Caroline Choquet

Pierre Sicard

Juliette Vahdat

Thi Hong

Minh Nguyen

Frank Kober

Isabelle Varlet

Monique Bernard

Sylvain Richard

Robert G Kelly

Nathalie Lalevée

Lucile Miquerol
email: lucile.miquerol@univ-amu.fr

Purkinje System Hampers Its Maturation and Leads to Mechanical Dysfunction

Keywords: His-Purkinje system, fast conduction markers, RSR' complex, Cx40, cell dropout, strain defects, cardiac dyssynchrony

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Electrical impulses delivered by specialized components of the conduction system (CS) ensure the rhythm and the coordination of cardiac contractions [START_REF] Boyett | The cardiac conduction system: The wiring system of the heart[END_REF][START_REF] Anderson | The morphology of the cardiac conduction system[END_REF]. Electrical activity initiates in the sinoatrial node (SAN) and then reaches the atrioventricular node (AVN), the unique electrical connection with the ventricles in the definitive heart. From the AVN, the electrical activity takes a fast-conducting route mediated by the His-Purkinje or ventricular conduction system (VCS). The VCS comprises the His or AV bundle (AVB), right and left bundle branches, and ends in a complex network of Purkinje fibers (PFs). The VCS represents only 1-2% of the cardiac volume but is responsible for the rapid cardiac conduction essential for the normal rhythm of cardiac contractions [START_REF] Miquerol | Biphasic development of the mammalian ventricular conduction system[END_REF][START_REF] Zaglia | Optogenetic determination of the myocardial requirements for extrasystoles by cell type-specific targeting of channelrhodopsin-2[END_REF]. The electrophysiological properties of these specialized conductive cardiomyocytes result from the expression of a large range of specific genes coding for ion channels enabling active conduction, and gap junctions responsible for passive conduction velocity and a low level of contractile proteins [START_REF] Van Kempen | Differential connexin distribution accommodates cardiac function in different species[END_REF][START_REF] Gros | Connexins in mammalian heart function[END_REF][START_REF] Marionneau | Specific pattern of ionic channel gene expression associated with pacemaker activity in the mouse heart[END_REF][START_REF] Gaborit | Regional and tissue specific transcript signatures of ion channel genes in the non-diseased human heart[END_REF]. Altered cardiac conduction can lead to ventricular arrhythmias and bundle branch blocks (BBB), which are associated with increased mortality in heart failure patients due to myocardial dyssynchrony [START_REF] John | Ventricular arrhythmias and sudden cardiac death[END_REF]. In addition, terminal conduction delay of left ventricular depolarization, showing abnormal QRS complex but different from either RBBB or LBBB, causes major ventricular arrhythmias due to impaired tissue surrounding old infarct scars [START_REF] Varriale | The RSR' complex not related to right bundle branch block: Diagnostic value as a sign of myocardial infarction scar[END_REF].

Nkx2-5 encodes for an essential homeobox transcription factor that orchestrates cardiac development [START_REF] Harvey | Homeodomain factor Nkx2-5 in heart development and disease[END_REF]. In humans, mutations of NKX2-5 with a high penetrance often promote the occurrence of severe and progressive AV conduction blocks and atrial septal defects [START_REF] Schott | Congenital heart disease caused by mutations in the transcription factor Nkx2-5[END_REF]. In transgenic mice, Nkx2-5 +/-embryos and mice progressively reproduce the phenotype observed in human patients, including atrial septal defects and conduction disturbances [START_REF] Tanaka | A mouse model of congenital heart disease: Cardiac arrhythmias and atrial septal defect caused by haploinsufficiency of the cardiac transcription factor Csx/Nkx2.5. Cold Spring Harb[END_REF].

Electrocardiogram analysis reveals that Nkx2-5 haploinsufficient mice present a prolonged PR interval and a progressive elongation of the QRS. This phenotypic evolution is associated with progressive loss of the AVB [START_REF] Jay | Nkx2-5 mutation causes anatomic hypoplasia of the cardiac conduction system[END_REF][START_REF] Pashmforoush | Nkx2-5 pathways and congenital heart disease; loss of ventricular myocyte lineage specification leads to progressive cardiomyopathy and complete heart block[END_REF]. Indeed, adipose tissue has been found to replace the AVB in a human patient with an NKX2-5 mutation [START_REF] Pashmforoush | Nkx2-5 pathways and congenital heart disease; loss of ventricular myocyte lineage specification leads to progressive cardiomyopathy and complete heart block[END_REF]. Moreover, patients carrying mutations in NKX2-5 present a large spectrum of congenital heart diseases frequently associated with conduction disturbances or arrhythmias, and long-term follow-up reveals a high incidence of sudden cardiac death in these patients with aging [START_REF] Ellesoe | Familial atrial septal defect and sudden cardiac death: Identification of a novel Nkx2-5 mutation and a review of the literature[END_REF][START_REF] Maury | Cardiac phenotype and long-term follow-up of patients with mutations in nkx2-5 gene[END_REF]. However, the underlying mechanisms remain unclear. In particular, whether Nkx2-5 plays a primary role within conductive cardiomyocytes remains an open question.

We have previously shown that Nkx2-5 plays a crucial role in Purkinje network patterning in mice by controlling the progressive recruitment of PF during ventricular development [START_REF] Choquet | Nkx2-5 defines distinct scaffold and recruitment phases during formation of the murine cardiac Purkinje fiber network[END_REF][START_REF] Choquet | Deletion of Nkx2-5 in trabecular myocardium reveals the developmental origins of pathological heterogeneity associated with ventricular non-compaction cardiomyopathy[END_REF].

In the present study, we characterized the role of Nkx2-5 in the mature VCS. We created a conditional knockout of Nkx2-5 within the VCS using a tamoxifen-inducible Cre mouse line [START_REF] Furtado | A novel conditional mouse model for Nkx2-5 reveals transcriptional regulation of cardiac ion channels[END_REF][START_REF] Beyer | Inducible Cx40-cre expression in the cardiac conduction system and arterial endothelial cells[END_REF]. Exploiting this strategy, we removed Nkx2-5 in the VCS without affecting Nkx2-5 expression in the contractile or working ventricular myocardium. Nkx2-5 VCS conditional deletion at birth provoked a mild VCS hypoplasia and progressive loss of expression of fastconduction markers, which resulted in the appearance of an altered ventricular activation sequence. Longitudinal analysis revealed that these conduction defects were associated with ventricular dyssynchrony. Nkx2-5 VCS-deleted mice developed contractility defects and early diastolic dysfunction associated with a decreased ejection fraction. This study reveals a cellautonomous role for Nkx2-5 in maintaining a functional conductive phenotype in the VCS and protecting against heart dysfunction.

Materials and Methods

Mouse Lines

The investigation was approved by the ethics committee for animal experimentation of the French ministry (no. 01055.02). Animal procedures conformed to the guidelines from Directive 2010/63/EU of the European Parliament for the Care and Use of Laboratory Animals. Cx40-CreERT2, R26-YFP and Nkx2-5-floxed mouse lines were genotyped as previously reported [START_REF] Furtado | A novel conditional mouse model for Nkx2-5 reveals transcriptional regulation of cardiac ion channels[END_REF][START_REF] Beyer | Inducible Cx40-cre expression in the cardiac conduction system and arterial endothelial cells[END_REF][START_REF] Srinivas | Cre reporter strains produced by targeted insertion of eYFP and eCFP into the Rosa26 locus[END_REF]. To conditionally delete Nkx2-5, tamoxifen was injected intraperitoneally into newborn pups (P0 or P1) in a single injection (10 L). Tamoxifen (T-5648, Sigma) was dissolved at a concentration of 20 mg/mL in ethanol/sunflower oil = (10/90). Nkx2-5-DNeo fl/fl ::Cx40 Cre/+ were designated as Nkx2-5DVCS mice and compared to control mice Nkx2-5 +/+ ::Cx40 Cre/+, both receiving Tam injection at birth.

Macroscopic and Histological Analyses

Mice were euthanized by cervical dislocation and hearts from young and old animals were rapidly collected in cold PBS (1m) at 2 and 12 months. Macroscopic examination of the internal surface of the ventricles was performed as previously described [START_REF] Miquerol | Architectural and functional asymmetry of the his-purkinje system of the murine heart[END_REF]. Whole-mount and histological immunofluorescence and image analysis were carried out as previously described [START_REF] Choquet | Deletion of Nkx2-5 in trabecular myocardium reveals the developmental origins of pathological heterogeneity associated with ventricular non-compaction cardiomyopathy[END_REF]. Antibodies used in this study are specific to Nkx2-5 (Sc8697 Santa-Cruz, Dallas, TX, USA), GFP (AbD Serotec, Purchheim, Germany), RFP (Rockland, Pottstown, PA, USA), Contactin-2 (AF1714 R&D system, Minneapolis, MN, USA), Pecam-1 (MEC13.3-BD Pharmingen, San Jose, CA, USA), HCN4 (Millipore, Burlington, MA, USA), ETV-1 (Abcam, Cambridge, U.K.), Troponin I (Abcam) and WGA-Cy3 (Sigma, St. Louis, MO, USA). Antibodies against Cx43 and CX40 are homemade and previously described [START_REF] Gros | Connexins in mammalian heart function[END_REF][START_REF] Miquerol | Architectural and functional asymmetry of the his-purkinje system of the murine heart[END_REF].

Cardiac Magnetic Resonance Imaging (MRI)

MRI was carried out every 2 months on the same animal groups of 2-12-monthold mice under isoflurane anesthesia (4% for the induction and 2% during the image recordings). The experiments were performed on a Bruker Biospec Avance 4.7 T/30 imager (Bruker Biospin GmbH, Ettlingen, Germany), and images were analyzed as previously described [START_REF] Choquet | Deletion of Nkx2-5 in trabecular myocardium reveals the developmental origins of pathological heterogeneity associated with ventricular non-compaction cardiomyopathy[END_REF].

Echocardiography

Echocardiography was performed for two groups of mice (3 and 12 months old) under isoflurane anesthesia (2%) using a Vevo 2100 ultrasound system (VisualSonics, Toronto, Canada) equipped with a real-time micro-visualization scan head probe (MS-550D) operating at a frame rate of 300 frames per sec (fps). Left ventricular (LV) characteristics were quantified according to the standards of the American Society of Echocardiology and the Vevo 2100 Protocol-Based Measurements and Calculations guide, as previously described [START_REF] Furtado | A novel conditional mouse model for Nkx2-5 reveals transcriptional regulation of cardiac ion channels[END_REF]. High-frequency speckle tracking for strain analysis was performed on parasternal longaxis B-mode loops. The standard deviation of time to peak strain corrected by the inter-beat interval was used to measure the intraventricular dyssynchrony [START_REF] Bhan | High-frequency speckle tracking echocardiography in the assessment of left ventricular function and remodeling after murine myocardial infarction[END_REF]. LV strain analysis was performed offline using Vevostrain analysis software 3.1.1 (VisualSonics). To quantify the peak longitudinal strain rate during early LV filling, the "reverse peak" option was used.

Surface Electrocardiography

Surface ECGs were performed on anesthetized mice. An induction with 5% isoflurane was followed by maintenance at 2% in a constant flow of oxygen at 1 L/min. ECGs were recorded every two months from 2 to 12 months using a bipolar system as previously described [START_REF] Choquet | Deletion of Nkx2-5 in trabecular myocardium reveals the developmental origins of pathological heterogeneity associated with ventricular non-compaction cardiomyopathy[END_REF].

Statistical Analysis

Data are expressed as means +/-standard error of the mean (SEM) for bar graphs and median with min./max. for box plots. Significant differences between groups were determined using one-way or two-way analysis of variance (ANOVA) followed by Sidak post hoc testing with PGraphPad Prism software (PGraphPad Prism 7.0, La Jolla, CA, USA). A p value < 0.05 was considered statistically significant.

Results

Conditional Deletion of Nkx2-5 in the Ventricular Conduction System

To conditionally delete Nkx2-5 gene in the VCS, we crossed mice containing Floxed-Nkx2-5-Dneo alleles with Cx40-CreERT2 mice. Cre recombinase activity was induced by tamoxifen injection into newborn pups, thereafter designated as Nkx2-5 VCS . The efficiency of Nkx2-5 deletion was verified by immunofluorescence on sections from control and Nkx2-5 VCS mice at P10. Nkx2-5-negative cardiomyocytes are restricted to subendocardial cells, representing a very low percentage of cells in the entire heart (Figure 1A). Using Contactin-2 (Cntn2) to identify cells of the VCS [START_REF] Pallante | Contactin-2 expression in the cardiac Purkinje fiber network[END_REF], we found that Nkx2-5-negative cells co-localize with Cntn2-positive cells (Figure 1B). These Nkx2-5-negative cardiomyocytes are also positive for Cx40 expression detected using a Cx40 antibody or through the expression of the Cx40-RFP allele (Figure 1B). As shown previously, a single postnatal injection of tamoxifen is sufficient to target a large proportion of cardiomyocytes of the VCS [START_REF] Beyer | Inducible Cx40-cre expression in the cardiac conduction system and arterial endothelial cells[END_REF] and efficiently delete Nkx2-5 in these cells.

Neonatal Loss of Nkx2-5 in the VCS Disturbs Its Maturation and Provokes Apical PF Hypoplasia

To investigate the phenotypic consequences of the loss of Nkx2-5 in the Purkinje fiber (PF) network, we performed whole-mount immunofluorescence using Cntn2 antibody on opened left ventricles from 3-month-old control and Nkx2-5 VCS mice (Figure 2A). Nkx2-5 VCS mutant mice present a reduced density of the PF network compared to control mice (Figure 2C), a defect that is more pronounced in the apical region (Figure 2A). Loss of Nkx2-5 in the VCS thus provokes a mild hypoplasia of PF, primarily affecting the apical part of the left ventricle. In contrast, prior work has shown that earlier deletion during development results in severe VCS hypoplasia [START_REF] Choquet | Deletion of Nkx2-5 in trabecular myocardium reveals the developmental origins of pathological heterogeneity associated with ventricular non-compaction cardiomyopathy[END_REF]. To understand the cellular mechanism responsible for this PF hypoplasia, we performed a genetic tracing analysis of Nkx2-5-deleted cardiomyocytes to follow their fate. We crossed Cx40-CreERT2::Nkx2-5 fl/+ with Rosa26-YFP::Nkx2-5 fl/+ mice and we induced Cre activity by tamoxifen injection into pregnant females at E18.5. We performed whole-mount immunofluorescence with Cntn-2 to compare the localization of YFP-positive cells in hearts from 3-week-old Nkx2-5 +/+ (WT) and Nkx2-5 VCS mice (Figure 2B). Cx40-derived (YFPpositive) cardiomyocytes are present in the same proportion in WT and Nkx2-5DVCS mutant hearts, demonstrating that those cells are not lost after Nkx2-5 deletion. In contrast to the situation in the control hearts, a large number of these YFP cells, mainly localized in the apex, do not express the conductive marker Cntn2, indicative of a maturation defect towards a conductive phenotype. We quantified this on sections and counted the percentage of YFP+ cells integrated in the VCS with the co-expression of Cntn-2 (Figure 2D). Only 50% of YFP+ cells co-expressed Cntn-2 in the apex of Nkx2-5 VCS mutant hearts compared to 70% in WT hearts (Figure 2D). The majority of YFP+/Cntn-2-cells were close to VCS (Cntn-2+) cells and maintained an elongated shape characteristic of PF cells (Figure 2B). This analysis demonstrates a failure of Nkx2-5-negative cardiomyocytes to robustly maintain a conductive phenotype and suggests that PF hypoplasia in Nkx2-5 VCS mutant hearts may arise by a mechanism of cell dropout.

Persistent Nkx2-5-Negative Conductive Cells Progressively Downregulate Fast-Conduction Markers

To further analyze the cellular phenotype of the cardiac conduction system after conditional deletion of Nkx2-5, we carried out immunostaining on sections from 3-monthold control and Nkx2-5 VCS mutant hearts. Nkx2-5 was not deleted in the AVN where Cx40-Cre was not expressed and only partially in the AVB (Figure S1). These two components expressed a high level of Hcn4, ETV1, and Cntn-2 in both control and Nkx2-5 VCS mutant hearts. The central part of the conduction system is thus largely unaffected in Nkx2-5 VCS mutant mice.

In the peripheral VCS, numerous Cntn-2+/Nkx2-5-cells were observed in Nkx2-5 VCS mutant hearts (Figure 3A). However, only very few of these Cntn-2+/Nkx2-5-cells express Cx40 and Hcn4; in contrast, these cells continue to express the conductive markers ETV1 and Cx43. Overall, these results show that the deficit of Nkx2-5 in the VCS induces the loss of a subset of markers important for electrical conduction such as Cx40 and Hcn4 but not ETV1 or Cntn-2, in line with the idea that Nkx2-5 promotes a conductive phenotype by directly controlling the expression of specific genes.

With aging, the density of Cntn-2 and Cx40 double-positive cells was very low in 10-monthold Nkx2-5DVCS mutant heart sections (Figure S2). High-magnification views showed a drastic reduction in these two markers in Nkx2-5 VCS mutant compared to the control hearts while the expression of Cx43 was preserved. Using WGA-Cy3 staining to label cell contours, we quantified the number of total cardiomyocytes per section and the percentage of Nkx2-5+ cells (Figure 3B). Quantification revealed a similar number of cardiomyocytes per field in control and Nkx2-5 VCS mutant mice, suggesting the absence of hypertrophy in these mutants. However, a reduced number of cardiomyocytes per field in old mice suggests a slight increase in cardiomyocyte size developing with age in both control and Nkx2-5VCS mutant mice. Moreover, the thin contour of WGA staining surrounding cardiomyocytes excludes any signs of fibrosis in both control and Nkx2-5 VCS mutant hearts. Quantification of the percentage of Nkx2-5+ cardiomyocytes per field revealed an increase in Nkx2-5 VCS mutant mice between 3 and 10 months of age, increasing from 60% to 75%. These data indicate that, in addition to premature cell dropout, a loss of Nkx2-5-deleted cardiomyocytes naturally occurs with age. Moreover, the loss of fast conduction markers suggests that persistent PFs in Nkx2-5 VCS mice are less conductive.

Conditional Deletion of Nkx2-5 in the VCS Leads to Cardiac Functional Defects

Surface electrocardiograms (ECGs) were recorded in control and Nkx2-5 VCS mutant mice to investigate cardiac electrical activity at 3, 6 and 9 months of age. ECG traces revealed a small increase in QRS complex duration in Nkx2-5 VCS mutant mice, which was significant at 6 months (Table 1). This increase was not representative of the defects observed in some Nkx2-5 VCS mutant mice. Indeed, the QRS complex had an RSR' shape in some mutant mice, the number of which increased with age from 28% at 3 months to 55% at 9 months (Table 1, Figure 4B). In these mice with an abnormal QRS complex, the average QRS duration was 3 ms longer than in the control mice. In addition, the amplitudes of the QRS complexes in derivation II were smaller in the mutant mice (Table 1, Figure 4A). This difference increased with aging and was due to the decrease in the amplitude of the R-wave (Figure 4C,D). No QRS defects were observed in control mice, and in contrast to ventricular activation, the repolarization phase, represented by QT intervals and T wave amplitude, and atrial activation, represented by PR interval, were unaffected in both groups of mice (Table 1). The cardiac function of individual 6-month-old mice was evaluated using cardiac MRI. Cardiac parameters calculated from MRI analysis showed a decrease in the ejection fraction (EF) and stroke volume (SV) in Nkx2-5 VCS mutant compared to control mice (Figure 4E,F). No differences were observed in morphological parameters between both groups or with aging (Table 2, Figure S3).

To better characterize this cardiac phenotype, we used a highly sensitive speckle trackingbased strain imaging technique to detect regional ventricular wall deformation. Nkx2-5 VCS mutant mice present reduced radial strain predominantly affecting the anterior wall region (Figure 4H). We found a strong correlation between reduced EF and radial strain (Figure 4G).

In addition, high-frequency speckle-tracking echocardiography was used to access subtle changes in diastolic function. Representative curves of longitudinal strain rate showed an important defect in the coordination of myocardial deformation and a reduced peak global strain rate during early diastole (SR E ) in Nkx2-5 VCS mutant compared to control mice (Figure 4I). The diastolic index E/SR E was increased in Nkx2-5 VCS mutant mice. Taken together, these data suggest a diastolic dysfunction in old mutant mice (Figure 4I). LV mechanical dyssynchrony was determined as time-to-peak variation, defined as the standard deviation of time to peak over all six segments. Control mice presented minimal longitudinal dyssynchrony (8.0+/-0.5% at 3 months and 8.5 +/-1.8% at 10 months), whereas Nkx2-5DVCS mice had significant dyssynchrony (13.9+/-1.4% at 3 months and 14.9 +/-1.9% at 10 months) compared to age-matched control mice (p < 0.05 at 3 months and p < 0.01 at 10 months). Defects of myocardial deformation and systolic/diastolic dysfunction suggest that premature VCS cell dropout and the loss of fast-conduction markers progressively lead to conduction defects associated with wall-motion abnormalities and ventricular dysfunction.

Discussion

In this study, we analyzed the cardiac function of mice lacking Nkx2-5 expression in the ventricular conduction system after conditional deletion in Cx40-positive cardiomyocytes one day after birth. Our data demonstrate that Nkx2-5 deletion restricted to the VCS induces progressive ventricular conduction defects that result in early intraventricular dyssynchrony followed by mechanical strain defects and decreased EF. Nkx2-5 is required in Cx40-positive cells to maintain a PF phenotype. Nkx2-5 VCS mutant hearts present a significant PF hypoplasia and loss of expression of a subset of conduction markers. These results highlight the importance of Nkx2-5 in the maturation and maintenance of the PF network and the role of loss of function of VCS cells in promoting conduction defects, ventricular mechanical dyssynchrony and left ventricular dysfunction.

Previous studies have shown an important role for Nkx2-5 in cardiac function in human patients as well as in Nkx2-5 haploinsufficient mice [START_REF] Benson | Genetic origins of pediatric heart disease[END_REF][START_REF] Prendiville | Insights into the genetic structure of congenital heart disease from human and murine studies on monogenic disorders[END_REF][START_REF] Benson | Genetics of atrioventricular conduction disease in humans[END_REF]. To date, the role of Nkx2-5 has been mainly studied in contractile cardiomyocytes, which represent the force generating myocardial component of the heart. Deletion of Nkx2-5 disturbed the expression of numerous cardiac genes, inducing calcium-handling defects and decreased contractibility, which impaired cardiac function [START_REF] Ashraf | A mouse model of human congenital heart disease: High incidence of diverse cardiac anomalies and ventricular noncompaction produced by heterozygous Nkx2-5 homeodomain missense mutation[END_REF][START_REF] Briggs | Perinatal loss of Nkx2-5 results in rapid conduction and contraction defects[END_REF][START_REF] Terada | Ablation of Nkx2-5 at mid-embryonic stage results in premature lethality and cardiac malformation[END_REF]. Here, Nkx2-5 deletion was restricted to a very small population of cells forming the VCS, representing only 1-2% of the total heart volume. ECG analysis revealed progressively reduced QRS amplitude and abnormal wave patterns, with RSR' complex associated with a wide QRS, in line with morphological and histological disturbance of the PF network in Nkx2-5DVCS mutant mice. In particular, the drastic decrease in R wave amplitude corresponding to a cutback of the parietal vector [START_REF] Boukens | Early repolarization in mice causes overestimation of ventricular activation time by the QRS duration[END_REF] is consistent with the hypoplasia of PF in the median and apical part of the myocardium. Mouse models in which Cx40 is abolished had a remodeling of passive conductance properties and developed prolonged intervals for all ECG parameters, including split QRS complexes, in line with uncoordinated ventricular activation [START_REF] Kirchhoff | Reduced cardiac conduction velocity and predisposition to arrhythmias in Connexin40-deficient mice[END_REF][START_REF] Simon | Mice lacking Connexin40 have cardiac conduction abnormalities characteristic of atrioventricular block and bundle branch block[END_REF][START_REF] Van Rijen | Impaired conduction in the bundle branches of mouse hearts lacking the gap junction protein Connexin40[END_REF]. Moreover, spatial propagation of electrical activity through the working myocardium, reflected by the mean frontal plane axis, is abnormal in Cx40 -/-mice [START_REF] Simon | Mice lacking Connexin40 have cardiac conduction abnormalities characteristic of atrioventricular block and bundle branch block[END_REF]. Our data on Nkx2-5 deletion in the VCS are consistent with these results and now confirm the role of a Cx40-deficient VCS in the occurrence of altered ventricular activation sequence [START_REF] Kirchhoff | Reduced cardiac conduction velocity and predisposition to arrhythmias in Connexin40-deficient mice[END_REF][START_REF] Simon | Mice lacking Connexin40 have cardiac conduction abnormalities characteristic of atrioventricular block and bundle branch block[END_REF][START_REF] Schrickel | Normal impulse propagation in the atrioventricular conduction system of Cx30.2/Cx40 double deficient mice[END_REF][START_REF] Tamaddon | Highresolution optical mapping of the right bundle branch in Connexin40 knockout mice reveals slow conduction in the specialized conduction system[END_REF].

Nkx2-5 regulates numerous cardiac genes involved in active conduction, such as HCN4, encoding the channel responsible for the I f current, and SCN5A, encoding the sodium channel Nav1.5 [START_REF] Terada | Ablation of Nkx2-5 at mid-embryonic stage results in premature lethality and cardiac malformation[END_REF][START_REF] Ye | A common Shox2-Nkx2-5 antagonistic mechanism primes the pacemaker cell fate in the pulmonary vein myocardium and sinoatrial node[END_REF]. Regarding the role of HCN4 in PF [START_REF] Mesirca | Cardiac arrhythmia induced by genetic silencing of 'funny' (f) channels is rescued by girk4 inactivation[END_REF], its decreased expression may also participate in the Nkx2-5 VCS phenotype, particularly the abnormal QRS pattern because of disturbed active ventricular conduction. Conversely, Nkx2-5-deleted conductive cardiomyocytes maintain expression of ETV1, Cx43 and Cntn-2, suggesting that these genes are not direct targets for this transcription factor. These data are consistent with Nkx2-5 operating downstream of ETV1, a transcription factor responsible for the activation of a rapid conduction genetic program [START_REF] Shekhar | Transcription factor Etv1 is essential for rapid conduction in the heart[END_REF]. PF hypoplasia and reduced expression of Nkx2-5 and fastconduction markers including Cx40 and Nav1-5 have been recently observed in cardiomyocyte-specific deletion of ETV1 [START_REF] Shekhar | Etv1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes[END_REF]. Besides this intrinsic role of Nkx2-5 in the maintenance of a fast-conductive PF phenotype, we observed cell dropout of a number of Nkx2-5-negative elongated cardiomyocytes associated with the loss of Cntn-2 expression. In patients, it was shown that the RSR' complex associated with a wide QRS, a unique mural conduction defect unrelated to right or left bundle branch block, was a sign of myocardial infarct scarring [START_REF] Varriale | The RSR' complex not related to right bundle branch block: Diagnostic value as a sign of myocardial infarction scar[END_REF]. Our data reveal that loss of VCS cells and their fast conduction capacity is similar to the terminal conduction delay of left ventricular depolarization within impaired tissue due to myocardial infarction [START_REF] Varriale | The RSR' complex not related to right bundle branch block: Diagnostic value as a sign of myocardial infarction scar[END_REF]. These data show that Nkx2-5 is required postnatally to retain cells in the VCS during maturation of the PF network and during aging. This provides new mechanistic insights into the previously defined requirement for maximal Nkx2-5 levels in VCS development [START_REF] Meysen | Nkx2.5 cell-autonomous gene function is required for the postnatal formation of the peripheral ventricular conduction system[END_REF].

Nkx2-5 VCS mice present conduction disturbances and ventricular dyssynchrony associated with LV dysfunction. Indeed, these mutant mice develop strain defects, which are correlated with a decreased EF and diastolic dysfunction. However, these mice exhibited no structural changes in the myocardium and developed no apparent signs of cardiac hypertrophy or fibrosis, known triggers of heart failure. In this respect, abnormal conduction and progressive mechanical dysfunction would result from intrinsic properties of the VCS rather than a secondary consequence of other cardiac defects. Our data strongly suggest a direct role for conduction defects in the appearance of cardiac dysfunction. In a previous study, we demonstrated that the conditional deletion of Nkx2-5 in trabecular cells during embryonic development provokes a hypertrabeculated phenotype associated with cardiac hypertrophy, subendocardial fibrosis and severe VCS hypoplasia, which induced heart failure with age [START_REF] Choquet | Deletion of Nkx2-5 in trabecular myocardium reveals the developmental origins of pathological heterogeneity associated with ventricular non-compaction cardiomyopathy[END_REF]. However, young mice developed LV dysfunction detected by a reduction in EF similar to Nkx2-5 VCS mice. Together, these data suggest that conduction defects represent a triggering factor for LV dysfunction in non-compaction cardiomyopathy and might explain the poor prognostic of patients with non-compaction cardiomyopathy associated with ECG defects [START_REF] Bhatia | Isolated noncompaction of the left ventricular myocardium in adults: A systematic overview[END_REF][START_REF] Brescia | Mortality and sudden death in pediatric left ventricular noncompaction in a tertiary referral center[END_REF][START_REF] Kimura | Prognostic impact of left ventricular noncompaction in patients with Duchenne/Becker muscular dystrophy-Prospective multicenter cohort study[END_REF]. Furthermore, our results highlight the important role of the ventricular conduction system in the origin of conduction defects and cardiac dysfunction in noncompaction cardiomyopathy. Likewise, the asymptomatic phenotype of numerous individuals with hypertrabeculated hearts may arise from a different etiology in which the VCS is not affected [START_REF] Towbin | Left ventricular non-compaction cardiomyopathy[END_REF]. Finally, this is the first study highlighting the direct effect of conduction defects on contractile function. Using recently developed technology, including high-frequency speckle tracking echocardiography [START_REF] Bhan | High-frequency speckle tracking echocardiography in the assessment of left ventricular function and remodeling after murine myocardial infarction[END_REF], we were able to detect mechanical dyssynchrony in Nkx2-5 VCS hearts. Mechanical dyssynchrony measures the variation in the timing of regional ventricular deformation during cardiac contraction and is altered in mouse models with contractile function defects or with age [START_REF] Bauer | Regional cardiac dysfunction and dyssynchrony in a murine model of afterload stress[END_REF][START_REF] De Lucia | Echocardiographic strain analysis for the early detection of left ventricular systolic/diastolic dysfunction and dyssynchrony in a mouse model of physiological aging[END_REF]. One of the explanations for age-related dyssynchrony is increased fibrosis and lipid content [START_REF] Crendal | Increased myocardial dysfunction, dyssynchrony, and epicardial fat across the lifespan in healthy males[END_REF][START_REF] Lin | Myocardial regional interstitial fibrosis is associated with left intra-ventricular dyssynchrony in patients with heart failure: A cardiovascular magnetic resonance study[END_REF]; however, in our mutants, the defects were restricted to the Purkinje fiber network. Recent studies have described dyssynchrony as an important prognostic factor in patients with heart diseases for cardiac resynchronization therapy (CRT) [START_REF] Cai | Left ventricular dyssynchrony by 3 dimensional echocardiography: Current understanding and potential future clinical applications[END_REF]. CRT is now an important therapy for heart failure patients with reduced EF and ventricular conduction delay [START_REF] Engels | Why qrs duration should be replaced by better measures of electrical activation to improve patient selection for cardiac resynchronization therapy[END_REF]. However, not all patients respond to this treatment, and those who do have more severe systolic dyssynchrony, suggesting that the VCS may be affected [START_REF] Suffoletto | Novel speckletracking radial strain from routine black-andwhite echocardiographic images to quantify dyssynchrony and predict response to cardiac resynchronization therapy[END_REF][START_REF] Yu | Usefulness of tissue doppler velocity and strain dyssynchrony for predicting left ventricular reverse remodeling response after cardiac resynchronization therapy[END_REF]. Our data strongly suggest that conduction defects are an important trigger of mechanical dyssynchrony that can be corrected by CRT and provide predictive insights into this responsiveness. If the VCS rather than the contractile compartment is primarily affected, as in our mutant mice, CRT may be more efficient. This assumption warrants further investigation in the future to establish clear diagnostic criteria for CRT.

Figure 1 .

 1 Figure 1. Condition deletion of Nkx2-5 in the ventricular conduction system. (A) Nkx2-5 immunofluorescence on transversal sections of P10 hearts from control (CTL) and Nkx2-5VCS mutant mice. High magnification of the squares is represented below. IVS: interventricular septum; pm: papillary muscles. Scale bars = 100m; high-magnification image bar = 25 m. (B) Co-immunofluorescence of Nkx2-5 and Cx40 or CNTN2 and Cx40-RFP on serial sections of P10 Nkx2-5VCS mutant hearts. Pecam1 and Dapi staining were used to delineate cardiac contours and nuclei. Scale bar = 50 m.

Figure 2 .

 2 Figure 2. Nkx2-5 VCS-conditional deletion mice present PF hypoplasia by cell dropout. (A) Wholemount immunofluorescence with Contactin-2 on opened LV from 3-month-old control and Nkx2-5 VCS hearts. Scale bar = 1 mm. (B) Genetic tracing of ventricular Cx40-positive cells after Cre induction at E18.5 in control and Nkx2-5 VCS mice showing the distribution of YFP+ cells in the PF network indicated by a coimmunofluorescence with CNTN2 at P20. High magnifications of peripheral PFs are presented in inserts. Scale bar = 0.5 mm. Below are sections from genetic tracing experiments stained with YFP and CNTN2 antibodies. Scale bars = 50 m. (C) Quantification of PF density and branching density from images treated with angiotool. (D) Quantification of the percentage of YFP+ cells included in the VCS. N = 3 hearts per group. Mean +/-SD; Student t-tests: * p < 0.05; ** p < 0.01; *** p < 0.001 Nkx2-5DVCS vs. control.

Figure 3 .

 3 Figure 3. Ventricular conduction defects in Nkx2-5VCS mutant hearts. (A) Immunofluorescence with Nkx2-5 and HCN4 or ETV1 and Contactin-2 or Cx40, CNTN2 and WGA or Troponin I (TrI) and Cx43 on serial sagittal sections at the level the left Purkinje fibers from control (CTL) and Nkx2-5 VCS hearts. Scale bar = 100 m. High magnifications of the selected area (rectangle) are presented below. Scale bar = 20m. (B) High magnifications at the level of LPF stained with WGA-cy3 and Nkx2-5 from 3-and 10-month-old control (CTL) and Nkx2-5 VCS hearts. Scale bar = 50m. (C) Nkx2-5 deletion was quantified by counting the percentage of Nk2-5-positive cardiomyocytes per frame at the subendocardial surface of transverse sections from 3-and 10-month-old control (CTL) and Nkx2-5 VCS (DVCS) mice. (D) Quantification of cardiomyocyte hypertrophy by counting the number of cardiomyocytes per frame on high-magnification images of transverse sections from 3-and 10-monthold control (CTL) and Nkx2-5  (DVCS) mice. n = 20-30 frames per heart; N = 3 mice per group; mean +/-SD; two-way analysis of variance (ANOVA) followed by Sidak post hoc tests: ** p < 0.01; **** p < 0.0001 DVCS vs. control; $ p < 0.05; $$ p < 0.01, 3-month-old vs. 10-month-old hearts.

Figure 4 .

 4 Figure 4. Nkx2-5 deletion in the conduction system of neonate mice leads to QRS defects associated with ventricular dysfunction. (A) Representative tracings from surface ECG measured in lead II in Tracings from Nkx2-5 VCS mice demonstrated ventricular conduction defects. (B) Representative tracings of a QRS pattern with RSR' shape observed in Nkx2-5DVCS mice. (C,D) Graphs representing the evolution of the QRS and R amplitudes measured in the same mice over a year. QRS and R amplitudes are lower in the Nkx2-5DVCS group compared to the control (N = 14-18 in the control and N = 12-18 in Nkx2-5DVCS). Mean+/-SD; two-way analysis of variance (ANOVA) followed by Sidak post hoc tests: * p < 0.05DVCS vs. control; § p < 0.05 3month-old vs. 9 month-old hearts. (E,F) Graphs representing MRI measurements of the ejection fraction (EF) and stroke volume (SV) in 6-month-old mice. Both parameters are significantly decreased in Nkx2-5DVCS mice (N = 11 in control and N = 17 for Nkx2-5DVCS). Mean +/-SD; Student t-tests: * p < 0.05 Nkx2-5DVCS vs. control. (G) Measurements of cardiac parameters by echocardiography in 10-month-old control and Nkx2-5 VCS mice show a strong correlation between ejection fraction and strain radial. Mean +/-SD; Student t-tests: ** p < 0.01 Nkx2-5 VCS vs. control. (H) Strain radial is decreased in 10-month-old Nkx2-5 VCS in comparison to control mice. (I) Representative curves of longitudinal strain rate in 10-month-old control and Nkx2-5 VCS mice. Diastolic function estimates by measurements of SR E and E/SR E . Mean+/-SD; Student t-tests: * p < 0.05; ** p < 0.01 Nkx2-5 VCS vs. control.

Acknowledgments:

We acknowledge the France-BioImaging/PICsL infrastructure (ANR-10-INSB-04-01), the Small animal imaging platform of Montpellier (IPAM) for echocardiography and France Life Imaging network (ANR-11-INSB-0006) for MRI.

Funding:

This work was supported by the Ligue contre la Cardiomyopathie, the Association Française contre les Myopathies (AFM-Téléthon) to L.M. and the Fondation pour la Recherche Médicale (DEQ20150331717) to R.G.K. C.C. was supported by the Groupe de Réflexion sur la Recherche Cardiovasculaire/Société Française de Cardiologie (GRRC/SFC). C.C. and J.V. were supported by the Association Française contre les Myopathies (AFM-Téléthon). N.L. and J.V. received support from the French government under the France 2030 investment plan, as part of the Initiative d'Excellence d'Aix-Marseille Université-A*MIDEX (AMX-19-IET-007).

Data Availability Statement:

Data are available from the authors upon request.

Institutional Review Board Statement:

The investigation was approved by the ethics committee for animal experimentation of the French ministry (no. 01055.02). Animal procedures conformed to the guidelines from Directive 2010/63/EU of the European Parliament for the Care and Use of Laboratory Animals.

Informed Consent Statement:

Not applicable.

Conflicts of Interest:

The authors declare no conflict of interest.


Iuliana M Andrei

Arnaud Chaix

∥ Belkacem

Tarek Benkhaled

Romain Dupuis

Chaimaa Gomri

Eddy Petit

Maurizio Polentarutti

Arie Van Der Lee

Mona Semsarilar

Mihail Barboiu

Selective Water Pore Recognition and Transport through Self-Assembled Alkyl-Ureido-Trianglamine Artificial Water Channels

In nature, aquaporins (AQPs) are proteins known for fast water transport through the membrane of living cells. Artificial water channels (AWCs) synthetic counterparts with intrinsic water permeability have been developed with the hope of mimicking the performances and the natural functions of AQPs. Highly selective AWCs are needed, and the design of selectivity filters for water is of tremendous importance. Herein, we report the use of self-assembled trianglamine macrocycles acting as AWCs in lipid bilayer membranes that are able to transport water with steric restriction along biomimetic H-bonding-decorated pores conferring selective binding filters for water. Trianglamine [(±)Δ, (mixture of diastereoisomers) and (R,R) 3 Δ and (S,S) 3 Δ], trianglamine hydrochloride (Δ.HCl), and alkyl-ureido trianglamines (n = 4, 6, 8, and 12) [(±)ΔC4, (±)ΔC8, (±)ΔC6, and (±)ΔC12] were synthesized for the studies presented here. The single-crystal X-ray structures confirmed that trianglamines form a tubular superstructure in the solid state. The water translocation is controlled via successive selective H-bonding pores (a diameter of 3 Å) and highly permeable hydrophobic vestibules (a diameter of 5 Å). The selfassembled alkyl-ureido-trianglamines achieve a single-channel permeability of 10 8 water molecules/second/channel, which is within 1 order of magnitude lower than AQPs with good ability to sterically reject ions and preventing the proton transport. Trianglamines present potential for engineering membranes for water purification and separation technologies.

■ INTRODUCTION

In the very near future, the world will face a global water depletion and the forecasts are increasingly alarming. Nowadays, 4 billion people suffer from a lack of access to clean water, a number that could reach 5.7 billion in 2050 according to the United Nations' report. [START_REF] Mekonnen | Sustainability: Four Billion People Facing Severe Water Scarcity[END_REF][START_REF] Boretti | Reassessing the Projections of the World Water Development Report[END_REF] Moreover, climate change, population growth, and the increase of industrial demand are accelerating the water scarcity. In order to counteract such factors and meet the needs of the population, innovative solutions are being researched within water purifications technologies. The past decade has seen the development of advanced membranes for the reverse osmosis (RO) as a major process for the production of millions of m 3 of desalinated water per day from oceans. Furthermore, a biomimetic approach to nature's functions in producing energy-efficient desalination membranes looking at artificial water channel (AWC)-based technologies appears as one of the solutions for low-energy water production. Nature has shown humankind its prowess; the goal would be to draw inspiration from the natural elements and design innovative, efficient, and less expensive technologies for water desalination. Aquaporins (AQPs) make up a family of protein-based membrane channels that play a crucial role in regulating the cellular osmotic pressure. AQPs efficiently conduct water across membrane bilayer while simultaneously exclude both hydrated ions (salts) and protons. [START_REF] Tajkhorshid | Control of the Selectivity of the Aquaporin Water Channel Family by Global Orientational Tuning[END_REF][START_REF] Carrillo | Crystallization and preliminary crystallographic analysis of human aquaporin 1 at a resolution of 3.28 angstrom[END_REF][START_REF] Kosinska Eriksson | Subangstrom Resolution X-Ray Structure Details Aquaporin-Water Interactions[END_REF] With very high selectivity and high rates of water transport (∼10 9 H 2 O molecules/s/channel), [START_REF] Agre | Aquaporin Water Channels (Nobel Lecture)[END_REF] AQPs have attracted interest in both academia and industries. Various attempts have been used to insert them into artificial membranes for water purification including medical and desalination applications. [START_REF] Shen | Biomimetic Membranes: A Review[END_REF][START_REF] Hélix-Nielsen | Biomimetic Membranes as a Technology Platform: Challenges and Opportunities[END_REF] However, manufacturing AQPbased membranes can be quite challenging because of their low stability to harsh pressure and salinity conditions as used in desalination. Bioinspired AWCs have been developed as an alternative strategy to overcome the limitations when using natural AQPs. [START_REF] Duc | Imidazole-Quartet Water and Proton Dipolar Channels[END_REF] On the other hand, AWCs address effective advantages due to a lower cost of production, high tunability, stability, and membrane compatibility, as well as high pore packing density with the surface membrane permeability. [START_REF] Shen | Achieving High Permeability and Enhanced Selectivity for Angstrom-Scale Separations Using Artificial Water Channel Membranes[END_REF][START_REF] Huang | Hydroxy Channels-Adaptive Pathways for Selective Water Cluster Permeation[END_REF] Interestingly, recent studies report the successful integration of AWCs into polyamide RO membranes, confirming the promising approach for highly efficient biomimetic membranes for water desalination. [START_REF] Licsandru | Salt-Excluding Artificial Water Channels Exhibiting Enhanced Dipolar Water and Proton Translocation[END_REF] In 2011, a series of pioneering work published by our group lead to the discovery of efficient AWCs such as I-quartet. [START_REF] Duc | Imidazole-Quartet Water and Proton Dipolar Channels[END_REF][START_REF] Licsandru | Salt-Excluding Artificial Water Channels Exhibiting Enhanced Dipolar Water and Proton Translocation[END_REF][START_REF] Murail | Water Permeation across Artificial I-Quartet Membrane Channels: From Structure to Disorder[END_REF][START_REF] Huang | Bilayer versus Polymeric Artificial Water Channel Membranes: Structural Determinants for Enhanced Filtration Performances[END_REF] Since this discovery, several studies reported the construction of new classes of AWCs such as carbon nanotube porins (CNTPs), [START_REF] Tunuguntla | Enhanced Water Permeability and Tunable Ion Selectivity in Subnanometer Carbon Nanotube Porins[END_REF] aquafoldamers, [START_REF] Shen | Aquafoldmer-Based Aquaporin-like Synthetic Water Channel[END_REF] pillararenes (PAs), [START_REF] Hu | Single-Molecular Artificial Transmembrane Water Channels[END_REF][START_REF] Yan | Artificial Aquaporin That Restores Wound Healing of Impaired Cells[END_REF][START_REF] Strilets | Biomimetic Approach for Highly Selective Artificial Water Channels Based on Tubular Pillar[5]Arene Dimers[END_REF][START_REF] Chen | Chiral Selective Transmembrane Transport of Amino Acids through Artificial Channels[END_REF] peptideappended Pillar [4]arenes (PAH [4]), [START_REF] Song | Artificial Water Channels Enable Fast and Selective Water Permeation through Water-Wire Networks[END_REF] porous organic cages (POCs), [START_REF] Yuan | Porous Organic Cages as Synthetic Water Channels[END_REF] and hydroxy channels (octyl-ureido-polyols) [START_REF] Huang | Hydroxy Channels-Adaptive Pathways for Selective Water Cluster Permeation[END_REF][START_REF] Mondal | Selective and rapid water transportation across a selfassembled peptide-diol channel via the formation of a dual water array[END_REF] and more recently were reported fluorous oligoamide nanorings. [START_REF] Itoh | Ultrafast water permeation through nanochannels with a densely fluorous interior surface[END_REF] Among these AWCs, few channels are able to translocate water with total salt rejection and the water vs ion/proton selectivity remains the actual challenge in creating novel designs. [START_REF] Huang | Artificial Water Channels: Towards Biomimetic Membranes for Desalination[END_REF] As an example, CNTPs with the highest water transport (six times the AQPs) have one of the lowest selectivities. [START_REF] Tunuguntla | Enhanced Water Permeability and Tunable Ion Selectivity in Subnanometer Carbon Nanotube Porins[END_REF] For this purpose, there is a high demand in searching for new AWCs and synthetic macrocycles appear to be one of the promising approaches due to their intrinsic porosity and multiple possibilities to generate nanochannels via self-assembly. Several types of synthetic macrocycles, [START_REF] Huang | Engineering Membranes with Macrocycles for Precise Molecular Separations[END_REF] such as crown ethers, 27 calixarenes, [START_REF] Atwood | Purification of C60 and C70 by Selective Complexation with Calixarenes[END_REF] cucurbituril, [START_REF] Lee | Cucurbituril Homologues and Derivatives: New Opportunities in Supramolecular Chemistry[END_REF] pillararenes, [START_REF] Ogoshi | Para-Bridged Symmetrical Pillar[5]Arenes: Their Lewis Acid Catalyzed Synthesis and Host-Guest Property[END_REF] and many others, [START_REF] Kaik | Unprecedented Selectivity in the Formation of Large-Ring Oligoimines from Conformationally Bistable Chiral Diamines[END_REF][START_REF] Dawn | Self-Assembled Phenylethynylene Bis-Urea Macrocycles Facilitate the Selective Photodimerization of Coumarin[END_REF][START_REF] Nalluri | Chiral Redox-Active Isosceles Triangles[END_REF][START_REF] Neira | Dissecting the "Blue Box": Self-Assembly Strategies for the Construction of Multipurpose Polycationic Cyclophanes[END_REF] have been designed for the development of applications such as separation, adsorption, catalysis, sensors, and drug delivery. Macrocycles can self-assemble through noncovalent interactions resulting in the formation of nonporous [START_REF] Wu | Synthetic Macrocycle-Based Nonporous Adaptive Crystals for Molecular Separation[END_REF][START_REF] Jie | Reversible Iodine Capture by Nonporous Pillar[6]Arene Crystals[END_REF] or porous supramolecular organic frameworks with linear, layered, or interconnected channels. [37][START_REF] Patil | Construction of Supramolecular Organic Frameworks Based on Noria and Bipyridine Type Spacers[END_REF][START_REF] Luo | A Microporous Hydrogen-Bonded Organic Framework: Exceptional Stability and Highly Selective Adsorption of Gas and Liquid[END_REF][START_REF] Chaix | Trianglamine-Based Supramolecular Organic Framework with Permanent Intrinsic Porosity and Tunable Selectivity[END_REF] Trianglamine macrocycles present several advantages due to their easy fabrication, purification, and scale-up at relatively low cost, making them an outstanding candidate so far for the construction of artificial aquaporins. Besides and since a recent decade, trianglamines have been used for several applications including gas capture/separation, [START_REF] Chaix | Trianglamine-Based Supramolecular Organic Framework with Permanent Intrinsic Porosity and Tunable Selectivity[END_REF] sensors, [START_REF] Chappanda | Trianglamine Hydrochloride Crystals for a Highly Sensitive and Selective Humidity Sensor[END_REF] membrane, [START_REF] Huang | Molecularly-Porous Ultrathin Membranes for Highly Selective Organic Solvent Nanofiltration[END_REF] and host-guest chemistry. [START_REF] Dey | Adsorptive Molecular Sieving of Styrene over Ethylbenzene by Trianglimine Crystals[END_REF][START_REF] Dey | Molecular Recognition and Adsorptive Separation Ofm-Xylene by Trianglimine Crystals[END_REF][START_REF] Hua | Tuning the Porosity of Triangular Supramolecular Adsorbents for Superior Haloalkane Isomer Separations[END_REF] To the best of our knowledge, no investigation was reported on the use of trianglamines as artificial aquaporins, emphasizing the novelty of this study.

Herein, we report the reference trianglamine (±)Δ (mixture of diastereoisomers), [START_REF] Gawronski | Conformationally Flexible Inclusion-Forming Chiral Hexamines[END_REF] (R,R) 3 Δ and (S,S) 3 Δ, [START_REF] Chadim | 3 + 3)-Cyclocondensation of the enantiopure and racemic forms of trans-1,2-diaminocyclohexane with terephthaldehyde. Formation of diastereomeric molecular triangles and their stereoselective solid-state stacking into microporous chiral columns[END_REF] and trianglamine hydrochloride (Δ.HCl) [START_REF] Chaix | Trianglamine-Based Supramolecular Organic Framework with Permanent Intrinsic Porosity and Tunable Selectivity[END_REF] previously reported macrocycles together with the novel alkyl-ureido-trianglamines porins [(±)ΔC4, (±)ΔC6, (±)ΔC8, and (±)ΔC12] (mixture of diastereoisomers) (Scheme 1), which were selected for transport studies as selective water channels through lipid bilayer membranes.

The alkyl-ureido-trianglamine channels present unprecedented well-established water H-bonding pores with a diameter of ∼3 Å (pore size of aquaporins) which is considered as an ideal size to selectively bind water and sterically reject bigger hydrated ions, successively superposed with an intrinsic porosity of ∼5 Å diameter that fastly translocate water. The successive distribution H-bonding and hydrophobic regions displays noncontinuous waters within trianglamine channels, allowing high water transport with ion/ proton rejection, making it a promising AWC platform.

■ RESULTS AND DISCUSSION Design Strategy. The trianglamine (±)Δ, (R,R) 3 Δ, (S,S) 3 Δ, and trianglamine hydrochloride [(±)Δ.HCl] macrocycles were prepared as previously reported [START_REF] Chaix | Trianglamine-Based Supramolecular Organic Framework with Permanent Intrinsic Porosity and Tunable Selectivity[END_REF] by reacting (±)-trans-1,2-diaminocyclohexane or (R,R)trans-1,2 diamino-cyclohexane or (S,S)trans-1,2 diaminocyclohexane (20 mmol) and terephthalaldehyde (20 mmol) in methanol in the presence of triethylamine (50 mmol), followed by reduction with sodium borohydride (3h) to afford (±)Δ consisting of a mixture of diastereomers composed of (R,R) 3 Δ, (R,R) 2 (S,S)Δ, (R,R)(S,S) 2 Δ, and (S,S) 3 Δ trianglamines and (R,R) 3 Δ and (S,S) 3 Δ trianglamines, respectively (Figure S1). S8). High-resolution mass spectrometry (ESI-MS +) and high-resolution elemental analysis were in agreement with the proposed formula (Figures S9-S14). Thermogravimetric analysis (TGA) confirms that trianglamine Δ starts to decompose at 350 °C with a complete degradation at 400 °C (Figure S15). Furthermore, differential scanning calorimetry (DSC) of the trianglamine (±)Δ showed crystallization and melting temperatures of 45 and 150 °C, respectively (Figure S16). We then synthesized four new lipophilic trianglamines in order to increase their solubility, insertion, and stabilization into the lipid bilayer membranes. The (±)ΔC4, (±)ΔC6, (±)ΔC8, and (±)ΔC12 were prepared through a one-step reaction in dry dichloromethane between the butyl-isocyanate C4, hexyl-isocyanate C6, octyl-isocyanate C8, and dodecylisocyanate C12, respectively, and the six secondary amines of the trianglamine, (±)Δ (Scheme 1). The obtained alkylureido-trianglamines were washed with hexane using centrifugation and without the need for further purification (yield ∼95%) (Figure S2). Based on water permeation results (see below the Water Transport Experiments) showing similar performances for racemic and enantiomeric trianglamines, we decided to use the racemic (±)Δ inside (R,R) 3 Δ and (S,S) 3 Δ enantiomers as a starting reagent for the synthesis of the hydrophobic (±)ΔCX compounds. Moreover, this is beneficial enough to produce at high scale and at lower cost stable active trianglamines for membrane engineering for water purification.

1 H and [START_REF] Murail | Water Permeation across Artificial I-Quartet Membrane Channels: From Structure to Disorder[END_REF] C NMR, ESI-MS, high-resolution elemental analysis, Fourier-transform infrared (FTIR), and TGA confirmed the successful functionalization of the trianglamine with alkyl-ureido-chains. Interestingly, the 1 H NMR spectra of the alkyl-ureido-trianglamines (±)ΔC4, (±)ΔC6, (±)ΔC8, and (±)ΔC12 performed in CDCl 3 showed broad signals, most probably due to the formation of highly self-aggregated oligomers fastly self-exchanging in solution (Figures S18-S21). In dimethyl sulfoxide (DMSO) and upon heating at 100 °C (Figures S22-S25), the 1 H NMR signals of the alkylureido-trianglamines (±)ΔC4, (±)ΔC6, (±)ΔC8, and Scheme 1. Chemical Structures of (±)Δ, (±)ΔC4, (±)ΔC6, (±)ΔC8, and (±)ΔC12

Journal of the American Chemical Society (±)ΔC12 become sharper and the proton resonances could be assigned as a mixture of diastereoisomers of trianglamines, as previously assigned for(±)Δ. The 13 C NMR (Figures S26-S29), ESI mass spectra, and high-resolution elemental analysis confirmed the successful functionalization with correct molecular weights for hexasubstituted compounds (±)ΔC4, (±)ΔC6, (±)ΔC8, and (±)ΔC12 (Figures S30-S37). Their structures were further supported by ATR-FTIR spectra where new intense bands at ν N-C = 1540 cm -1 and ν C�O = 1620 cm -1 and ν CH2as = 2852 cm -1 and ν CH2sim = 2925 cm -1 assigned to the stretching vibrations of urea bonds and methylene moieties of alkyl chains appeared (Figures S38-S39). TGA analysis shows that the self-assembled alkyl-ureatrianglamines decomposed at higher temperature 400 °C compared to that at 350-400 °C for the unfunctionalized trianglamine (Figure S40).

Self-Aggregation in Solution.

To gain insight into the mechanism of formation of AWC aggregates via self-assembly, we monitored, by dynamic light scattering (DLS) experiments, Δ and ΔCX samples that were dissolved in an ethanol, followed by sonication. The aggregation behaviors were strongly dependent on the concentration of (±)Δ, (±)ΔCX, and the solvent (EtOH)/nonsolvent (H 2 O) volume ratios. Herein, for 0.5 and 0.9 wt % solutions of Δ, (±)ΔCX, a linear increase of the hydrodynamic diameter D h of colloidal aggregates was observed after adding a certain amount of water, from D h = 400 to D h = 1200 nm for (±)Δ, (±)ΔC4 and from D h = 500 to D h = 2000 nm for the more lipophilic ones (±)ΔC6 and (±)ΔC8 (Figure S41), without any visible precipitation. The constant increase of the dimension of D h particles shows an increased aggregation behavior for longer alkyl tails such as (±)ΔC12 for which the aggregation was very fast in comparison, the trianglamine (±)Δ where smaller layered particles are observed.

X-Ray Single-Crystal Structure. In order to have more precise information about possible molecular packing and their self-assembly behaviors, compounds ± Δ and (±)ΔC4 were crystallized by slow evaporation (3 days) of solvents from ethyl acetate/benzene mixtures, resulting in the formation of good quality crystals useful for X-ray structure determination (Figure 1). The single structures of (±)Δ.HCl have previously been reported (Figure S42). [START_REF] Chaix | Trianglamine-Based Supramolecular Organic Framework with Permanent Intrinsic Porosity and Tunable Selectivity[END_REF] The single-crystal X-ray structures of (±)Δ (Figure 1a) confirm the macrocyclic trianglamine superstructure in which the central phenyl rings are slightly twisted, closing a free water hydrophobic pore of 4 Å diameter (Figure 1). It consists of the selective crystallization of a mixture of (R,R) 3 Δ and (S,S) 3 Δ to form a single crystal of (±)Δ. This suggests a possibility that the solubility in solvents is different among the diastereomers and that the specific diastereomers (R,R) 3 Δ and (S,S) 3 Δ tend to form a possible self-assembled structure in the lipid bilayers. In the crystal, each macrocycle interacts with two neighboring ones so cyclohexyl and phenyl groups fill the interstices so that all available void space between the components is filled. Accordingly, arrays of closed packed trianglamines are generated in the solid state. The alternative packing (±) Δ single-crystal structure is different from previously reported (R,R)Δ single-crystal tubular packing which is strongly dependent on the used crystallization solvent. [START_REF] Gawronski | Conformationally Flexible Inclusion-Forming Chiral Hexamines[END_REF][START_REF] Chadim | 3 + 3)-Cyclocondensation of the enantiopure and racemic forms of trans-1,2-diaminocyclohexane with terephthaldehyde. Formation of diastereomeric molecular triangles and their stereoselective solid-state stacking into microporous chiral columns[END_REF] The single-crystal X-ray structures of (±)ΔC4 confirm the selective crystallization of a mixture of (R,R) 3 ΔC4 and (S,S) 3 ΔC4 (Figure 2) and confirm the coupling of six alkylureido groups to the trianglamine backbone, resulting in the formation of a "bouquet"-molecule conformation. Three amide groups of lateral alkyl-urea chains display two donor N-H and one acceptor C�O groups optimally fitting the space to form anchoring H-bonds around one water molecule. Importantly a single water molecule is strongly encapsulated in the pocket jointly formed by the lateral arms (Figure 2a,b), which induce the constriction of the triangular backbone of the central phenyl segment toward the narrowest twisted water binding pores of 3 Å diameter, superposed with larger hydrophobic pockets of 5 Å diameter between the macrocycles (Figure 2c).

The other three lateral arms adaptively bind to neighboring arms bridging different trianglamines to form a network of four water channels in the solid state via hydrophobic packing of cyclohexane rings of monomers in the center of the tetramer (Figure 2d), reminiscent with human AQP1 tetrameric structure (Figure 2e). [START_REF] Carrillo | Crystallization and preliminary crystallographic analysis of human aquaporin 1 at a resolution of 3.28 angstrom[END_REF] This amazing biomimetic directional pore offers a combined size restriction of 3 Å H-bonding pore and a large hydrophobic pocket of 5 Å reminiscent to natural AQPs that selectively allows water molecules for a fast transit of the overall structure and together with a selective rejection of the hydrated cations. Finally, this structure does not allow any water wire formation in the solid state that could be an

D

important premise for proton rejection as observed for AQPs. [START_REF] Kosinska Eriksson | Subangstrom Resolution X-Ray Structure Details Aquaporin-Water Interactions[END_REF][START_REF] Agre | Aquaporin Water Channels (Nobel Lecture)[END_REF] Liposome Transport Experiments. In order to check the transport performances of the trianglamine channels, the compounds (±)Δ, (R,R) 3 Δ and (S,S) 3 Δ, (±)Δ.HCl, (±)ΔC4, (±)ΔC6, and (±)ΔC8 were tested for water, ion, and proton transport. The water transport experiments were assessed by standard stopped-flow technique. [START_REF] Licsandru | Salt-Excluding Artificial Water Channels Exhibiting Enhanced Dipolar Water and Proton Translocation[END_REF] The method is based on mixing two osmolyte D(+)-sucrose solutions of different concentrations; thus, the instrument detects the shrinkage rate of vesicles under osmotic pressure determined by the induced outward water permeability through the trianglamine channels. The ion transport was conducted for different cations (Li + , Na + , K + , Rb + and Cs + ) and was assessed by using a pH-sensitive HPTS fluorescence assay. [START_REF] Matile | Transport Experiments in Membranes[END_REF] Moreover, the use of the FCCP and valinomycin as assisting H + and K + carriers, respectively, determines the intrinsic selectivity of the channels based on deconvoluted transport rates.

Water Transport Experiments. The trianglamine channels were reconstituted into a phosphatidylcholine (PC)/ phosphatidylserine (PS)/cholesterol (Chl) (PC/PS/Chl) mixture with a molar ratio of 4/5/1 obtaining lipid vesicles with a 100 mm in diameter. The trianglamines (±)Δ, (R,R) 3 Δ and (S,S) 3 Δ, (±)Δ.HCl, (±)ΔC4, (±)ΔC6, (±)ΔC8, and (±)ΔC12 were added from their DMSO solutions to the preformed vesicle suspension at different molar compound to lipid ratios (mCLRs) (see Supporting Information for details). The water transport measurements were performed under hypertonic conditions driven by an outwardly 10 mM PBS solution (pH = 6.4) of 200 mM D(+)-sucrose osmolyte. The shrinkage of the liposomes produced an increase of the lightscattering signal (Figure S49). In order to facilitate the description of the influence of the chemical structure of the trianglamine correlated with their water transport performances, the compounds were grouped in two categories, as they presented different behaviors. For the first category containing macrocyclic (±)Δ, (R,R)Δ and (S,S)Δ, and (±)Δ.HCl, the water transport rates were dependent on the concentration used of the channels; the net permeabilities show a continual increase showing a saturation behavior when mCLRs increase (Figure 3). The range of the permeabilities obtained with Δ.HCl and (±)Δ, (R,R) 3 Δ, and (S,S) 3 Δ is from 6.91 to 10.5 μm/s at high mCLR = 0.231 (Figure S50).

Interestingly, the net permeabilities of racemic (±)Δ and enantiopure (R,R) 3 Δ and (S,S) 3 Δ trianglamines as well as a equimolar mixture of enantiomers at a channel-to-lipid weight ratio of 0.2309 (CLR = 0.2309) are almost the same order of magnitude around 8 μm/s (Figure 3b). This observation suggests that the lipids do not exert a conformational preference, stabilizing the chiral superstructures over the racemic superstructures for which the insertion and the water transport activity do not depend on the chirality. Therefore, we pursued our studies on hydrophobic less expensive compounds (±)ΔC4, (±)ΔC6, (±)ΔC8, and (±)ΔC12. When considering the second category of hydrophobic alkylurea-trianglamines (±)ΔC4, (±)ΔC6, (±)ΔC8, and (±)ΔC12, the water transport rates are strongly dependent on the variations of the length of the alkyl chain. (±)ΔC4 and (±)ΔC12 presented lower water permeabilities regardless of the concentrations used; thus, it can be stated that a more compact packing into the membrane may interfere with the water transport rates. Shorter alkyl chains provide less insertion of (±)ΔC4 within membrane, while sterical impediments of longer chains lead to a dysfunctional insertion of (±)ΔC12 into the membrane. The net permeabilities for (±)ΔC4 and (±)ΔC12 are almost the same: 2.45 to 2.85 μm/s at mCLR = 0.0014 and 2.58 to 3.68 μm/s at high mCLR = 0.0231 (Figure S40), respectively. The (±)ΔC6 and (±)ΔC8 trianglamines presented higher water permeabilities. For (±)ΔC6, the net permeability is almost constant over lower or higher mCLR (5.30 μm/s at mCLR = 0.0014 and 6.32 μm at mCLR = 0.0231). For the (±)ΔC8 at low mCLR = 0.007-0.0014, the net permeabilities are ∼3.4-3.7 μm/s, then we observed a saturation behavior reached at mCLR = 0.0115 with a maximal net permeability of 6.38 μm/s (Figure S49).

We further estimated the single-channel permeability (P s ) of the trianglamine channels, based on their total insertion, lipid concentration, and the channel self-assembly into the lipid bilayers.

To be able to determine the single-channel permeabilities, we considered that the number of stacked molecules needed to form a channel that crosses the entire length of 35 Å of the bilayer is equal to 5 molecules, equal to the number of molecules found in the single-crystal structure of (±)ΔC4 (Figure 2c). The single-channel permeability values varied from 2.0 × 10 7 water molecules/s/channel at mCLR = 0.19 for (±)Δ and (±)Δ.HCl (Table S1), while single-channel permeability values between 4 × 10 8 at 10 times more diluted mCLR = 0.029 were determined for lipophilic (±)ΔC4, (±)ΔC8, (±)ΔC6, and (±)ΔC12 (Table S2), which are 1 or 2 orders of magnitude lower than those of AQPs (∼10 9 water molecules/s/channel). 6 Δ and Δ.HCl showed higher water net transport permeabilities for 10 times more concentration in the bilayer membranes, when compared with more lipophilic (±)ΔC4, (±)ΔC6, (±)ΔC8, and (±)ΔC12. These higher net permeabilities are reminiscent of the increased solubility of (±)Δ and (±)Δ.HCl in the membrane. The lipophilic (±)ΔCX generates stabilized percolated channels with higher single channel permeability, but they are less soluble in the membrane due to their aggregation. The single channel permeability is strongly enhanced for the compounds presenting higher hydrophobicity, as they can form stable cluster-type membrane aggregation that allows for fast and selective water single-channel translocation.

Cation and Proton Transport Experiments. The cation transport activity of all of the compounds was tested using the standard HPTS fluorescence assays. The large unilamellar vesicles containing PC were hydrated with a PBS buffer solution (10 mM, pH 6.4) containing an alkaline salt (LiCl, NaCl, KCl, RbCl, and CsCl) with a concentration of 100 mM. Cation (M + = Li + , Na + , K + , Rb + , and Cs + ) transport activities across the bilayer membrane incorporating (±)ΔC4, (±)ΔC6, (±)ΔC8, and (±)ΔC12 channels were tested at the same mCLRs used for water transport (see Supporting Information for details). When tested with MCl on external buffer (±)ΔC4, (±)ΔC6, (±)ΔC8, and (±)ΔC12 do not present a dose-response-type activity, showing constant nearly zero transport activity demeanor, regardless of the concentration used. Δ.HCl and Δ showed a slight change in the fluorescence intensity when base was added into the extravesicular media reminiscent of HPTS deprotonation and a rapid proton transport via the -NH groups of trianglamines. However, after the equilibrium was reached, the dose-response profile displays constant absorbance behaviors (Figures S51-S55). It is clear that M + /H + antiport conductance of the trianglamine channel states across the lipid bilayer membrane is not observed for any of the studied concentrations (Figure 4).

To complement these results and check whether H + transport occurs through the assembled channels, the compounds were tested for K + /H + antiport activity. We conducted standard HPTS assays with (±)ΔC4, (±)ΔC6, (±)ΔC8, and (±)ΔC12 in the presence of cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) as a H + carrier and valinomycin as a K + carrier (Figure S56).

The membrane polarization via FCCP and valinomycin for (±)ΔC4, (±)ΔC6, (±)ΔC8, and (±)ΔC12 does not generate an increase in fluorescence intensity, meaning that their channel structuration is not suitable to accommodate K + /H + antiport (Figure 5). In the case of (±)Δ and (±)Δ.HCl, it generated a strong initial increase in fluorescence intensity, but after pH equilibrium was reached, the dose-response experiments had a constant absorbance acquisition. Overall, all tested compounds had very low total inactivity for K + and H + transport in the presence of FCCP and valinomycin.

Molecular Dynamic Simulations. Molecular dynamics simulations (see Supporting Information for details) have been used to test the stability of self-assembled superstructures F formed by stackings of eight (±)ΔC4 macrocycles as extracted from its crystal structure of eight bundles of a mixture of (R,R) 3 ΔC4 and (S,S) 3 ΔC4 and immersed in water at room temperature. We noted that the stabilization effect of the stackings by the alkyl chains via H-bonding and hydrophobic effects prevents exfoliation of the layers (Figure 6). This confirms that it is thermodynamically possible to obtain stable regular self-assembled channels as observed experimentally. The monomer highlighted in orange, while other monomers have a semitransparent shading in order to increase visual contrast in Figure 6a, has three lateral ureido-alkyl arms pointing up and the other three pointing down.

The ureido-alkyl arms appear to interpenetrate with each other, creating a locking mechanism of these monomers that stabilizes the overall structure. This highlights the importance of the interplay between the different monomers and their alkyl arms in creating a functional and stable trianglamine channel.

The (±)ΔC4 stackings that contain eight layers have a length of approximately 50 Å; as a result, the interlayer distance is 6 Å, which is in agreement with the experimental expectations of having 5 molecules stacking to form a 35 Å channel. The narrowest pore size, perpendicular to the channel length, is 3 Å in accordance with the obtained X-ray results. Thus, the channel can accommodate water molecules with a kinetic diameter of 2.65 Å. Further molecular simulations allowed us to assess whether the water-charged superstructures may selectively accommodate water translocation. These simulation results taken together suggest that at some stage, the lateral amide arms may rearrange so water is oscillating into a sponge like form (green trajectories shown in Figure 6b,c), to give rise to a limited water flow stabilized via amide H-bonding in this narrowest region of the pore. Radial distribution function (Figure S57) between water molecules and the channel shows a peak at a distance of 1.8 Å, suggesting that strong H-bonding between water molecules is occurring within the channel. With such a stabilized water cluster, water transport is indeed observed between different clusters and it is occurring in the external hydrophobic pocket of the Δ platforms in-between the alkyl arms (red trajectories shown in Figure 6b,c). The distribution of restrictive pore sizes along a variable porous structure reflects the transient appearance of water clusters between these regions inside the channel. For such narrow pores, the entrance of hydrated ions into the channels is restricted by the dehydration energy penalty arising from their incomplete hydration within (±)ΔC4 channels. The water cluster stabilization via H-bonding to amide bonds confers to these channels selective behaviors that make the intrinsic features of small pores particularly self-protective against ions.

We show that systematic functionalization of trianglamine macrocycles with alkyl-ureido chains leads to highly effective and selective AWCs. Simple use of such lateral amide arms led to the formation of an unexpected binding/recognition of one water molecule within a selective pocket of the molecular receptor via a combination of three donor-acceptor H-bonds of lateral amide arms. It results in an increased water-transport activity at lower concentration of the alkyl-ureido-trianglamine channels within membranes. The stacking of several trianglamines led to the formation of such pocket-decorated pores conferring selective binding for water accompanied by the formation of larger pores between trianglamines favorable for fast water translocation.

From a mechanistic point of view, the H-bonding network offered by the six-armed alkyl-ureido arms can act itself as a recognition-based barrier with high selectivity for water translocation. Moreover, the H-bonding of water molecules can intervene with both donor and acceptor H-bonds, satisfying the full number of hydrogen bonds within the pocket and making possible its facilitated translocation through the gate. Oppositely, the energy penalty for binding either cations or anions would be too high, as the entrance of hydrated ions into the channels may be restricted by the dehydration energy penalty arising from the preference of ions to be surrounded by water molecules rather than incomplete hydration within trianglamine channels. The distribution of pockets along the channel is not continual and reflects a transient appearance of single water molecules inside the channel, so proton translocation through such discontinuous waters is prohibited. It is very tempting to equate that selective water binding pockets can form permeable waters in the bilayer membrane, so that triangle channels feature this rather unique selective permeability of water, rejecting ions and protons.

■ CONCLUSIONS

In summary, we have developed a new class of AWCs based on self-assembled trianglamines and alkyl-ureido trianglamines. They self-assembled in directional channels that present enhanced selective water permeability with almost total proton rejection across a lipid bilayer. More importantly, the structuration of these channels is not suitable to accommodate hydrated cations and presents total salt rejection, which can offer a close approach in mimicking the functions of natural AQPs. The results presented here are of great importance, as these AWCs showed adaptive behaviors of water inside the channels being strongly dependent on the dimension and structuration of the channels and being active at the very low concentration used. The trianglamine channels described here, which are capable of mimicking the natural functions of AOPs, are holding significant promise for further developing energyeffective membranes for water desalination.

■ ASSOCIATED CONTENT

* sı Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.3c02815. Materials and methods, NMR and ESI MS spectra analysis of studied compounds, TGA and DSC analyses, FTIR, DLS, X-ray diffraction powder, SCXRD, XRD1 beamline of Elettra synchrotron, and transmembrane transport experimental and molecular dynamic simulation (PDF)

1 H

 1 and 13 C NMR spectra in CDCl 3 confirm the chemical structure of the macrocycles (±)Δ, (R,R) 3 Δ, and (S,S) 3 Δ (Figures S3-

Figure 1 .

 1 Figure 1. (a) Trianglamine macrocycle Δ with a 4 Å inner diameter (N, blue; C, gray; H, white) and (b) crystal packing in stick representation.

Figure 2 .

 2 Figure 2. (a) Top and (b) lateral view of crystal structures in stick representation of (±)ΔC4 encapsulating a water molecule inside a 3 Å triamide H-bonding pocket (N, blue; C, gray; O, red; H, white). (c) Lateral view channel type packing of (±)ΔC4 composed of external hydrophobic platform (blue) of 5 Å diameter and of narrowest twisted triamide pore (red) of 3 Å diameter encapsulating water in CPK representation. (d) Tetrameric crystal packing of alkyl-ureido-trianglamine channels, reminiscent with (e) tetrameric protein structure of human AQP1 (Reproduced with permission from ref 48. Copyright 2001, Springer Nature).

Figure 3 .

 3 Figure 3. Water transport activity in liposomes through Δ-channels. Comparison of net permeability values for (a) Δ-channels at different mCLRs and for (b) commercial diasteroisomeric(±)Δ, enantiomeric (R,R) 3 Δ and (S,S) 3 Δ, and their racemic mixture (MCLR = 0.231) determined by stopped-flow light scattering experiments under hypertonic conditions abruptly exposed to a solution of 200 mM D(+)-sucrose as osmolyte (Δ osm = 212 mOsm/kg).

Figure 4 .

 4 Figure 4. Comparison of Na + transport activity expressed as normalized fluorescence intensity of 1 mM Δ-alkyl channels containing in the extravesicular media 100 mM NaCl in PBS (pH 6.4) together with a schematic representation of the M + /H + antiport mechanism that should occur through the assembled channels.

Figure 5 .

 5 Figure 5. Coupled transport experiments of (±)ΔCX channels (1 mM) with (a) FCCP (50 μM) as H + carrier and (b) valinomycin K + carrier (50 μm) and containing 100 mM KCl in PBS (pH 6.4) in the extravesicular media.

Figure 6 .

 6 Figure 6. (a) Lateral view of the (±)ΔC4 water channel, where 8 layers stacking after 50 ps of simulation at 300 K are shown in different snapshots obtained simulating a periodic channel structure. Snapshots (b) side view and (c) top view of a water molecule trajectory in (±)ΔC4 channels obtained by NPT simulation at room temperature and ambient pressure. Two successive Δ platforms are represented in cyan and yellow. Each green or red sphere represents a position in time of the same single water molecule center of mass (over a trajectory of 25 ns). Green represents the instants in which the water molecule is H-bonded via amide bonds, while diffusion is limited, red represents instants of diffusion between layers occurring in the external pocket of the Δ in-between the alkyl arms.

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