current voltage was  6 mV more positive and its amplitude was larger ( 2.5 fold) as well as its ratio to I Camax recorded at +20 mV ( 2 fold; Table 1). The apparent reversal potential was  11.3 mV positively shifted, as expected for a current carried only by Ca 2+ (theoretical shift of 11.7 mV at 25°C). Neither the peak current amplitude nor its ratio to I Camax depended on the CM size approximated by its membrane capacitance in both conditions (Fig. S2). There was also no correlation between peak FLVA-I Ca and I Camax density. FLVA-I Ca was also found in 50 out of 79 LACM in 2 mM Ca 2+ . Its activation threshold, peak voltage and apparent reversal potential were not significantly different but its amplitude was smaller than in PVCM (Table 1 and Fig. S1b). In both PV and LA CM, the presence of FLVA-I Ca could not be ascribed to a specific CM phenotype.

Steady-state availability of the current was determined with a classical double-pulse voltageclamp protocol (Fig. S3) . In the presence of 5 mM extracellular Ca 2+ , the availability curve determined with a test potential to -40 mV (~ maximum FLVA current amplitude) was best described with a double Boltzmann equation in 4 out of 6 PVCM and a single Boltzmann in the two others. As there was no difference in the half inactivation voltages V 0.5 of the more negative component between the two groups of cells, values were pooled. In these conditions, the mean V 0.5 values were -85.57 ± 0.90 mV (n=6) and -65.31 ± 2.19 mV (n=4), with respective slope factors of -4.38 ± 0.23 mV and -8.00 ± 0.70 mV (Fig. S3). When present, the less negative component accounted for 21.89 ± 7.56 % of the total current at -40 mV. These results suggest either that FLVA-I Ca presented two voltage-dependent inactivating components or that a second smaller Ca 2+ current, with less negative V 0.5 , was concurrently activated during the test pulse to -40 mV (see below).

Figure 2a1 shows that FLVA-I Ca was entirely blocked by 10 µM TTX over the whole range where the current could be recorded (Fig. 2a3). Thus, it might correspond to the TTX-sensitive calcium current, I Ca(TTX) , as already suggested by the shape of its IV curve (Fig. 1b). In some CM, TTX application unveiled a slower activating and inactivating calcium current component (Fig. 2a2).

Ranolazine, another Na + channel blocker, had no effect at 10 µM (Fig. 2b and Table S2), a concentration that we have shown to block 28 % of the fast Na v 1.5 current in rat PVCM [START_REF] Malécot | A TTX-sensitive resting Na + permeability contributes to the catecholaminergic automatic activity in rat pulmonary vein[END_REF].

FLVA channel permeability to divalent cations

Figure 3a shows that removal of external Ca 2+ completely suppressed FLVA-I Ca , thus confirming its Ca 2+ nature, as already suggested by the shift of its apparent reversal potential when the extracellular Ca 2+ concentration was increased from 2 to 5 mM. If the FLVA channel was a Ca 2+ selective ion channel, then the classical Ca 2+ substituting ions should also permeate the channel and give rise to a sizable current. As shown in Fig. 3b and3c1, equimolar substitution of Ca 2+ by Sr 2+ or Ba 2+ entirely suppressed the fast current(n=6 and 10, respectively). In some CM, a small slower activating and inactivating current component remained in the presence of Ba 2+ (Fig. 3c2) or Sr 2+ , suggesting that it might correspond to a T-type Ca 2+ current. Thus, FLVA-I Ca and T-type Ca 2+ current could be simultaneously present and/or activated in some PVCM (see also Fig. 2a2).

The FLVA channel is a non-selective cationic channel: permeability to monovalent cations

The negative apparent reversal potential and voltage range of activation of the FLVA current suggested that the underlying channel might be non-selective for Ca 2+ . Therefore, we next examined whether monovalent cations naturally present in blood or physiological solutions could permeate the channel in the presence of Ca 2+ . Figures 4a and4b respectively illustrate that the addition of either Na + (1 and 3 mM) or K + (5 and 10 mM) to the extracellular medium actually increased FLVA current.

As shown Fig. S4, it should be noted that, for a given monovalent ion concentration, increase of the current amplitude was greater with Na + than with K + . Shifts of the apparent reversal potentials of the currents induced by increasing either Na + or K + concentrations (Na + from 1 to 3 mM:  13 mV and K + from 5 to 10 mM:  6 mV) in the presence of 5 mM Ca 2+ were much less than expected for a current carried only by Na + or K + (Table S1; theoretical shifts of 28.2 mV and 17.8 mV at 25°C, respectively), thus suggesting that Ca 2+ and Na + or K + can simultaneously flow through the channel. Monovalent cations alone could also permeate the channel as shown Fig. 4c for K + . In these experiments, when 5 mM Ca 2+ was replaced by equimolar Mg 2+ to keep the divalent ions concentration constant, the current recorded in the presence of both Ca 2+ and K + was rapidly reduced and only an inward current carried by K + remained (n=8). This current was similar in amplitude to that carried by Ca 2+ alone.

The relative permeability for Ca 2+ over Na + or K + were therefore calculated using a modified GHK equation for bi-ionic conditions (see Supplementary Material) to take into account the presence of the divalent cation Ca 2+ . P' Ca /P K values were not significantly different when calculated from the shift of the apparent reversal potential (see Supplementary Material) induced by the addition of either 5 or 10 mM KCl (respectively: 1.944 ± 0.447 and 1.812 ± 0.299, n=7 for each; Table S1; Mann-Whitney rank sum test, P=0.795). Therefore, when all values were pooled, the mean P' Ca /P K was equal to 1.878 ± 0.249 (n=14). The relative permeability P' Ca /P Na calculated after adding 1 mM NaCl amounted to 2.254 ± 0.508 (n=6), and was not significantly different from the calculated P' Ca /P K (Mann-Whitney rank sum test, P=0.483). Both values were not significantly different from a value of 2 (z-test; P=0.291 for P' Ca /P Na and 0.694 for P' Ca /P K ). Thus, P' Ca /P K = P' Ca /P Na  2. It should be noted that in the presence of 3 mM Na + , P' Ca /P Na markedly decreased to  0.2 (Table S1), likely because of Na + flow through the fast Na v 1.5 channel whose activation voltage overlaps with that of FLVA.

Channel conductance in the presence of 5 mM Ca 2+ alone (control conditions) or 5 mM Ca 2+ plus Na + or K + were calculated from the peak currents and apparent reversal potentials for each CM, as described in the Supplementary Material (Eq. S2). In control conditions and with 1 mM NaCl added (Fig. 5a), single Boltzmann functions were sufficient to best describe the conductance (control: V 0.5 of -39.86 ± 2.90 mV with k= 2.72 ±0.24 mV, n=5; +1 mM NaCl: V 0.5 of -40.85 ± 1.32 mV, with k = 3.62 ± 0.23 mV, n=5; P<0.05 for k values). However, a double Boltzmann function was necessary with 3 mM NaCl added (respectively, V 0.5 of -44.01 ± 1.84 mV and -32.87 ± 2.48 mV, k values of 3.05 ± 0.12 mV and 3.44 ± 0.53 mV, and amplitudes of 0.37 ± 0.04 nS/pF and 0.12 ± 0.02 nS/pF, n=5), likely because of the overlap of Na v 1.5 and FLVA channels activation voltages. On the contrary, in control conditions and in the additional presence of either 5 or 10 mM KCl, channel conductance was always best described with a single Boltzmann function (Fig. 5b) with respective V 0.5 of -44.15 ± 1.56 mV, -41.33 ± 1.75 mV and -43.88 ± 1.97 mV (n=8). Only k values were significantly different from control (respectively: 2.51 ±0.12 mV, 4.35 ± 0.16 mV and 3.91 ± 0.24 mV).

Figure 5c shows that the main negative component of channel conductance (V 0.5 around --40 to -45 mV) saturated for added monovalent concentration as low as 5 mM. From the Langmuir isotherm (Y = Y max /(1 + (K D /[Ion]) p ) adjusted to the mean data points, a K D value of 0.78 mM with p = 1.24 could be determined as well as a maximum total conductance of 0.43 nS/pF in the presence of 5 mM Ca 2+ and either Na + or K + .

Activation and inactivation kinetics of FLVA and fast Na current I Na

To determine whether FLVA-I Ca might just correspond to Ca 2+ flowing through fast Na + channels since there is overlap of their activation voltage ranges, their kinetics of activation and inactivation were compared. As shown in Fig. S5a1, activation of FLVA-I Ca assessed by its time to peak (n=14) was significantly slower than that of I Na (n=12) at all voltages. Inactivation time course of I Na was best described by the sum of two exponentials whose time constant voltage relationships are shown in Fig. S5a2. The major component of I Na was also the faster. On the contrary, the FLVA current had a mono exponential decay at all voltages. The inactivation rate of FLVA-I Ca lay between those of I Na , as shown in Fig. S4a2. The addition of either Na + or K + did not significantly modify the time to peak current (Fig. S5b1) or decay time constants (Fig. S4b2). Although these parameters values were slightly decreased, they remained statistically different from those of I Na .

Pharmacological characterization of the FLVA channel

The pharmacological data presented in Table S2 and Fig. 6 show that beside its inhibition by TTX, FLVA-I Ca was sensitive to dihydropyridines, being markedly reduced by 5 µM nifedipine (L-type Ca channel blocker; Fig. 6a) and increased by 300 nM (±)-BayK 8644 (L-type Ca channel activator; Fig. 6b). On the other hand, it was not reduced by 200 nM (+)-cis-diltiazem, a benzothiazepine derivative Ca 2+ channel blocker (n=6). Norepinephrine (10 µM) also increased the current (Table S2), an effect being reversed by 5 µM atenolol. Cirazoline (1 µM), a specific α 1 -adrenergic agonist, had no effect on the current (n=5). All these effects were reversible upon washout.

Multiple components of LVA Ca 2+ current in rat PVCM

Study of the steady-state availability of the FLVA determined with a test pulse to -40 mV has revealed the possible existence of the simultaneous activation of a second type of Ca 2+ current representing about 20 % of the total current with a less negative half inactivation voltage V 0.5 . (~-65 mV compared to ~-85 mV; Fig. S3). To confirm the presence of this current, we used a test pulse of -25 mV, close to FLVA apparent reversal potential. Two Boltzmann functions were still necessary to best describe the current availability (Fig. S3). The main component, representing about 92 % of the total current, had a mean V 0.5 . value of -69.34 ± 0.14 mV (n=4) which was not statistically different from that of the small component found with the test pulse of -40mV. These V 0.5 .values are those expected for T-type Ca 2+ channels. The minor component, with a mean V 0.5 . value of -26.73 ± 3.20 mV (n=4) corresponded to activation of L-type Ca 2+ channels, as shown with a test pulse to +15 mV (mean V 0.5 . values of -66.78 ± 1.44 mV and -24.38 ± 1.16 mV (n=10) for the small and major components, respectively; Fig. S3). Similar results were also obtained in LACM. No current component with a mid-inactivation voltage compatible with that of Ca v 1.3 channel (~-40 to -45 mV; [START_REF] Mangoni | Functional role of L-type Cav1.3 Ca 2+ channels in cardiac pacemaker activity[END_REF]) could be isolated.

To test whether the Ca 2+ current which overlapped with the non-selective cationic FLVA current was indeed a T-type Ca 2+ current, as suggested by the V 0.5 .values of the steady-state inactivation curves, we used two reportedly specific blockers of Ca v 3.1 and Ca v 3.2. Surprisingly, FLVA-I Ca was significantly increased by TTA-A2 (10 nM, n=11 and 100 nM, n=6 -Table S2 and Fig. 6c) and NiCl 2 (40 µM, n=9, Table S2 and Fig. 6d). Similar results were also obtained in LACM (not shown).

Nevertheless, although the number of PVCM presenting a marked shoulder in their current-voltage (IV) relationships at negative potentials (maximum around -25 mV; see Fig. 1b, filled circles and Fig. S6) is quite low, we were able to apply 100 nM TTA-A2 to two such PVCM. Fig. S6 shows that TTA-A2 significantly reduced the shoulder amplitude at -25 mV, suggesting the effective presence of an underlying T-type Ca 2+ current. The Ca 2+ current was also markedly reduced by 40 µM NiCl 2 (-84.0 ± 4.5 % and -78.6 ± 4.8 % at -25 mV and +15 mV, respectively, n=9).

To confirm the presence of Ca v 3.x T-type Ca 2+ current in PVCM, besides the TTX-sensitive FLVA-I Ca , currents recorded with 200 ms pre-pulses to -40 mV were subtracted from the whole Ca 2+ current recorded from -100 mV in the same cells. In both PV and LA CM, three groups of CM were first visually distinguished on the basis of the shape of their IV curve from -100 mV (see Fig. 1b), before subtraction. Figure 7 shows that the IV curves of the -40 mV pre-pulse difference currents markedly differ in PV (Fig. 7a) and LA (Fig. 7b) CM. In PVCM, a small voltage-dependent pre-pulsesensitive Ca 2+ current could be recorded positive to -50 mV (Fig. 7a, open circles). As expected from its inactivation curve, FLVA-I Ca was sensitive to the -40 mV pre-pulse and its IV relationship globally retained its "V"shape (filled triangles). These currents were much smaller in LACM (Fig. 7b). Finally, a large prepulse-sensitive current was present in PVCM which presented a shoulder in their -100 mV IV curve (filled circles). This last current was barely detectable in LACM. Thus, three types of LVA Ca 2+ currents exist among the PVCM population and are much larger than those found in the LACM population.

DISCUSSION

Three principles points arise from this investigation. (1) Cardiomyocytes (CM) isolated from rat pulmonary vein (PV) and left atrium (LA) present a fast low voltage-activated non selective cationic current (carried by Na + , K + and Ca 2+ ) which is sensitive to TTX and dihydropyridines. However, neither Ba 2+ nor Sr 2+ could substitute as permeant species for Ca 2+ . (2) A large T-type Ca 2+ current was found in a subpopulation of rat PVCM but not in LACM. (3) There was no evidence for the presence of a Ca 2+ current through Ca v 1.3 channels.

A low voltage activated Ca 2+ current blocked by TTX (I Ca(TTX) ) has been described in rat hippocampal CA1 neurones [2,[START_REF] Takahashi | Effects of Ca 2+ antagonists and antiepileptics on tetrodotoxin-sensitive Ca 2+ -conducting channels in isolated rat hippocampal CA1 neurons[END_REF] and cardiac tissues from the ventricle of rat [1,3,[START_REF] Bodi | Tetrodotoxin-sensitive Ca 2+ currents, but no T-type currents in normal, hypertrophied, and failing mouse cardiomyocytes (2016)[END_REF][START_REF] Chen-Izu | I Ca(TTX) channels are distinct from those generating the classical Na + current[END_REF][START_REF] Heubach | T-type and tetrodotoxin-sensitive Ca 2+ currents coexist in guinea pig ventricular myocytes and are both blocked by mibefradil[END_REF][START_REF] Pidoplichko | Two different tetrodotoxin-separable inward sodium currents in the membrane of isolated cardiomyocytes[END_REF], guinea-pig [START_REF] Cole | Ca 2+ permeation through Na + channels in 0.guinea pig ventricular myocytes[END_REF][START_REF] Heubach | T-type and tetrodotoxin-sensitive Ca 2+ currents coexist in guinea pig ventricular myocytes and are both blocked by mibefradil[END_REF], mouse [START_REF] Bodi | Tetrodotoxin-sensitive Ca 2+ currents, but no T-type currents in normal, hypertrophied, and failing mouse cardiomyocytes (2016)[END_REF][START_REF] Wang | Regional differences in the expression of tetrodotoxin-sensitive inward Ca 2+ and outward Cs + /K + currents in mouse and human ventricles[END_REF], and from human atrium [START_REF] Lemaire | Tetrodotoxin-sensitive Ca 2+ and Ba 2+ currents in human atrial cells[END_REF] and ventricle [START_REF] Wang | Regional differences in the expression of tetrodotoxin-sensitive inward Ca 2+ and outward Cs + /K + currents in mouse and human ventricles[END_REF]. To our knowledge, our study reports for the first time its existence in rat PVCM and LACM. The ionic nature of this current is still a matter of debate: a novel Ca 2+ channel [START_REF] Lemaire | Tetrodotoxin-sensitive Ca 2+ and Ba 2+ currents in human atrial cells[END_REF], a Na + channel distinct from Na v 1.5 [3,[START_REF] Chen-Izu | I Ca(TTX) channels are distinct from those generating the classical Na + current[END_REF], Na + channels permeant to Ca 2+ [START_REF] Guatimosim | Molecular identification of a TTX-sensitive Ca 2+ current[END_REF][START_REF] Wang | Regional differences in the expression of tetrodotoxin-sensitive inward Ca 2+ and outward Cs + /K + currents in mouse and human ventricles[END_REF].

Although blocked by TTX, the FLVA-I Ca reported in our study markedly differs from I Ca(TTX) described in human atrial cells [START_REF] Lemaire | Tetrodotoxin-sensitive Ca 2+ and Ba 2+ currents in human atrial cells[END_REF] in that neither equimolar Ba 2+ nor Sr 2+ could substitute for Ca 2+ . However, Ba 2+ was also found to be unable to replace Ca 2+ in rat ventricular cells [START_REF] Guatimosim | Molecular identification of a TTX-sensitive Ca 2+ current[END_REF], as well as in HEK-293 cells expressing the cardiac Na v 1.5 channel (SCN5A; [START_REF] Guatimosim | Molecular identification of a TTX-sensitive Ca 2+ current[END_REF]), two preparations in which I Ca(TTX) was present. In rat CA1 hippocampal neurones, replacement of 10 mM Ca 2+ by equimolar Sr 2+ and Ba 2+ induced much smaller currents (amplitude ratio Ca 2+ :Sr 2+ :Ba 2+ = 1.00:0.33:0.005; [2]). Because Cs + can permeate I Ca(TTX) channel [START_REF] Chen-Izu | I Ca(TTX) channels are distinct from those generating the classical Na + current[END_REF][START_REF] Guatimosim | Molecular identification of a TTX-sensitive Ca 2+ current[END_REF][START_REF] Wang | Regional differences in the expression of tetrodotoxin-sensitive inward Ca 2+ and outward Cs + /K + currents in mouse and human ventricles[END_REF] and that our pipette solution was Cs + -based, we cannot exclude the possibility that a very small current carried by either Sr 2+ or Ba 2+ could have been masked by an overlapping outward Cs + current, though inspection of the current records (Fig. 3) makes this unlikely. FLVA-I Ca differs also from I Ca(TTX) found in guinea-pig ventricular cells [START_REF] Cole | Ca 2+ permeation through Na + channels in 0.guinea pig ventricular myocytes[END_REF] as it was sensitive to dihydropyridines. There again, FLVA-I Ca shared similitudes with I Ca(TTX) found in rat CA1 hippocampal neurones which are concentration-dependently inhibited by several dihydropyridines [START_REF] Takahashi | Effects of Ca 2+ antagonists and antiepileptics on tetrodotoxin-sensitive Ca 2+ -conducting channels in isolated rat hippocampal CA1 neurons[END_REF]. Thus, since the reported I Ca(TTX) properties seem to be tissue-dependent, it is not possible to affirm that FLVA-I Ca is really I Ca(TTX) but only that it shares some of its reported properties. Therefore, in this study, the term of FLVA current will be retained.

The negative apparent reversal potential of FLVA-I Ca is in agreement with the non-selective nature of the channel. Indeed, we found that Na + or K + ions can simultaneously flow through FLVA channels with Ca 2+ and that extracellular K + ions alone were also able to carry an inward current through the channel. All these observations are also in agreement with results for I CaTTX , showing that monovalent cations can also give rise to sizable currents, either inwardly (Na + , Cs + ) or outwardly (Cs + , K + ) directed [1,2,[START_REF] Chen-Izu | I Ca(TTX) channels are distinct from those generating the classical Na + current[END_REF][START_REF] Guatimosim | Molecular identification of a TTX-sensitive Ca 2+ current[END_REF][START_REF] Wang | Regional differences in the expression of tetrodotoxin-sensitive inward Ca 2+ and outward Cs + /K + currents in mouse and human ventricles[END_REF]. However, since K + is only weakly permeant through Na + channels (P K /P Na ~0.03 to 0.08; [START_REF] Favre | On the structural basis for ionis selectivity among Na + , K + , and Ca 2+ in the voltage-gated sodium channel[END_REF][START_REF] Hille | Ion channels of excitable membranes (3rd edition)[END_REF][START_REF] Sun | On the structural basis for size-selective permeation of organic cations through the voltage-gated sodium channel. Effects of Alanine mutations at the DEKA locus on selectivity, inhibition by Ca 2+ and H + , and molecular sieving[END_REF], FLVA-I Ca is unlikely to result from Ca 2+ flow through classical Na + channels. Moreover, the FLVA current had slower activation and inactivation kinetics than I Na , even in the presence of Na + or K + .

To our knowledge, a FLVA channel permeant to Ca 2+ , Na + and K + giving rise to inward currents has not been reported in the literature. Thus, it would be tempting to classify this channel as a kind of voltage-gated transient receptor potential (TRP) channel, as it is now admitted that some of them present a voltage-dependent activation (for review, [START_REF] Nilius | Gating of TRP channels: a voltage connection?[END_REF]). The fact that we found in our experimental conditions that Ca 2+ was two times more permeant than Na + or K + suggests that the channel might derivate from a Ca 2+ channel rather than from a Na + channel [1,[START_REF] Chen-Izu | I Ca(TTX) channels are distinct from those generating the classical Na + current[END_REF][START_REF] Guatimosim | Molecular identification of a TTX-sensitive Ca 2+ current[END_REF][START_REF] Sha | An antisense oligonucleotide against H1 inhibits the classical sodium current but not I Ca(TTX) in rat ventricular cells[END_REF][START_REF] Wang | Regional differences in the expression of tetrodotoxin-sensitive inward Ca 2+ and outward Cs + /K + currents in mouse and human ventricles[END_REF]. This hypothesis is also supported by the saturation of the channel conductance for monovalent cation concentrations as low as 5 mM. Another possibility would be that Ca 2+ has partially blocked Na + and K + permeation through the channel.

Taking into account the screening effect of Ca 2+ , the mid-activation voltage (V 0.5 ) of FLVA conductance we determined in the presence of 5 mM Ca 2+ (-42.3 ± 1.5 mV, n=13) is similar to that of the negative ranolazine-sensitive component of Na + conductance we have found in PVCM in the presence of 1 mM Ca 2+ (~-50 mV; [START_REF] Malécot | A TTX-sensitive resting Na + permeability contributes to the catecholaminergic automatic activity in rat pulmonary vein[END_REF]). Since we show here that 10 µM ranolazine had no effect on FLVA-I Ca , these results strongly suggest that two types of channels with identical mid voltage of activation, i.e., FLVA channels and ranolazine-sensitive Na v 1.5 channels, contribute to the negative component of Na + channel conductance we have found [START_REF] Malécot | A TTX-sensitive resting Na + permeability contributes to the catecholaminergic automatic activity in rat pulmonary vein[END_REF]. This hypothesis is also supported by the observation that currents recorded in the presence of both Ca 2+ and Na + were larger than those in the presence of Ca 2+ and K + . Indeed, if only one type of channel were involved, current amplitudes in the presence of additional Na + or K + should be identical for a given monovalent concentration since Na + and K + permeabilities through FLVA channels are identical and that both ions have the same activity coefficient in solution. These findings also support the idea that FLVA channels are not just Na v 1.5 channels permeant to Ca 2+ and K + .

After rupture of the membrane patch, we noticed that FLVA-I Ca needed some time to appear (diffusion of the pipette solution into the cell) and to be stable in amplitude (not shown). Moreover, it was subject to use-dependent potentiation after a rest period, as found for Na v 1.6 [START_REF] Zhou | Use-dependent potentiation of the Na v 1.6 sodium channel[END_REF] and Ca 2+ channels [START_REF] Fedida | Mechanism of the use-dependence of Ca2+ current in guinea-pig myocytes[END_REF]. Taking into consideration these peculiarities during our experiments, we found that FLVA-I Ca was nevertheless not present in all CM, and that it was more frequently found in LACM (in 50 out of 79 CM in the presence of 2 mM Ca 2+ -63.3 %) than in PVCM (in 122 out of 228 CM in the presence of either 2 or 5 mM Ca 2+ -53.5 %). Lemaire et al [START_REF] Lemaire | Tetrodotoxin-sensitive Ca 2+ and Ba 2+ currents in human atrial cells[END_REF] also found that I Ca(TTX) was present in only 40 % of the human atrial cells they studied. This contrasts with the recently published observation that I Ca(TTX) was recorded in all myocytes isolated from adult mouse interventricular septum [START_REF] Wang | Regional differences in the expression of tetrodotoxin-sensitive inward Ca 2+ and outward Cs + /K + currents in mouse and human ventricles[END_REF], but with considerable variability in peak current densities among cells. We also found a great variability of peak FLVA-I Ca density in PVCM, especially in the presence of 5 mM Ca 2+ , which could not be explained by the CM size approximated by its membrane capacitance. Moreover, it was not possible to visually distinguish CM with or without FLVA current. Different levels of expression of the channel within the CM population might possibly contribute to the observed variability of FLVA-I Ca density.

Our study of LVA Ca 2+ channels in a large number of PV and LA CM has revealed that an important T-type Ca 2+ current was present in only 13.5 % of PVCM (31 out of 228 CM) and barely visible in LACM. Although the only T-type Ca 2+ channels mRNA found in rat atria are those of Ca v 3.1 and Ca v 3.2, Ca v 3.1 being the predominant form [START_REF] Lee | Cloning and expresssion of a novel member of the low voltage-activated T-type calcium channel family[END_REF][START_REF] Leuranguer | T-type calcium currents in rat cardiomyocytes during postnatal development: contribution to hormone secretion[END_REF], it was not possible to determine from our results to which type of Ca v 3.x channel this large current might correspond in PV. Indeed, using three different test potentials, we determined in the presence of 5 mM Ca 2+ V 0.5 values of -65 to -69 mV which were not statistically different. Taking into account the Ca 2+ screening effect, these values are similar to those reported for Ca v 3.1 and Ca v 3.2 (V 0.5 of ~-70 mv in the presence of 2 mM Ca 2+ ; [START_REF] Chemin | Specific contribution of human T-type calcium channel isotypes (α 1G , α 1H and α 1I ) to neuronal excitability[END_REF]).

The presence of this large T-type current only in PVCM contributes to define a large Ca 2+ window current in these cells and thus to facilitate Ca 2+ dependent arrhythmogenic activity. Our results from Ca 2+ channel availability are also not in favour of the presence of Ca v 1.3 channels which inactivate at less negative potentials than the T type channels [START_REF] Chemin | Specific contribution of human T-type calcium channel isotypes (α 1G , α 1H and α 1I ) to neuronal excitability[END_REF].

Testing the potential role of FLVA-I Ca as a trigger of CAA is not possible in the absence of specific blockers. In a previous study, we have shown that TTX was able to decrease CAA incidence, burst duration and to increase burst interval [START_REF] Malécot | A TTX-sensitive resting Na + permeability contributes to the catecholaminergic automatic activity in rat pulmonary vein[END_REF] and we concluded that all these effects could be attributed to Na + channels blockade. At present, we cannot exclude the participation of FLVA-I Ca to CAA. Indeed, at the concentration of 10 µM used to induce CAA [START_REF] Doisne | Catecholaminergic automatic activity in the rat pulmonary vein: electrophysiological differences between cardiac muscle in the left atrium and pulmonary vein[END_REF][START_REF] Malécot | A TTX-sensitive resting Na + permeability contributes to the catecholaminergic automatic activity in rat pulmonary vein[END_REF][START_REF] Maupoil | Ectopic activity in the rat pulmonary vein can arise from simultaneous activation of alpha1-and beta1-adrenoceptors[END_REF], NE increased both FLVA-I Ca (~2.5 times) and I Camax (~+31%, n=10) and this might also contribute to facilitate CAA. Although diltiazem did not block FLVA-I Ca , it suppressed CAA in a concentration-dependent manner probably by a direct action on I CaL [START_REF] Bredeloux | Contribution of calcium to the catecholaminergic automatic activity of rat pulmonary veins[END_REF]. Thus although FLVA-I Ca could modulate CAA, it is probably not the main trigger as it is also present in LA (albeit smaller) in which CAA cannot be induced [START_REF] Doisne | Catecholaminergic automatic activity in the rat pulmonary vein: electrophysiological differences between cardiac muscle in the left atrium and pulmonary vein[END_REF][START_REF] Maupoil | Ectopic activity in the rat pulmonary vein can arise from simultaneous activation of alpha1-and beta1-adrenoceptors[END_REF].

The PV myocardium is characterized by a marked structural heterogeneity in terms of tubular system, Ca v 1.2 channels localization and Ca v 1.2-ryanodine receptor coupling [START_REF] Pasqualin | Structural heterogeneity of the rat pulmonary vein myocardium: consequences on intracellular calcium dynamics and arrhythmogenic potential[END_REF] with functional consequences on Ca 2+ cycling. Our present study indicates that it might also be heterogeneous in terms of FLVA and Ca v 3.x channels distribution, which could therefore contribute to enhance Ca 2+ influx near the resting potential and to induce arrhythmias in some parts of the PV. Moreover, the presence of a large Ca v 3.x current in a small number of PVCM might underlie localization of ectopic foci in the PV myocardial sleeve, as reported in humans [START_REF] Arentz | High-density mapping of spontaneous pulmonary vein activity initiating atrial fibrillation in humans[END_REF][START_REF] Haïssaguerre | Spontaneous initiation of atrial fibrillation by ectopic beats originating in the pulmonary veins[END_REF] and more recently in rat PV [START_REF] Pasqualinc | Mapping of the catecholaminergic automatic activity from its foci within the pulmonary veins to the left atria in rat[END_REF]. Representative traces of Na + (a1) and K + (b1) permeation in the presence of 5 mM Ca 2 . Test pulses applied from the pre-pulse of -100 mV to -40 mV (a1) and -35 mV (b1). a2 and b2, mean IV curves.

Na + : n=6. K + : n=8. c, time course of a representative experiment illustrating permeation of K + ions through FLVA channel in the presence or absence of 5 mM Ca 2+ (Ca 2+ replaced by equimolar Mg 2+ ).

Test pulses applied from the pre-pulse of -100 mV to -35 mV at 0.2 Hz. Mean IV curves of the currents sensitive to 200 ms pre-pulses to -40 mV recorded in the presence of 2 mM Ca 2+ in the three groups of PVCM (a) and LACM (b). Each of these groups had been visually distinguished on the basis of the shape of their IV curve recorded from the normal pre-pulse to -100 mV (Fig. 1b). SLVA current () PVCM, n=4 and LACM, n=5. FLVA current () PVCM, n=7 and LACM, n=4. FSLVA current () PVCM, n=5 and LACM, n=10. through a Digidata 1200A interface (Axon Instruments) was used to control voltage and record currents. Cells were continuously held at -70 mV (holding potential, HP) and activating pulses were preceded by a 200 ms hyperpolarising pulse to -100 mV. The data, acquired at 5 kHz and filtered with an 8-pole lowpass Bessel filter at 2 kHz, were analysed offline with Clampfit.

The pipette and cell capacitances were compensated by 80%. In order to have an adequate control of the membrane voltage and to improve the clamp speed, care was taken to select relatively small cells (C m : 87.2±1.8 pF, n=148 for PV CM and 50.7±2.1 pF, n=79 for LA CM, in 2mM extracellular Ca 2+ ).

Chemicals and Reagents

Chemicals were of reagent grade and obtained from Sigma-Aldrich (Saint Quentin Fallavier, France). Ranolazine (Tocris, R&D Systems, Lille, France), tetrodotoxin (TTX; Alomone Labs Ltd, Jerusalem, Israel), (+)-cis-diltiazem and norepinephrine (NE; both from Sigma-Aldrich) were prepared as stock solutions in distilled water and kept frozen (-20°C) until just prior to dilution in saline solutions. Nifedipine, (±) BayK 8644, and TTA-A2 (Alomone Labs Ltd) were prepared as 10 -2 M stock solutions in DMSO. An equivalent concentration of DMSO was added to control solutions (the maximal final DMSO concentration of 0.05 % reached in the presence of 5 µM nifedipine had no effect per se). Liberase TM Research Grade and Protease XXIV were purchased from Sigma-Aldrich.

Current apparent reversal potential and channel conductance determinations

The apparent reversal potentials V rev of LVA currents were determined by linear extrapolation of the descending limb of the current-voltage relationship to zero current.

The theoretical variation V rev of the reversal potential of the current carried solely by ion X + (see Table S1) when X + extracellular concentration was increased from concentration 1 to concentration 2 was calculated with the equation:

𝑉 𝑟𝑒𝑣 = 𝑅𝑇 𝐹 𝑙𝑜𝑔 𝑒 [𝑋 + ] 2 [𝑋 + ] 1

Eq. S1

with the assumption that the intracellular X + concentration (due to X + influx) remained identical in both conditions.

Channel conductance was classically calculated from the peak current amplitude at each potential V and corresponding driving force with the equation:

𝑔 𝑖𝑜𝑛 = 𝐼 𝑖𝑜𝑛 (𝑉-𝑉 𝑟𝑒𝑣 )
Eq. S2

Conductance-voltage relationships were described by adjusting a single or double Boltzmann equation to the data points, as needed:

𝑔 𝑖𝑜𝑛 = 𝐴 1 (1+𝑒𝑥𝑝( 𝑉-𝑉 0.5,1 𝑘 1
))

+ 𝐴 2 (1+𝑒𝑥𝑝( 𝑉 -𝑉 0.5,2 𝑘 2
))

Eq. S3

with A i the amplitude, V 0.5,i the mid activation voltage and k i , the slope factor of component i (1 or 2).

Relative permeability ratios, ion activities and liquid junction potential calculations

Relative permeability ratios for Ca 2+ over Na + (P' Ca /P Na ) or K + (P' Ca /P K ) were calculated from the shifts of the apparent reversal potential of the Ca 2+ current induced by the addition of Na + or K + to the extracellular solution, using a modified Goldman, Hodgkin and Katz equation (GHK) to take into account both divalent and monovalent ions. Thus, with a new Ca 2+ permeability

𝑃 𝐶𝑎 ′ = 𝑃 𝐶𝑎 1+exp( 𝐹𝑉 𝑅𝑇 )
Eq. S4

as described by Lewis [1], the apparent reversal potential V o of the current in the presence of both Ca 2+ and a monovalent cation X + is given by the following equation:

𝑉 𝑜 = 𝑅𝑇 𝐹 𝑙𝑜𝑔 𝑒 [𝑋 + ] 𝑜 +4 ( 𝑃 ′ 𝐶𝑎 𝑃 𝑋 ) [𝐶𝑎] 𝑜 [𝑋 + ] 𝑖 +4 ( 𝑃 ′ 𝐶𝑎 𝑃 𝑋 ) [𝐶𝑎] 𝑖 exp( 𝐹𝑉 𝑜 𝑅𝑇 )
Eq. S5

where [ion] o and [ion] i are the extracellular and intracellular ion activities, respectively, and R, T and F have their usual meaning. Since thermodynamic ion activity rather than concentration should be used in this equation and considering the ionic strength of the extracellular solution, activity coefficients of Ca 2+ , Na + and K + were calculated in each condition with the following Davies equation:

𝑙𝑜𝑔 10  𝑖 = -0.509 𝑧 𝑖 2 √𝐼 1+ √𝐼 -0.3 I Eq. S6
with  i and z i the activity coefficient and the valence of the ion i, respectively, and I, the ionic strength of the extracellular solution.

From Eq. S4 and assuming that [Ca] i was negligible due to the presence of 10 mM EGTA in the pipette solution and that [X + ] i due to X + influx remained constant when X + concentration was increased from concentration 1 to concentration 2, the relative permeability P' Ca over P X can be calculated with the equation:

𝑃 𝐶𝑎 ′ 𝑃 𝑋 = [𝑋 + ] 𝑜,2 𝑒𝑥𝑝( 𝑉 𝑟𝑒𝑣 𝐹 𝑅𝑇 ) -[𝑋 + ] 𝑜,1 4 [𝐶𝑎] 𝑜 (1-𝑒𝑥𝑝( 𝑉 𝑟𝑒𝑣 𝐹 𝑅𝑇 ))
Eq. S7

Liquid junction potential was determined with the calculator provided in pClamp 9. Its variation due to solutions changes were less than 1 mV (and also less than the error possibly made on the apparent current reversal determination) and were therefore ignored.

Supplementary figures S1 to S6

  µm above the cell and allowing fast solution changes) with a solution containing (in mM): 144 TEACl, 2 or 5 CaCl 2 , 1 MgCl 2 , 5 4-aminopyridine, 10 HEPES buffer, and 11 glucose (pH 7.4 with TEAOH). The pipette solution contained (in mM): 115 DL-aspartic acid, 10 TEACl, 2 MgCl 2 , 10 HEPES buffer, 10 EGTA, 5 Mg-ATP, and 5 diTris-phosphocreatine (pH 7.25 with CsOH leading to a final Cs + concentration of 137 mM). Cells were continuously held at -70 mV (holding potential, HP) and activating pulses were preceded by a 200 ms hyperpolarising pulse to -100 mV. The stimulation frequency was 0.2 Hz.

  . The extracellular solution contained (in mM): 110 TEACl, 30 NaCl, 1 CaCl 2 , 1 MgCl 2 , 2 CoCl 2 ; 0.1 GdCl 3 , 5 4aminopyridine, 10 HEPES buffer, and 11 glucose (pH 7.4 with TEAOH). The pipette solution contained (in mM): 110 CsCl, 30 TEACl, 7 NaCl, 10 HEPES buffer, 10 EGTA, and 5 Mg-ATP (pH 7.3 with CsOH).

Figure 1 .

 1 Figure 1. Low voltage activated (LVA) Ca 2+ currents in PVCM in the presence of 5 mM Ca 2+ . a1, fast activating and inactivating (FLVA) current; a2, slow activating and inactivating current (SLVA); a3, fast activating and slow inactivating current (FSLVA). The test pulses were applied from the prepulse voltage of -100 mV to -35 mV (a1 and a3) and -40 mV (a2). The symbols above each trace refer to the mean current voltage (IV) relationships shown in b. Data points are mean ± SE of n PVCM. (), n=12; (), n=37; (), n=10.

Figure 2 .

 2 Figure 2. TTX-sensitive, ranolazine insensitive FLVA current in PVCM. a, representative recordings of the effects of 10 µM TTX on the FLVA current recorded in the presence of 5 mM Ca 2+ . a1, complete block and a2, unveiling of a slower activating and inactivating Ca 2+ current component. Test pulses applied from the pre-pulse of -100 mV to -30 mV (a1) and -35 mV (a2). a3, mean current voltage (IV) relationships of the global calcium current in control conditions and in the presence of 10 µM TTX, n=8. b, lack of effect of 10 µM ranolazine (Rano; b1, test pulse from -100 mV to -35 mV) in the presence of 5 mM Ca 2+ . b2, mean IV curves in control conditions and in the presence of 10 µM Rano (n=6).

Figure 3 .

 3 Figure 3. Divalent ion permeation through LVA channels in PVCM. a, external Ca 2+ -dependence of the FLVA current. b and c, representative traces of the effects of Sr 2+ (b) or Ba 2+ (c) equimolar substitution for Ca 2+ on the current. In c2, Ba 2+ substitution for Ca 2+ reveals a slower activating and inactivating current component. Test pulses applied from the pre-pulse of -100 mV to -40 mV (a) or -35 mV (b and c).

Figure 4 .

 4 Figure 4. Permeability to monovalent cations of the FLVA channel in PVCM.

Figure 5 .

 5 Figure 5. Conductance of the FLVA channel. a and b, voltage-dependence of the FLVA channel conductance in 5 mM Ca 2+ control conditions and in the additional presence of either Na + (a, n=6) or K + (b, n=8). Smooth lines represent adjustments of Boltzmann functions to the mean data points, as described in the text. c, channel conductance to Na +

Figure 6 .

 6 Figure 6. Sensitivity of the FLVA current in PVCM to L-and T-type Ca 2+ channel blockers. a and b, dihydropyridines sensitivity. a1 and b1, representative traces of the effects of 5 µM Nifedipine (Nif) and 0.3 µM BayK8644 (BayK) on the currents recorded from the pre-pulse of -100 mV to -35 mV and -40 mV, respectively. a2 and b2, mean IV curves in control conditions with 5 mM Ca 2+ + 5 mM K + and then in the additional presence of 5 µM Nif (n=5) or 0.3 µM BayK (n=6), respectively. (*): P< 0.05 vs. control. c and d, representative traces of the effects of 0.1 µM TTA-A2 and 40 µM NiCl 2 , respectively, on the FLVA current recorded in the presence of 2 mM Ca 2+ . Test pulses applied from the pre-pulse of -100 mV to -45 mV. In c, C and T represent control and TTA-A2 traces, respectively.

Figure 7 .

 7 Figure 7. -40 mV pre-pulse-sensitive Ca 2+ currents.

  Figure 4

Figure 5

 5 Figure 5graphics program OriginPro 7

Figure 7 graphics

 7 Figure 7

Fig

  Fig S1 Low voltage activated (LVA) Ca 2+ currents from -100 mV in PVCM and LACM in the presence of 2 mM Ca 2+ . a, Mean current voltage (IV) relationships of the fast activating and inactivating (FLVA) current (, n=63), slow activating and inactivating current (SLVA; , n=43) and fast activating and slow inactivating current (FSLVA; , n=21) in PVCM. b, same as a, but for LACM (, n=29; , n=33; , n=11. Data points are mean ± SE of n PVCM or LACM.

Fig S2 :

 S2 Fig S2: Peak FLVA-I Ca in rat PVCM vs. CM size.Peak FLVA-I Ca does not depend on the CM size approximated by the membrane capacitance (a), as well as its ratio to maximum peak I Ca , I Camax (b), either in the presence of 2 mM (black symbols) or 5 mM (red symbols) Ca 2+ .

Fig. S3 :Fig. S4 :

 S3S4 Fig. S3: Steady-state availability of the Ca 2+ current recorded at three different test potentials in PVCM. The protocol used is shown on the upper part of the figure. Test potentials correspond to nearly maximum FLVA current (-40 mV, n=6), apparent reversal potential of FLVA current and prominent shoulder of the current-voltage relationship (-25 mV, n=4 -see Fig.S6) and maximum peak I Ca current (+15 mV, n=10). Smooth lines represent fit to the mean data points of double Boltzmann equations with respective half-voltage of inactivation V 0.5 indicated near each curve.

Fig. S5 .

 S5 Fig. S5. Kinetics of activation and inactivation of the fast Na (I Na ) and FLVA currents. Time to peak (a1) and inactivation time constants (a2) voltage relationships of the fast Na (I Na ) and FLVA currents recorded in the presence of 5 mM Ca 2+ . Data are mean values ± SE (I Na : n=12; FLAV-I Ca : n=14). (*) denote statistical difference between I Na and FLVA current (Mann-Whitney rank sum test or Student's t-test where appropriate). Time to peak (b1) and inactivation time constants (b2) voltage relationships of the FLVA current in control conditions (5 mM Ca 2+ ) and in the additional presence of Na + (n=6) or K + (n=8).

  Figure S3

Table 1 : Fast LVA current characteristics in PV and LA CM

 1

				PVCM			LACM
		2 mM [Ca	2+ ] o (n)	5 mM [Ca	2+ ] o (n)	P (2 vs 5 mM)	2 mM [Ca	2+ ] o (n)	P (LA vs PV CM)
	Threshold (mV)	-53.85 ± 0.61 (63)	-53.22 ± 0.95 (45)	0.337	-54.64 ± 0.55 (50)	0.437
	Peak voltage (mV)	-43.33 ± 0.54 (63)	-37.55 ± 0.96 (45)	< 0.001	-41.10 ± 2.55 (50)	0.251
	Peak amplitude
	(pA/pF) -0.584 ± 0.067 (63)	-1.556 ± 0.201 (45)	< 0.001	-0.353 ± 0.035 (50)	0.012
	(% of I Camax )	13.45 ± 1.61 (63)	26.1 ± 3.6 (45)	< 0.001	9.95 ± 1.07 (50)	0.189
	Apparent V rev (mV)	-31.58 ± 0.56 (58)*	-20.22 ± 1.03 (45)	< 0.001	-32.10 ±0.64 (50)	0.542
	PVCM: pulmonary vein cardiomyocytes; LACM, left atrial cardiomyocytes. V rev : current reversal
	potential. P values for Mann-Whitney Rank Sum test. (*) Apparent V rev was too small in 5 PV CM to
	be accurately determined

ACKNOWLEDGMENTS

The author is indebted to Dr Ian Findlay for helpful discussions and critical comments on the manuscript.

SOURCES OF FUNDING

This work was financially supported by the University of Tours and the Centre National de la Recherche Scientifique.

DISCLOSURES

None

Supplementary Material Low voltage-activated channels in rat pulmonary vein cardiomyocytes: Coexistence of a non-selective cationic channel and of T-type Ca channels

Claire O. Malécot (1) (1) Centre National de la Recherche Scientifique Université de Tours, Université de Poitiers, Laboratoire Signalisation et Transports Ioniques Membranaires, EA 7349, 37200 Tours, France e-mail: malecot@univ-tours.fr

Methods

Male Wistar rats (350-480g; CER Janvier, Le Genest St Isle, France) were anesthetized by intraperitoneal injection of pentobarbital (60 mg/kg). After intravenous injection of heparin (500 IU/kg), the heart and lung block was rapidly removed and placed in a dissecting dish that contained cold (4°C) cardioplegic solution (in mM: 110 NaCl, 1.2 CaCl 2 , 16 KCl, 16 MgCl 2 , 10 NaHCO 3 , and 10 glucose). Left and right superior PVs were dissected out under a stereomicroscope, just below the first bifurcation to exclude the junctional area with the left atrial wall. All protocols have been approved by the local ethical committee (Comité d'Ethique en Expérimentation Animale Val de Loire n°019, Tours, France; approval reference number APAFIS#6649-2016090711251954 v2) and conform to the guidelines from Directive 2010/63/EU of the European Parliament.

Cardiomyocytes Isolation

Cardiac myocytes were enzymatically (liberase plus protease) isolated as previously described [2,3] at 37°C from the left and right superior pulmonary veins (PV) and from the left atrial appendage (LA). Liberase TM research grade (0.6 U/mL) and protease (Type XXIV, 1U/mL) were dissolved in a modified Krebs-Ringer-Bicarbonate (KRB) solution (pH 7.4 equilibrated with 95% O 2 -5% CO 2 ) and containing (in mM): [START_REF] Pasqualinc | Mapping of the catecholaminergic automatic activity from its foci within the pulmonary veins to the left atria in rat[END_REF]

Patch-clamp recording of the calcium current I Ca

Patch pipettes (2.5 to 3.5 MΩ) were pulled from thick wall borosilicate glass capillaries (Clark Electromedical Instruments, Harvard Apparatus, UK). An Axopatch 200A amplifier (Axon Instruments Inc., USA) connected to a PC computer running Clampex (pClamp9 software, Axon Instruments) Supplementary Tables S1 andS2 Table S1 V rev : current reversal potential. P' Ca /P X : relative permeabilty of Ca 2+ over cation X + . Ionic strength I of the extracellular recording solution was 0.162 mol/L in 5 mM CaCl 2 control conditions. With 1, 3, 5 and 10 mM added XCl (with X representing Na + or K + ), I increased to 0.163, 0.165, 0.167 and 0.172 mol/L, respectively.


Maxime Fuster

Hermine Billard

Marie Mandart

Johannes Steiger

Télesphore Sime-Ngando

Jonathan Colombet
email: jonathan.colombet@uca.fr

Trophic conditions influence widespread distribution of Aster Like Nanoparticles within aquatic environments Authors

Aster Like Nanoparticles (ALNs) are newly described femto-entities. Their ecology (e.g. geographic distribution, spatial dynamic, preferences, forcing factors) is still unknown. Here we report that these entities, which have largely been ignored until now, can develop or maintain themselves in most aquatic environments in the Loire River catchment, France. We observed a significant influence of the trophic state on ALN ecological distributions. A positive relationship between prokaryotic abundance and ALN (r² =0.72, p<0.01) has been identified, but its exact nature remains to be clarified. Combined with their ubiquitous distribution and high abundances (up to 7.9 x 10 6 ALNs.mL -1 ) recorded in our samples, this probably makes ALNs an overlooked functional component in aquatic ecosystems.

The discovery of the importance of extracellular vesicles [START_REF] Soler | Membrane vesicles in natural environments: a major challenge in viral ecology[END_REF], bacteria CPR (Candidate Phyla Radiation), archaea DPANN (Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanoarchaeota, Nanohaloarchaea) [START_REF] Castelle | Major New Microbial Groups Expand Diversity and Alter our Understanding of the Tree of Life[END_REF][START_REF] Ghuneim | Nano-Sized and Filterable Bacteria and Archaea: Biodiversity and Function[END_REF] and 'biomimetic mineralo-organic particles' [START_REF] Wu | Formation and characteristics of biomimetic mineralo-organic particles in natural surface water[END_REF] permitted to reconsider the diversity and ecological role of the femtoplankton, hitherto confined to the sole viruses, some <0.2µm filterable prokaryotes [START_REF] Sieburth | Pelagic ecosystem structure: Heterotrophic compartments of the plankton and their relationship to plankton size fractions[END_REF]. The recent discovery of "Aster Like Nanoparticles" (ALNs) in pelagic aquatic environments showed that the femtoplankton still hosts many unknown and undervalued entities [START_REF] Colombet | Discovery of High Abundances of Aster-Like Nanoparticles in Pelagic Environments: Characterization and Dynamics[END_REF]. In a previous, unprecedented study on these entities, the authors have shown that ALNs are amorphous star-shaped entities suspected to be organic in nature (composed mainly of carbon, oxygen, calcium and nitrogen with traces of potassium). Pleomorphic, these entities present three main morphotypes, with 4, 11 and 20 arms, and a reduced biovolume, lower than of the smallest known prokaryote. These original characteristics, combined with their sensitivity to biocidal treatments and their ability to grow in the absence of cells, raise questions about their exact nature and origin [START_REF] Colombet | Discovery of High Abundances of Aster-Like Nanoparticles in Pelagic Environments: Characterization and Dynamics[END_REF]. Their high seasonal abundances (up to 9.0 ± 0.5 x 10 7 entities•mL -1 ), in the range of virus-like particles, may exceed those of prokaryotes by up to about one order of magnitude [START_REF] Colombet | Discovery of High Abundances of Aster-Like Nanoparticles in Pelagic Environments: Characterization and Dynamics[END_REF]. Added to observations of phenotypic close contact with prokaryotes [START_REF] Colombet | Discovery of High Abundances of Aster-Like Nanoparticles in Pelagic Environments: Characterization and Dynamics[END_REF], such numerical abundance levels probably make ALNs a significant actor in the functioning of aquatic systems. For example, its calcium composition [START_REF] Colombet | Discovery of High Abundances of Aster-Like Nanoparticles in Pelagic Environments: Characterization and Dynamics[END_REF] could have a significant impact on calcium homeostasis of aquatic environments. Without yet knowing their full biological nature, we hypothesized that their distribution and dynamics in a wide range of aquatic environments will open a window on the understanding of their ecological significance.

Except for first findings obtained from lake samples [START_REF] Colombet | Discovery of High Abundances of Aster-Like Nanoparticles in Pelagic Environments: Characterization and Dynamics[END_REF], we still lack information on the ecological distribution and spatio-temporal dynamics of ALNs, in relation to the potential environmental forcing factors.

Results acquired in this second study demonstrate that ALNs are overlooked femto-entities, widely distributed and that their dynamics are controlled by trophic status, in particular by the biological environment. Identification and quantification of ALNs and of their morphological variants are described in Colombet and collaborators [START_REF] Colombet | Discovery of High Abundances of Aster-Like Nanoparticles in Pelagic Environments: Characterization and Dynamics[END_REF]. Twenty-five study sites distributed over the Loire River catchment (France) were selected to represent different aquatic systems (rivers, canal, marshes, lakes, mudflat, and coastal ocean) as well as longitudinal gradient from the source to the estuary (Figure 1a). Sampling sites were classified into three trophic states according to all abiotic and biotic parameters measured in-situ. Further details on the methodology used are provided in the supplementary text and data.

ALNs have the ability to colonize different aquatic environments: lakes, rivers, marshes, coastal oceanic waters, except source areas and altitude lakes (Figure 1). They were found in environments with contrasting physico-chemical (e.g. conductivity, oxygen, nutrients) and biological (e.g. virus, prokaryotes, algae) characteristics (Supplementary Table S1). This defines them as tolerant and ubiquitous entities capable of maintaining themselves in the majority of continental and coastal aquatic environments. The abundances of ALNs recorded (1.1 x 10 7 ± 0.17 x 10 7 ALNs.mL -1 ) indicate preferential development in plain lakes, marshes, Loire estuary and in lesser extent within free-flowing river channels (Figure 1b-c). Detection of ALNs has also been mentioned in the tropical estuarine system of Ha Long Bay (Vietnam) and in the Saloum River in Senegal, thus broadening the habitat preferences and potential distribution of these entities [START_REF] Colombet | Discovery of High Abundances of Aster-Like Nanoparticles in Pelagic Environments: Characterization and Dynamics[END_REF]. However, sampling of these studies was sporadic, and these conclusions must be weighted by the high seasonal or even daily fluctuations of ALNs recorded previously [START_REF] Colombet | Discovery of High Abundances of Aster-Like Nanoparticles in Pelagic Environments: Characterization and Dynamics[END_REF]. The sampling period, i.e. spring, was based on the results obtained from the unique study on seasonal monitoring of ALNs in a eutrophic lake [START_REF] Colombet | Discovery of High Abundances of Aster-Like Nanoparticles in Pelagic Environments: Characterization and Dynamics[END_REF]. We recognize the need of additional data on the seasonal variability of ALNs in different aquatic environments in order to optimize sampling periods.

The presence of ALNs in the coastal ocean samples raises questions about their potential for development in the marine environment and their possible transfer from continental waters to the oceans. We suggest that the gradual and significant increase of ALN abundance (undetectable to 8.8 x 10 5 ALNs.mL -1 ) from the source to the estuary (Figure 1b) can be explained through the ALN downstream transfer along the river continuum, as observed for other entities or pollutants [START_REF] Harvey | Upstream trophic structure modulates downstream community dynamics via resource subsidies[END_REF][START_REF] Jeffries | Persistence, loss and appearance of bacteria upstream and downstream of a river system[END_REF][START_REF] Cho | Modeling fate and transport of fecally-derived microorganisms at the watershed scale: State of the science and future opportunities[END_REF]. The occurrence of 11-budding-arm forms in the downstream area suggests that ALNs could be transported from the upstream catchment to more favorable conditions for their development downstream. The occurrence of the different forms of ALN indicates dominance of 20-arm forms (69 % of total ALNs) in marshes, while 11-arm forms dominate in lakes and rivers (38 % and 47 % of total ALNs, respectively; Figure 1c).

Environmental conditions probably select and/or favor the development of one type in respect to another, through a process that remains unexplained and which could either reflect the gradual development from one form to another or the occurrence and development of rather independent forms. In order to better understand spatial distributions of ALNs, future investigations will have to demonstrate if a form of resistance distinct from forms of propagation able to develop over time (i.e. assuming distinct growth stages of the same organism) [START_REF] Colombet | Discovery of High Abundances of Aster-Like Nanoparticles in Pelagic Environments: Characterization and Dynamics[END_REF], exist.

These observations also point to the hypothesis that ALNs could be present in all aquatic environments but are selected by environmental factors (Baas Becking hypothesis) [START_REF] Baas Becking | On the ecology of a Sphagnum Bog[END_REF]. Such an assumption is supported by the capacity of ALNs to develop in unrelated biomes, from temperate regions separated by hundreds of kilometers as in our study, to tropical regions [START_REF] Colombet | Discovery of High Abundances of Aster-Like Nanoparticles in Pelagic Environments: Characterization and Dynamics[END_REF].

According to the parameters recorded, we reported a significant influence of the trophic state on ecological distributions of ALNs(Fig. 2a) with a significant increase from undetectable ALNs.mL -1 in the environments classified in the first trophic state, to 2.2 x 10 6 ALNs.mL -1 in those classified in the third trophic state [START_REF] Carlson | A Coordinator's Guide to Volunteer Lake Monitoring Methods[END_REF]. In this study, the trophic state was estimated according to the Trophic State Index, a reference method for the classification of aquatic environments [START_REF] Carlson | A trophic state index for lakes[END_REF]. Therefore, we suggest that ALNs, in addition to certain microbial components such as phytoplankton communities [START_REF] Martinet | Phytoplankton community and trophic status assessment of a newly impounded sub-tropical reservoir: case study of the Nam Theun 2 Reservoir (Lao PDR, Southeast Asia)[END_REF], may serve as a putative indicator of trophic conditions of aquatic environments. Whatever the aquatic system considered, it seems that biological features prevail over physico-chemical ones in the regulation of the distribution and abundances of ALNs (Fig. 2b). The major correlative factor identified is the abundance of prokaryotes (r² =0.72, p< 0.01; Fig. 2b). A significant statistical correlation and the observation of direct physical contact between prokaryotes and ALNs [START_REF] Colombet | Discovery of High Abundances of Aster-Like Nanoparticles in Pelagic Environments: Characterization and Dynamics[END_REF], suggest an interaction between these two components. Previous study showed that ALNs can grow without a potential host [START_REF] Colombet | Discovery of High Abundances of Aster-Like Nanoparticles in Pelagic Environments: Characterization and Dynamics[END_REF], however, the presence of prokaryotes could promote their development through mechanisms that remain unknown [START_REF] Colombet | Discovery of High Abundances of Aster-Like Nanoparticles in Pelagic Environments: Characterization and Dynamics[END_REF]. Therefore, future investigations are needed to elucidate the nature of this mutual relationship which, perhaps, could be related to either bottom-up resource-driven or to topdown host-symbiont interactions. [START_REF] Sieburth | Pelagic ecosystem structure: Heterotrophic compartments of the plankton and their relationship to plankton size fractions[END_REF][START_REF] Colombet | Discovery of High Abundances of Aster-Like Nanoparticles in Pelagic Environments: Characterization and Dynamics[END_REF][START_REF] Jeffries | Persistence, loss and appearance of bacteria upstream and downstream of a river system[END_REF][START_REF] Carlson | A Coordinator's Guide to Volunteer Lake Monitoring Methods[END_REF][START_REF] Sime-Ngando | Virus and prophages in aquatic ecosystems[END_REF][START_REF] Brussaard | Viral Control of Phytoplankton Populations-a Review[END_REF]16,18,19,20,24,25) and high (state 3 = sites [START_REF] Harvey | Upstream trophic structure modulates downstream community dynamics via resource subsidies[END_REF][START_REF] Cho | Modeling fate and transport of fecally-derived microorganisms at the watershed scale: State of the science and future opportunities[END_REF][START_REF] Baas Becking | On the ecology of a Sphagnum Bog[END_REF][START_REF] Carlson | A trophic state index for lakes[END_REF][START_REF] Martinet | Phytoplankton community and trophic status assessment of a newly impounded sub-tropical reservoir: case study of the Nam Theun 2 Reservoir (Lao PDR, Southeast Asia)[END_REF]17,21,22,23) trophic levels of the different aquatic environments. b. Correlation analysis (Spearman test, p<0.01) between the different physical chemical and biological parameters of the different sampling points. T°C = temperature, O2=dissolved oxygen, P= total organic phosphorus, N= total organic nitrogen, C= total organic carbon.

a b

Our findings reinforced the hypothesis of the biological origin of ALNs [START_REF] Colombet | Discovery of High Abundances of Aster-Like Nanoparticles in Pelagic Environments: Characterization and Dynamics[END_REF] and the hypothesis that ALNs are ubiquitous and widespread within continental freshwaters and coastal brackish waters. The estimation of the ecological importance of femtoplanktonic entities depends closely on their abundance and functions. This is demonstrated for viruses [START_REF] Sime-Ngando | Virus and prophages in aquatic ecosystems[END_REF][START_REF] Brussaard | Viral Control of Phytoplankton Populations-a Review[END_REF], extracellular vesicles, ultramicro-prokaryotes [START_REF] Castelle | Major New Microbial Groups Expand Diversity and Alter our Understanding of the Tree of Life[END_REF][START_REF] Ghuneim | Nano-Sized and Filterable Bacteria and Archaea: Biodiversity and Function[END_REF], and suspected for non-biological entities such as 'biomimetic mineralo-organic particles' [START_REF] Wu | Formation and characteristics of biomimetic mineralo-organic particles in natural surface water[END_REF]. The average abundances of 9.9 x 10 5 and 7.9 x 10 6 we recorded respectively in plain lakes and marshes area (see fig. 1), are in the range of those of prokaryotes or viruses. The strong empirical relationship with prokaryotes and the regulation by their environment implies that ALNs are additional and so far, ignored actors in aquatic ecosystems. Furthermore, to fully understand the ecological significance of ALNs in plankton dynamics, it is crucial to validate their biological nature.

Fig 1 .

 1 Fig 1. Sampling site locations (1-25) and distribution of ALNs in different aquatic environments along the watershed of Loire River (France). a. Site locations are plotted on the map of the Loire watershed. Each site or river continuum is characterized by ALN concentration range. b. Distribution of the ALN concentrations and their different forms along the river continuum. c. Comparisons of the ALN concentrations and their different forms between the different aquatic environments sampled. Statistical differences between aquatic environments (test U, *** = p<0.01) are provided in the inserted table (S= Source area, I= Intermediate Loire, D= Downstream Loire, A= Altitude lakes, P=Plain lakes, M=Marshes, NS=Not significant). m= mean.

Fig 2 .

 2 Fig 2. Relationships between ALNs and physical-chemical and biological environment. a. Comparison of the average (test U, p<0.01) of ALNs according to the low (state 1 = sites 1, 2, 3, 4), medium (state 2 = sites 5, 6, 8, 11, 14, 15, 16, 18, 19, 20, 24, 25) and high (state 3 = sites 7, 9, 10, 12, 13, 17, 21, 22, 23) trophic levels of the different aquatic environments. b. Correlation analysis (Spearman test, p<0.01) between the different physical chemical and biological parameters of the different sampling points. T°C = temperature, O2=dissolved oxygen, P= total organic phosphorus, N= total organic nitrogen, C= total organic carbon.

Acknowledgments

This study was supported by the CYSTEM platform UCA-PARTNER (University of Clermont Auvergne UCA), Clermont-Ferrand, France) and the Laboratory Microorganisms: Genome and Environment (LMGE, UMR6023 CNRS-UCA, Clermont-Ferrand, France).

Fundings MF was supported by a PhD fellowship from the CPER 2015-2020 SYMBIOSE challenge program (French Ministry of Research, UCA, CNRS, INRA, Auvergne-Rhône-Alpes Region, FEDER). This study is a contribution to the "C NO LIMIT" project funded by the Interdisciplinary Mission of the French National Center of Scientific Research (CNRS) Program X-life, 2018 edition. This research was also financed by the French government IDEX-ISITE initiative 16-IDEX-0001 (CAP 20-25).

Conflict of Interest

The authors declare no conflict of interest.

Authors contribution

MF, HB, MM, JC designed the study and analyzed data. All authors wrote the manuscript, contributed critically and gave final approval for publication.