Dr Emilie Mari

Pr Yasmina M Bousmah

Dr Céline Boutin

Dr Estelle Léonce

Dr Gaelle Milanole

Thierry Brotin

Patrick Berthault
email: patrick.berthault@cea.fr

Marie Erard
email: marie.erard@u-psud.fr

Erard

Bimodal detection of proteins by 129 Xe NMR and fluorescence spectroscopy

The full understanding of biological phenomena involves sensitive and non -invasive detection. Here we report the optimization of a probe for intracellular proteins that combines the advantages of fluorescence and hyperpolarized 129 Xe NMR detection. The fluorescence detection part is composed of six residues containing a tetracysteine tag (-CCXXCC-) genetically incorporated into the protein of interest and of a small organic molecule, CrAsH. CrAsH becomes fluorescent when it binds to the tetracysteine tag. The part of the biosensor that enables 129 Xe NMR detection, linked to the CrAsH moiety by a spacer, is based on a cryptophane core fully suited to reversibly host xenon. We benchmark three different peptides containing the tetracysteine tag and four organic biosensors of different stereochemistry to propose the best couple, fully suited for the in vitro detection of proteins.

Introduction

The study of intracellular biological mechanisms requires sensitive and non-invasive techniques. The combined use of several analytical methods makes it possible to overcome the limitation of each of them. This is the viewpoint of the multimodal approaches, highly developed in recent years, in particular for in vivo molecular imaging. [1] Fluorescence imaging techniques are easy to implement and widely used due to their high sensitivity and excellent spatial and temporal resolution. For the imaging of samples such as tissues or live organisms, the observation depth in fluorescence microscopy is limited by light scattering. This drawback could be overcome by the complementary use of magnetic resonance imaging if the inherent lack of sensitivity of this technique is circumvented for instance by using hyperpolarized 129 Xe NMR. We report here the optimization of a synthetic biosensor for a specific protein of interest based on the dual detection by fluorescence imaging and hyperpolarized 129 Xe NMR (Figure 1). The fluorescence detection part involves a genetically encoded target peptide fused to the protein of interest which contains a tetracysteine tag (TC-tag: -CCXXCC-). The biosensor ligand moiety, CrAsH, is a biarsenical fluorogenic compounds [2] that is a carboxy derivative of the FlAsH probe, initially developed by Tsien and coworkers. [3] The two pairs of thiol groups present in the target peptide react with the arsenic atoms of the CrAsH ligand to form a covalent bond. The high affinity of CrAsH to the tetracysteine tag of the target peptide leads to the specific detection of the protein of interest. [4] In addition, upon binding, CrAsH switches from a dark to a strongly fluorescent state allowing a very good signal to noise ratio in fluorescence imaging.

The interest of magnetic resonance imaging (MRI) for molecular imaging has experienced a renewed enthusiasm 20 years ago with the development of the first activatable contrast agents that act as biosensors in response to a specific biological activity. [5] However, this technique suffers from poor sensitivity due to the very low population differences between the nuclear spin energy levels at Boltzmann equilibrium. A way to tackle this lack of sensitivity consists in transiently unbalancing the nuclear spin repartition of the energy level populations initially at the Boltzmann distribution, by polarization transfer from a more ordered system such as photons or electrons. Among the species that can be spin-hyperpolarized, xenon is of high interest, due to its exogenous nature leading to the absence of background signal and the fact that it can act as a spy of biological events without interfering on them. Moreover owing to the high deformability of its large electron cloud, xenon is deeply sensitive to its local environment and constitutes a powerful NMR probe for various biological interactions. Soluble in most biological fluids, xenon can cross the plasma membrane in a few tens of milliseconds without losing its hyperpolarization. [6] All these properties led to 129 Xe NMR-based biosensors whose concept is based on reversible encapsulation of the noble gas into dedicated molecular hosts that specifically alter the resonance frequency of the xenon nuclei. [7] In our case the host is based on a cryptophane cage and combines 129 Xe NMR-based and fluorescence detection (Figure 1A). Other dual fluorescence / Xe NMR probes have been proposed in the literature. [8] Here however the concept is extended to the conception and use of a probe activatable both in fluorescence and 129 Xe NMR (i. e. the responses of these modalities change upon binding to the target). With respect to our previous study in ref 9, [9] the reported chemical shift modifications of caged xenon induced by the binding of the probe to the target should be improved further for in cellulo biosensing applications. In this study, a set of four biosensors and three targeted peptide motifs have been investigated in order to optimize the 129 Xe NMR response. The most promising couple has then used for the 129 Xe NMR detection of purified proteins in vitro. At each step, the response in fluorescence spectroscopy of the best couple has been evaluated.

Results and Discussion

Biosensors are synthesized from the combination of a watersoluble hexacarboxylate cryptophane-222 [10] and a CrAsH moiety (Figure 1B). The choice of a hydrophilic cryptophane core avoids aggregation or formation of self-organized systems. [11] Two enantiomers according to the helicity of the linkers between the triveratrylene bowls of the cryptophane (M;M or P;P) have been purified from the initial synthesis. [12] The CrAsH moiety has been obtained after addition of an anhydride (4-carboxyphtalic anhydride) on resorcinol. It leads to an equimolar mixture of two isomers of carboxyfluorescein. Then, the two atoms of arsenic and the ethylene diamine group in the para or meta positions toward the carboxyl function are introduced (Figure 1B). [13] The coupling of both CrAsH isomers to each enantiomer of cryptophane leads to two products labelled 1 or 2 (see supporting information for experimental details and product characterization). MM-1, MM-2 and PP-1 have been isolated for further investigation.

It has been impossible to properly isolate a sufficient amount of PP-2 from PP-1 due to the low yield of the coupling reaction (< 6%). Note that, as no stereogenic center is present on the CrAsH moiety, MM-1 and PP-1 are enantiomers and therefore xenon in their cavities exhibits the same 129 Xe NMR spectrum. It is the same for MM-2 and PP-2. The aromatic rings of the cryptophanes create a large shielding of the encapsulated Xenon. As a consequence, the expected NMR signals are upfield shifted from the dissolved xenon signal by more than 120 ppm. We observed a clear 129 Xe NMR response for the different biosensors MM-1 and MM-2 (Figure 2). This proves that the cavity of the biosensor is able to cage the noble gas. We have also checked by fast repetition of the sequence (soft pulse at the Xe@cryptophane frequencyacquisition) [14] that the xenon exchange in and out of the cavity is not impaired by the spacer nor by the CrAsH moiety. As MM-1 and MM-2 are both products of the same chemical reaction, traces of one isomer are still remaining in the other compound (indicated by an asterisk in Fig. 2). The minor peak in the MM-1 129 Xe NMR spectrum corresponds to xenon in MM-2 and vice versa (Figure 2A andB). Both signals are separated by less than 1 ppm (67.1 ppm and 66.3 ppm for MM-1 and MM-2 respectively) showing the high environmental sensitivity of 129 Xe NMR. We did not observe the signature of xenon encapsulated in the hexacarboxylate cryptophane at 64 ppm in the 129 Xe NMR spectra of MM-1 and MM-2 (Figure 2C): all the biosensors in the solution are thus chemically intact, composed of the cryptophane cage, the spacer and the CrAsH ligand.

The NMR responses of the three biosensors have been evaluated in the presence of three model peptides possessing the tetracysteine motif, -CCXXCC-. The three peptide sequences have been chosen in such a way that the distance between the two pairs of cysteines matches the spacing between the two arsenics. As a consequence, the two dithiol-arsenic interactions are highly cooperative and entropically favorable. The first peptide (P1) has two pairs of cysteines spaced by an arginine and a glutamic acid (WEAAAREACCRECCARA). [3a] This peptide has been selected for its ability to promote the formation of an α-helix -due to the EAAAR sequence -, the four thiols then forming a parallelogram on one of its faces. The second peptide (P2), has a proline and a glycine between the two pairs of cysteines (FLNCCPGCCMEP). [15] NMR studies have shown that P2 adopts a non-traditional β-hairpin structure in complex with bi-arsenical compounds, strongly different from the α-helix structure of complexed P1. [16] The amino acids before and after the two pairs of cysteines have shown their influence on the complexation properties. [3b] This led to the study of the third peptide P3 (AREACCPGCCK), which is an optimized version of P2 fully adapted for CrAsH. [2] The chemical shift of caged xenon is strongly modified upon binding of the biosensors to the peptide target (Figure 3). When MM-1 is bound to the three different peptidesthe flexible strand P3 (Figure 3A), the β-hairpin P2 (Figure 3B), or the α-helix peptide P1 (Figure 3C)xenon encapsulated in the cryptophane exhibits different signals. For instance δ = 67.7 ppm for P3 and δ = 74.1 ppm for P1 (Figure 3A,C). For P2 several signals are observed (Figure 3B). They are shifted in comparison to the signal of the free biosensor (dashed line). This is likely the consequence of multiple grafting configurations of the biosensor on the hairpin. Indeed, even if the caged xenon is distant from the peptide, the structure of the peptide may lead to significantly different xenon chemical shifts. The other enantiomeric form of the hexacarboxylate cryptophane, PP-1, leads to a 129 Xe chemical shift of δ= 76.3 ppm when bound to the flexible strand P3 (Figure 3D). Whereas MM-1 and PP-1 are enantiomers, as soon as they are grafted on a peptide, the 129 Xe NMR chemical shifts of caged xenon are significantly different (Figure 3A,D). Note that for PP-1 in interaction with P3, as for MM-1 bound to P2, the presence of anotherbroaderxenon signal witnesses the multiple grafting configurations of the biosensor. In this case, we have checked by dilution experiments that it could be due to intermolecular grafting (e. g. one biosensor molecule bound to two peptides). The interaction of P1 with MM-2 leads to a xenon resonance frequency of δ = 84.5 ppm (Figure 3E). The exchange of the grafting position of the spacer on CrAsH from MM-1 to MM-2 results in a large xenon chemical shift splitting of 10.4 ppm (comparison between spectra in Figures 3C and3E). Due to the extreme responsiveness of xenon towards perturbation in its environment, every modification of the biosensor structure results in new caged xenon chemical shift. The three biosensors lead to a single 129 Xe signal depending on the cryptophane enantiomeric form, the CrAsH ligand grafting position and the peptide conformation. The strongest influence on the xenon magnetic shielding is due to the structure of the cryptophane cage itself. Nevertheless, it is not possible to pinpoint the structural feature of the molecular assembly responsible of the variation of the caged xenon chemical shift itself. The largest chemical shift variations with respect to the free biosensors have been observed for the combination PP-1 / P3 and MM-2 / P1 (9.2 ppm and 18.2 ppm respectively, Figure 3 D,E). Furthermore, the peak at δ = 76.3 ppm for the couple PP-1 / P3 is thinner than the signal obtained with MM-2 / P1 at δ = 84.5 ppm (FWMH ca. 50 Hz versus 85 Hz, values extracted from a Lorentzian deconvolution). Tsien and coworkers suggest that P1 could adopt multiple grafting configurations with CrAsH. [3a] This would lead to a different environment for the xenon and a slight broadening of the signal as it is observed for the MM-2 / P1 combination. With PP-1 / P3, the main peak is likely the result of only one grafting configuration, explaining the thinner signal whatever the concentration. As a consequence, we have kept the PP-1 / P3 combination in the next experiments. We have further studied the coupling of PP-1 with P3 by monitoring the fluorescence properties of the CrAsH moiety in PP-1 upon binding. Its fluorescence increases 19 times upon binding to the peptide P3 and reaches its maximum at 10 equivalents (Figure 4A). This maximum is obtained after 50 minutes of reaction and remains constant beyond. In order to estimate an apparent dissociation constant for the complexation of P3 with PP-1, we plotted the variations of fluorescence intensity with the amount of peptide. They can be fitted by a saturation binding curve, leading to an apparent dissociation constant 8 ± 1 µM (Figure 4B). This affinity is lower than the Kd value of ~0.5 µM found for CrAsH with the same peptide in the same experimental conditions. [2] This is due to the presence of the cryptophane cage fused to the ligand moiety. We have further investigated the evolution of the fluorescence emission spectra of PP-1 upon addition of the peptide P3 using normalized spectra. For ratios 0.1 to 1, the shape of the spectra widens and, in the meantime, the wavelength of the maximal fluorescence shifts to higher wavelengths, from 520 nm to 540 nm depending on the fraction of PP-1 bound to the peptide (Figure 4C). When there is a significant excess of peptide, the fluorescence emission spectrum sharpens again. The shape widens at first most likely due to the simultaneous presence of at least 3 populations, (i) free PP-1 -whose emission wavelength is centered at 520 nm, (ii) PP-1 / P3 complex whose emission wavelength is centered at 540 nm and (iii) intermediate structures such as monoarsenical complexes or inter-peptide complexation. The shifts depend on the proportion of PP-1 / P3 complex. As there is only a very little evolution of the fluorescence signal between 5 and 10 equivalent of P3, we assume that at 10 equivalents of peptide, the PP-1 / P3 complex is most likely present alone in solution, all PP-1 biosensors being bounded to P3. The fluorescence intensity is very low in absence of peptide and its increase is proportional to the fraction of bound PP-1 when P3 is added (Figure 4A). So, the fluorescence intensity at the plateau for each ratio allows us to determine the fraction of bound PP-1. The wavelength of the maximum of fluorescence intensity varies linearly with the apparent fraction PP-1 in complex with P3 (Figure 4D). This linear dependence shows that, in addition to being a fluorescent sensor that can be switched on, the monitoring of the position of the fluorescence spectrum of the biosensor indicates the fraction of bound biosensor. In conclusion, the fluorescence increase upon CrAsH complexation with P3 is significant and easily detectable in fluorescence spectroscopy.

The ability of the biosensor to detect a protein of interest has been further evaluated with a purified protein genetically fused to P3. We have chosen the red fluorescent protein, mCherry, as a target. It was modified by fusion of the P3 tetracysteine tag at its Nterminus with either a 4 or 18 amino-acid linker leading to constructions I and II respectively (Figure 5A). The unmodified form of mCherry was used as a reference. In the presence of an excess of mCherry, the NMR signal of xenon inside PP-1 is not shifted (Figure 5B,C) showing that there is no non-covalent interaction between the biosensor and the fluorescent protein. Upon addition of 2 or 8 equivalents of construction I, the NMR signal is strongly modified. With 2 equivalents, the signal of xenon in the free PP-1 is still present whereas two peaks upfield shifted by 2.5 ppm and 7.5 ppm appear (Figure 5D). With 8 equivalents, the signal of xenon in the free PP-1 disappears completely to the benefit of the two upfield shifted peaks already observed at the lower ratio (Figure 5E). They likely correspond to different xenon environments and thus to two different orientations of the cryptophane cage with respect to the protein. When 8 equivalents of the construction II were added to PP-1, no net 129 Xe NMR frequency for caged xenon could be identified (not shown). Nevertheless, we controlled the fluorescence emission of the mixture before the NMR experiment and we checked that the complexation was efficient (see below). The lack of a clear NMR signal is thus most likely due to the very high flexibility of the linker leading to a distribution of relative positions of the cryptophane cage with respect to the protein. Altogether, these observations demonstrate again the very high sensitivity of xenon to its environment, even at such large distances. In addition, the chemical shift variations observed upon reaction of PP-1 with the construction I (2.5 and 7.5 ppm) are smaller than the 9.2 ppm principal value obtained for the same peptide in its free version. This confirms a posteriori the necessity to optimize at first the amplitude of the chemical shifts for biosensor bound to the free peptide alone. We have investigated by fluorescence spectroscopy the reaction of the constructions I and II with PP-1 (Figure 6). mCherry is a fluorescent protein with an absorption spectrum centered at 587 nm. It is very close to the maximum of fluorescence emission of CrAsH at 540 nm. The spectral overlap and the spatial proximity of both fluorophores upon the coupling of PP-1 to the protein lead to an efficient Förster resonance energy transfer (FRET) from CrAsH to mCherry, CrAsH being the donor and mCherry the acceptor. Upon reaction of both constructions I and II by PP-1, we made several observations (Figure 6A,B). First, the wavelength of the CrAsH band shifts from below 520 nm to 540 nm. Second, the amplitude of the later band increases at 540 nm. Third, we observed the presence of a band at 610 nm corresponding to the stimulated fluorescence of the bound mCherry by FRET. Even if the evolution of these three parameters is slightly different for constructions I and II, it proves the efficient detection of the tetracysteine sequence on both constructions by PP-1.

The total fluorescence that is proportional to the amount of bound biosensor was calculated by integration of the two bands of the fluorescence emission spectra. It increases gradually with the ratio [mCherry]/[PP-1] for both constructions (Figure 6E). The maximum integrated fluorescence is 8-10 times the one of the biosensor alone. Nevertheless, photons are directly emitted from CrAsH or through the FRET by mCherry. This contribution of mCherry to the fluorescence signal is presented Figure 6F. The quantum yield of mCherry is lower than the one of CrAsH (0.22 ; [17] vs ~ 0.8 deduced form [2] respectively). The contribution of mCherry, whose quantum yield is lower, decreases the effective integrated fluorescence detected. We estimated at 5 10 6 cps the maximum integrated fluorescence without FRET by replacing the contribution of mCherry to the signal (60 % with a quantum yield of 0.22) by an equivalent fraction of CrAsH. This is approximately 20 times the initial fluorescence (Figure 6E) consistently with the 19 times-increase obtained with the peptide alone. The maximum of fluorescence is reached at a lower ratio for construction I than for construction II (ratio of 2 vs. 4) showing a better affinity of the biosensor for the first construct (Figure 6E). The wavelength shift of the CrAsH band with the concentration of mCherry is maximum for a 1:1 ratio for construction I that has the shorter linker whereas it increases stepwise for the construction II (Figure 6C andD). The gradual shift of the emission band of CrAsH upon binding suggest a binding mechanism of the protein to the biosensor likely involving several steps. This could be due to the flexibility of the long linker in construction II that allows conformations in which the TC-tag is not fully involved in a concerted reaction of the two arsenic atoms. In addition the final coupling product of the construction II with PP-1 cannot be detected in 129 Xe NMR. Again, the high flexibility of the long linker in construction II could be responsible of this observation. In conclusion, the length of the amino-acid linker that anchors the tetracysteine tag on the protein of interest influences the response in fluorescence spectroscopy as well as in 129 Xe NMR. This length need to be optimized. The optimization of amino-acid linkers is a very general concern in the genetically encoded biosensors field. This has already been discussed for other type of biosensors such as the FRET-based biosensors. [18]

Conclusions

Using hyperpolarized 129 Xe NMR and fluorescence spectroscopy, we showed the detailed optimization of a bimodal biosensor. Each component influences the efficacy of the detection and has to be chosen carefully from the design of the organic moiety, the structure of the peptide and the length of the peptide between the specific protein of interest and the target peptide. The couple P3 / PP-1 was the best combination for NMR and fluorescence detection. We also showed that the fusion of P3 to the protein of interest can be optimized. The quality of the NMR signals was here correlated to an efficient cooperative binding leading to a clear shift of CrAsH fluorescence band to 540 nm. The suitability of this biosensor in live cell imaging could now be evaluated in a next step.

Experimental Section

All chemicals were purchase at Sigma. The peptide P1, P2 and P3 were ordered at BioMat k (Delaware, USA) All experiments were performed in Phosphate-Buffered Saline (PBS) solution at pH 7.4. It guarantee a stable ionic strength during all the measurements. Coupling experiments were performed in reducing conditions: triscarboxyethylphosphine (TCEP) at 1 mM, βmercaptoethanol at 1 mM and 10 µM of 1,2-Ethanedithiol (EDT) for 1µM of biosensor. Such strategy prevents non-specific complexation. Monothiols such as 2-mercaptoethanol are helpful to catalyse equilibration but do not compete themselves. EDT is exchanged by the peptide. [3a] In order to guarantee that the cysteines are in the thiolate form, the pH of the medium should be in the region of 7. Indeed, pKa of thiols in cysteines has been found to be between 7.6 and 9.6. [19] The reducing reactive were first mixed to the biosensor and then added to the target. For end point experiments, the proteins and the biosensors in the reducing medium are incubated 60 min at 37°C. Hyperpolarized xenon was produced in the batch mode via SEOP (Spin Exchange Optical Pumping) using a home-built setup [20] . Five minutes suffice to produce five millilitres of xenon at a polarization ranging from 15 to 25%. Frozen hyperpolarized xenon immersed in a bath of liquid nitrogen and subjected to a magnetic field of 3 kG provided by a solenoid is transported near the NMR magnet. There it is sublimated in the fringe field of the magnet and transferred thanks to a vacuum line to the NMR tube. A hollow spinner enabled us to condense it in the upper part of the NMR tube without cooling the solution.

The hyperpolarized 129 Xe NMR experiments were run at 11.7 T and 293 K using a 5mm-broadband inverse probehead. 129 Xe NMR spectra were recorded at micromolar concentrations of peptides inducing no interaction or ionic strength modification. Actually, it has been proved during our experiments that small modification of salt concentrations -and therefore the ionic strength -can alter significantly the resonance frequency of dissolved xenon. They were all recorded in 64 scans, and the free induction decays were apodized with a Lorentzian broadening of 10 Hz before Fourier transformation. The spectra were cal brated with respect to the peak corresponding to xenon dissolved into PBS referenced at 196 ppm. The spectra can be then compared to each other.

Absorption and fluorescence spectra were recorded with a SynergyH1 plate reader (Biotek, USA) in 96-well plate (clear bottom, black wall). The plate reader is equipped with monochromators and allows the precise tuning of excitation and emission wavelengths. The temperature is fixed at 37°C. For the spectra presented figure 6, the direct contribution of mCherry to the fluorescence was taken in account: spectra of the same mixture but without PP-1 were systematically subtracted (PP-1 was replaced with PBS buffer). Fluorescence spectra of constructions I and II were recorded before the NMR experiments in order to verify that the binding of the biosensor to the protein was effective.

The total fluorescence plotted Figure 6A and B is the sum of the two contr butions of CrAsH and mCherry: 𝐹 𝑡𝑜𝑡𝑎𝑙 = 𝐹 𝐶𝑟𝐴𝑠𝐻 + 𝐹 𝑚𝐶ℎ𝑒𝑟𝑟𝑦 . The contr bution, α, of mCherry to the spectra was calculated as: 𝛼 = 𝐹 𝑚𝐶ℎ𝑒𝑟𝑟𝑦 /𝐹 𝑡𝑜𝑡𝑎𝑙 , 𝐹 𝑡𝑜𝑡𝑎𝑙 being the integrated fluorescence (surface of the whole spectrum). In order to calculated 𝐹 𝑚𝐶ℎ𝑒𝑟𝑟𝑦 , we used a fluorescence spectrum of mCherry whose surface was normalized to 1, 𝐹 𝑚𝐶ℎ𝑒𝑟𝑟𝑦 𝑁𝑂𝑅𝑀 𝑡𝑜 1 with 𝐹 𝑚𝐶ℎ𝑒𝑟𝑟𝑦 = 𝑥 × 𝐹 𝑚𝐶ℎ𝑒𝑟𝑟𝑦 𝑁𝑂𝑅𝑀 𝑡𝑜 1 . It was subtracted to 𝐹 𝑡𝑜𝑡𝑎𝑙 as following: 𝐹 𝐶𝑟𝐴𝑠𝐻 = 𝐹 𝑡𝑜𝑡𝑎𝑙 -𝑥 × 𝐹 𝑚𝐶ℎ𝑒𝑟𝑟𝑦 𝑁𝑂𝑅𝑀 𝑡𝑜 1 . We optimized the parameter x in order to obtain a pure spectra for CrAsH centred at 540 nm and with no fluorescence above 665 nm.

The synthesis of the biosensor, the cloning, the expression and the purification of mCherry are detailed in supplementary information. The sequence of the linker in the construction I is -TFGSand in construction II -TFGSFTDVMSTGTGSTGS-.

Figure 1 .

 1 Figure 1. Structure of the biosensor. A) Concept of the combined fluorescence and hyperpolarized 129 Xe NMR detection biosensor. B) Structures of the biosensors obtained from CrAsH moieties (ligands) fused to water-soluble hexacarboxylate cryptophanes (cages) by a spacer: MM-1, MM-2, PP-1 and PP-2. R = CH2COOH

Figure 2 .

 2 Figure 2. One-scan 129 Xe NMR spectra of the biosensors (enlargement of he high field region). A) MM-1 B) MM-2 and C) hexacarboxylate cryptophane-222. Concentration c.a. 30 µM, pH = 7.4. For A) and B), the asterisk indicates the presence of a residual of the other form. The difference of signal-to-noise ratio of these spectra only reflects variation in xenon polarization.

Figure 3 .

 3 Figure 3. 129 Xe NMR spectra of the biosensors bound to the peptides. MM-1 bound to P3 in A), to P2 in B) and to P1 in C), PP-1 bound to P3 in D) and MM-2 bound to P1 in E). The dashed lines represent the chemical shift of the corresponding free biosensors. Yellow spheres symbolized the cysteine residues. For all spectra, the peptides are in large excess. The difference of signal-to-noise ratio of these spectra only reflects variation in xenon polarization.

Figure 4 .

 4 Figure 4. Characterization of the reaction of PP-1 with P3 in fluorescence spectroscopy. 0 to 10 equivalents of P3 peptide were added to 10 µM PP-1 and illuminated at 480 nm. A) Maximum of the fluorescence intensity against time, B) Maximum of the fluorescence intensity at 90 min against the concentration ratio peptide/biosensor, C) Evolution of the normalized fluorescence emission spectra monitored between 520 nm and 580 nm at 90 min, D) Evolution of the wavelength of the maximum fluorescence emission with the proportion of bound biosensors. Reaction performed at 37°C.

Figure 5 .

 5 Figure 5. 129 Xe NMR spectra of he biosensors bound to the protein of interest, mCherry. A) Scheme of the modified protein. 129 Xe NMR spectra of PP-1 (2µM) in the presence of reducing buffer (B), wi h 8 equivalents of unmodified mCherry (C), with 2 (D) or 8 (E) equivalents of construction I.

Figure 6 .

 6 Figure 6. Characterization of the reac ion of construc ions I or II with PP-1 via fluorescence spectroscopy. A representative set of data among 3 independent experiments is presented. 0 to 8 equivalents of construction I (A and C) and II (B and D) were added to 0.5 µM of PP-1 and illuminated at 490 nm. Fluorescence emission spectra were recorded from 520 nm to 700 nm (A and B) and normalized to the amplitude of the fluorescence of the CrAsH band (C and D). (E) Integrated fluorescence of the spectra shown in A and B. (F) The fraction of the integrated fluorescence corresponding to the emission from mCherry itself was calculated for each ratio [mCherry]/[PP-1].

Acknowledgements

Emilie Mari's PhD fellowship was funded by the IDEX Paris Saclay. Keywords: 129 Xe NMR • fluorescence • biosensor • tetracyteinetag • fluorescent protein • cryptophane


Calypso Mathieu

Martine Desrois

Frank Kober

Nathalie Lalevée

Carole Lan

Natacha Fourny

Magali Iché-Torres

Thi Thom Tran

Linh Thuy Lê

Mervyn Singer

Jean-Louis Mege

Monique Bernard

MD Marc Leone
email: marc.leone@ap-hm.fr

Sex-mediated response to the beta-blocker landiolol in sepsis: an experimental, randomized study

Keywords: Wistar rats sepsis, sex differences, adrenergic beta-blockers, cardiac dysfunction, magnetic resonance imaging, energy metabolism

Objective: To investigate any gender effect of the beta1-adrenergic blocker, landiolol, on cardiac performance and energy metabolism in septic rats, and to explore the expression of genes and proteins involved in this process.

INTRODUCTION

The adrenergic system plays a key role in septic shock [START_REF] De Montmollin | Bench-to-bedside review: Betaadrenergic modulation in sepsis[END_REF]. However, prolonged and excessive adrenergic stimulation is cardiotoxic [START_REF] Dünser | Sympathetic overstimulation during critical illness: adverse effects of adrenergic stress[END_REF]. In critically ill patients, the administration of dobutamine to maintain an elevated cardiac index is associated with increased mortality [START_REF] Hayes | Elevation of systemic oxygen delivery in the treatment of critically ill patients[END_REF].

In a previous animal study, we showed that use of a pure ß-agonist led to hypertrophic cardiac failure [START_REF] Desrois | Effect of isoproterenol on myocardial perfusion, function, energy metabolism and nitric oxide pathway in the rat heart -a longitudinal MR study[END_REF]. In experimental models of sepsis esmolol, a selective ß1-adrenergic blocker, improved cardiac contractility, stroke volume (SV) and vascular responsiveness to norepinephrine [START_REF] Kimmoun | β1-Adrenergic Inhibition Improves Cardiac and Vascular Function in Experimental Septic Shock[END_REF][START_REF] Aboab | Effects of esmolol on systemic and pulmonary hemodynamics and on oxygenation in pigs with hypodynamic endotoxin shock[END_REF][START_REF] Suzuki | Infusion of the beta-adrenergic blocker esmolol attenuates myocardial dysfunction in septic rats[END_REF]. In patients in established septic shock, Morelli et al. also showed that esmolol infusion reduced heart rate (HR), increased SV and decreased norepinephrine requirements [START_REF] Morelli | Effect of heart rate control with esmolol on hemodynamic and clinical outcomes in patients with septic shock: a randomized clinical trial[END_REF].

Experimentally, the host response to an inflammatory insult differs according to sex [START_REF] Angele | Gender differences in sepsis: cardiovascular and immunological aspects[END_REF][START_REF] Zhu | Rac1 mediates sex difference in cardiac tumor necrosis factoralpha expression via NADPH oxidase-ERK1/2/p38 MAPK pathway in endotoxemia[END_REF].

We showed that male mice were at higher risk of developing severe forms of acute Q fever [START_REF] Leone | Effect of sex on Coxiella burnetii infection: protective role of 17beta-estradiol[END_REF][START_REF] Textoris | Sex-related differences in gene expression following Coxiella burnetii infection in mice: potential role of circadian rhythm[END_REF]. In an endotoxin murine model, ex-vivo cardiac performance was more impaired [START_REF] Zhu | Rac1 mediates sex difference in cardiac tumor necrosis factoralpha expression via NADPH oxidase-ERK1/2/p38 MAPK pathway in endotoxemia[END_REF]. In humans, sex seems to affect the response to ß-blockers [START_REF] Kneale | Gender differences in sensitivity to adrenergic agonists of forearm resistance vasculature[END_REF][START_REF] Hart | Sex and ageing differences in resting arterial pressure regulation: the role of the β-adrenergic receptors[END_REF].

To our knowledge, in sepsis, the gender-dependent response to ß-blockers remains undetermined. Our hypothesis was that beta-blockade would prevent cardiac dysfunction in males but not in females. Landiolol has a high affinity for ß1-adrenergic receptors (ß1-AR), with a ß1: ß2 ratio of 255, compared to 33 for esmolol [START_REF] Iguchi | Development of a highly cardioselective ultra short-acting beta-blocker, ONO-1101[END_REF].

We thus assessed the sex-dependent effects of landiolol on in-vivo cardiac performance during sepsis using magnetic resonance imaging (MRI). We further evaluated the effects of landiolol on ex-vivo cardiac performance, energy metabolism and oxidative stress on an isolated perfused heart preparation. Finally, we investigated the expression of candidate genes involved in various pathways and known to be involved in cardiac function to elicit potential mechanisms of action during sepsis.

MATERIALS AND METHODS

Animals and cecal ligation and puncture model

Male and female Wistar rats (9-12 weeks old, Charles River, Saint-Germain sur l'Arbresle, France) were housed for a 5-7-day acclimatization period in a temperature and lightcontrolled room with free access to water and food. All animal procedures were conducted in accordance with national Guidelines for Care and Use of Laboratory Animals in conformity with the 2010/63 EU directive and with the approval of the Institutional Animal Care Committee of Aix-Marseille University (APAFIS#3746-201601221813985). The anesthetic and surgical procedure is reported in Supplemental Digital Content 1.

Experimental protocol

Supplemental Digital Content 2 summarizes experimental protocol. One hour after cecal ligation and puncture (CLP), males and females were randomized to receive landiolol (AOP Orphan, Vienna, Austria) diluted in n-saline and infused at 0.1 mg/kg/min or n-saline (10 mL/kg/h). The infusion volume was similar in all groups. Six groups were studied: sham male (n=6), CLP male (n=8), CLP+landiolol male (n=7), sham female (n=7), CLP female (n=8) and CLP+landiolol female (n=7). Eighteen hours after surgery, rats were anesthetized using 1.5 2% isoflurane. The caudal ventral artery was cannulated (24-gauge catheter) for blood pressure recording (TruWave pressure transducer, Edwards Lifesciences, Irvine, CA). Systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean blood pressure (MBP) were recorded at H18. Body weight variations between H0 and H18 were measured. Cardiac MRI was performed 18 hours after CLP to assess in-vivo cardiac function (cine-MRI) and myocardial perfusion (Arterial spin labeling, ASL MRI). Ex-vivo cardiac function measurement and 31P magnetic resonance spectroscopy (MRS) were performed at H19 using isolated heart preparation. At H20, hearts were frozen for subsequent biochemical analyses and quantification of gene expression.

MRI assessment of in-vivo cardiac function

MRI was performed 18 hours after CLP, immediately after arterial cannulation. During MRI, inhalation anesthesia was maintained with 1.4-1.8% isoflurane in 1.5 L/min oxygen continuously delivered through a face mask. Details are provided in Supplemental Digital Content 1.

End-diastolic volume (EDV), end-systolic volume (ESV), stroke volume (SV), left ventricle ejection fraction (LVEF), mean wall thicknesses in diastole (WTd) and systole (WTs), and systolic wall thickening (WTn) were calculated from the volume measurements, as described previously [START_REF] Kober | Cine-MRI assessment of cardiac function in mice anesthetized with ketamine/xylazine and isoflurane[END_REF]. Cardiac output (CO) was calculated as CO=HRxSV. EDV (EDVi), ESV (ESVi), SV (SVi) and cardiac index (CI) were indexed to weight.

In-vivo myocardial perfusion was quantified using the cine-ASL technique, as previously described [START_REF] Kober | High-resolution myocardial perfusion mapping in small animals in vivo by spin-labeling gradient-echo imaging[END_REF]. Image analyses were performed using a home-made program running in an IDL environment which generated absolute myocardial blood flow (MBF) maps. MBF was determined by manually delineating regions of interest in the entire left ventricular myocardium on the corresponding MBF maps.

Isolated rat heart preparation for ex vivo cardiac function and 31P MRS

At H19, rats were anesthetized by intraperitoneal injection of 50 mg/kg pentobarbital sodium. Blood samples were taken through the arterial catheter and immediately centrifuged. After bilateral thoracotomy, the heart was quickly excised and mounted onto an isolated perfused heart apparatus according to the Langendorff method (4). Details are provided in Supplemental Digital Content 1.

A latex balloon was introduced into the left ventricle to evaluate ex vivo cardiac function.

End-diastolic pressure was set to 10 mmHg. Left ventricular developed pressure (LVDP) and HR were monitored [START_REF] Desrois | Limitation of myocardial and endothelial injury of the rat heart graft after preservation with Centre de Résonance Magnétique Biologique et Médicale (CRMB) solution[END_REF]. The rate-pressure product (RPP), calculated as the product of LVDP and HR, was used as an index of cardiac function. Coronary flow was measured by timed collection of the coronary venous effluent, and indexed by weight of heart.

The perfused rat heart was placed in a 20-mm MR sample tube and inserted into a 31P probe seated in the bore of a superconducting wide-bore (89-mm) 4.7-T magnet (Oxford Instruments, Oxford, UK) interfaced with a Bruker-Nicolet WP-200 spectrometer (Bruker, Karlsruhe, Germany). The appropriate conditions for acquiring 31P magnetic resonance spectra and the quantification of phosphorus metabolites have been detailed previously [START_REF] Desrois | Effect of isoproterenol on myocardial perfusion, function, energy metabolism and nitric oxide pathway in the rat heart -a longitudinal MR study[END_REF][START_REF] Desrois | Limitation of myocardial and endothelial injury of the rat heart graft after preservation with Centre de Résonance Magnétique Biologique et Médicale (CRMB) solution[END_REF]. Details are provided in Supplemental Digital Content 1.

Ribonucleic acid (RNA) extraction and quantitative reverse transcription-polymerase chain reaction (RT-PCR), Immunoblotting and Western-blot, and Biochemical analyses in plasma

For more details, see Supplemental Digital Content 1.

Statistical analysis

Data are expressed as means±standard error of the mean (SEM). After statistical evaluation, the number of rats required was 6 per group to show a 30% increase in SV by landiolol with a power and an alpha risk of 80% and 5%, respectively. Significant differences between groups were determined using two-way analysis of variance (ANOVA) followed by Sidak post-hoc testing with Graphpad Prism software (Graphpad Prism 7.0, La Jolla, CA, USA). A p value <0.05 was considered statistically significant. Isolated heart preparation data from 6 rats (CLP male (n=2), sham female (n=2), CLP female (n=2)) were not determined due to traumatic removal.

RESULTS

Effects of CLP and landiolol on weight variation and temperature after 18 hours

In both males and females, CLP was associated with weight gain (p<0.001 for both) and hypothermia (p<0.001 and p=0.003, respectively), as compared with sham animals (Table 1).

The CLP+landiolol male group had a lower weight gain (p=0.001) and less hypothermia (p=0.015) than untreated CLP males. In septic females, landiolol did not impact upon either weight or temperature.

Effects of CLP and landiolol on hemodynamic variables after 18 hours

Landiolol decreased HR from 404±9 to 330±11 b/min in septic males (p<0.001), and from 388±14 to 345±14 b/min in septic females (p=0.024) (Table 1). Landiolol did not affect blood pressure in males, whereas DBP and MBP decreased in landiolol-treated females (p=0.002; p=0.013). Plasma lactate concentrations were similar in all groups.

Effects of CLP and landiolol on in vivo cardiac function and perfusion using MRI

Cardiac MRI variables are reported in Table 2 and indexed parameters in Figure 1. In males, SVi, CI and EDVi decreased after CLP (p<0.001 vs. sham) whereas only CI and EDVi decreased in females (p=0.020 and p=0.046 vs. sham).

In septic males, landiolol administration increased SVI, ESVi and EDVi (all p<0.05) compared with untreated animals (Figure 1). LVEF and systolic wall thickening (sWtn) were similar between groups (Table 2). In females, landiolol increased ESVi (p<0.001) and decreased LVEF (p<0.001) and sWtn (p=0.009) compared to untreated animals. Myocardial blood flow was not different between groups.

Effects of CLP and landiolol on ex vivo cardiac function in the isolated heart preparation

Table 3 shows myocardial function and coronary flow in the isolated rat heart preparation at H19. Heart weight to body weight ratios were similar between groups. CLP decreased LVDP and RPP in both males and females (all p<0.05). LVDP, RPP and coronary flow index were similar between landiolol-treated and untreated CLP groups.

High-energy phosphate compounds and pHi measured by 31P MRS are shown in Supplemental Digital Content 3. Data represent the average of the 4 last spectra.

Phosphocreatine (PCr), adenosine triphosphate (ATP), phosphomonoesters (PME), inorganic phosphate (Pi), intracellular pH (pHi) and PCr/ATP ratio were similar in males and females.

Effects of CLP and landiolol on myocardial gene expression

Genes encoding signaling proteins known for their role in cardiac and immune function are detailed in Supplemental Digital Content 4. IL-10 increased in males and females after CLP (p<0.001 and p<0.01, respectively), without any effect of landiolol. IL-15 was lower in CLP females (p=0.01 vs. sham) and landiolol reversed this effect (p=0.032 vs. CLP female group).

IL-1ra was higher in the CLP female group compared to sham (p=0.016) and landiolol attenuated this effect. These variations were not found in males. More details are available in Supplemental Digital Content 1.

CLP decreased NPR3 (natriuretic peptide receptor 3) transcript levels in both males and females (p<0.001). Landiolol reversed this decrease in males (p>0.05 vs. sham males) but not in females (p=0.002 vs. sham females). In males, no effect of CLP was observed on transcript levels of ryanodine receptor 2 (RYR2). In females, RYR2 expression was decreased after CLP with landiolol reversing this effect.

Effects of CLP and landiolol on myocardial protein expression

In CLP males, the pAKT:AKT ratio decreased (p=0.04 vs. sham males) (Figure 2) and this was unaffected by landiolol. In CLP females, the pAKT:AKT ratio was similar to sham and landiolol increased this ratio (p=0.004 vs. CLP females) (Figure 2). In males, eNOS expression was higher in CLP than in sham (p=0.004). Landiolol attenuated this effect (Figure 2). In females, CLP and landiolol did not affect eNOS expression, as compared with sham females. As compared with males, eNOS expression was higher in sham (p=0.028) and landiolol females (p=0.026).

DISCUSSION

To our knowledge, this is the first comparison of response to ß-blockers in males and females with sepsis-induced myocardial dysfunction. Our results highlight disparities between the sexes. While landiolol improved cardiac performance in the septic males, it was associated with a reduction in LVEF and sWtn in the septic females. A strength of our study relies on the assessment of cardiac function using MRI, providing an in-vivo assessment of cardiac function in addition to ex-vivo evaluation. At variance with previous models based on the isolated perfused heart [START_REF] Suzuki | Infusion of the beta-adrenergic blocker esmolol attenuates myocardial dysfunction in septic rats[END_REF][START_REF] Ackland | Cardioprotection, attenuated systemic inflammation, and survival benefit of beta1adrenoceptor blockade in severe sepsis in rats[END_REF][START_REF] Hagiwara | Landiolol, an ultrashort-acting beta1adrenoceptor antagonist, has protective effects in an LPS-induced systemic inflammation model[END_REF], the in-vivo study incorporates heart-vessel interactions in the analysis of cardiac performance. As in septic patients, ß-blockers were associated with reduced ventriculo-arterial decoupling [START_REF] Morelli | Heart rate reduction with esmolol is associated with improved arterial elastance in patients with septic shock: a prospective observational study[END_REF][START_REF] Mathieu | Beta-blockers in septic shock: a magnifying glass on the relation heart vessel[END_REF], underlining the clinical relevance of the model.

In males, landiolol improved SVi, probably by reversing the reduction in EDVi after CLP, but without any significant effect on LVEF, CI and Wtn. This finding is in line with previous experimental studies [START_REF] Suzuki | Infusion of the beta-adrenergic blocker esmolol attenuates myocardial dysfunction in septic rats[END_REF][START_REF] Hagiwara | Landiolol, an ultrashort-acting beta1adrenoceptor antagonist, has protective effects in an LPS-induced systemic inflammation model[END_REF]. Using a conductance catheter in male rats undergoing CLP, Kimmoun et al. also found improved myocardial function with ß-blockers [START_REF] Kimmoun | β1-Adrenergic Inhibition Improves Cardiac and Vascular Function in Experimental Septic Shock[END_REF]. A metaanalysis has shown an association between diastolic dysfunction and mortality in septic patients so this may represent an important protective effect of ß-blockers [START_REF] Sanfilippo | Diastolic dysfunction and mortality in septic patients: a systematic review and meta-analysis[END_REF].

In contrast to septic males, septic females however maintained SVi at the level of sham females. Landiolol did not improve SVI, CI and EDVi but decreased LVEF and sWtn. Cardiac performance ex-vivo was more impaired in males than in females after endotoxin administration [START_REF] Zhu | Rac1 mediates sex difference in cardiac tumor necrosis factoralpha expression via NADPH oxidase-ERK1/2/p38 MAPK pathway in endotoxemia[END_REF]. In our in-vivo study, landiolol improved cardiac performance in septic males but seemed deleterious in females.

While not yet formally examined in septic patients, women undergoing high-risk surgery did not benefit from perioperative ß-blockers, a finding at variance with men [START_REF] Matyal | Sex-related differences in outcome after high-risk vascular surgery after the administration of betaadrenergic-blocking drugs[END_REF]. With exercise, the hemodynamic response to metoprolol in women resulted in a larger reduction of HR and blood pressure [START_REF] Luzier | Gender-related effects on metoprolol pharmacokinetics and pharmacodynamics in healthy volunteers[END_REF]. Lindenfeld et al.

showed that sex affects age-related ß-AR downregulation in normal human ventricles, suggesting an effect of sex hormones [START_REF] Khaliq | Beta-blockade in experimental fluid-resuscitated sepsis : acute haemodynamic effects of esmolol differ in predicted survivors and non-survivors[END_REF]. Downregulation of ß-AR by estrogen may be a protective strategy in the case of hyperstimulation [START_REF] Dhainaut | Coronary hemodynamics and myocardial metabolism of lactate, free fatty acids, glucose, and ketones in patients with septic shock[END_REF][START_REF] Rudiger | Mechanisms of sepsis-induced cardiac dysfunction[END_REF][START_REF] Smeding | Structural changes of the heart during severe sepsis or septic shock[END_REF]. The interaction between estrogen-receptors and ß-AR may explain the sex-dependent response to ß-blockers [START_REF] Smeding | Structural changes of the heart during severe sepsis or septic shock[END_REF].

In our model, septic myocardial dysfunction was more pronounced in males. In an experimental model of fluid resuscitated sepsis, Khaliq et al. compared the hemodynamic effects of esmolol in predicted survivors and predicted non-survivors in a fecal peritonitis model using male rats only [START_REF] Cimolai | Mitochondrial Mechanisms in Septic Cardiomyopathy[END_REF]. In the predicted non-survivors, esmolol infusion was associated with an increase in SV, while CO remained stable, whereas SV and CO decreased with esmolol in predicted survivors. Thus, the underlying severity of the septic myocardial dysfunction may impact upon the response to ß-blockers. In our study landiolol did not alter myocardial blood perfusion nor indexed coronary flow in either gender. In septic patients, coronary flow is typically normal or increased [START_REF] Singer | The role of mitochondrial dysfunction in sepsis-induced multi-organ failure[END_REF].

Mitochondria are the predominant generators of ATP in most cell types. Cardiomyocyte mitochondrial dysfunction is characterized by ultrastructural damage, decreased activity of electron transport chain enzyme complexes, and an increase in reactive oxygen species production [START_REF] Sikes | Sodium/hydrogen exchange activity in sepsis and in sepsis complicated by previous injury: 31P and 23Na NMR study[END_REF][START_REF] Solomon | Myocardial energy metabolism and morphology in a canine model of sepsis[END_REF][START_REF] Escames | Attenuation of cardiac mitochondrial dysfunction by melatonin in septic mice[END_REF]. In sepsis, mitochondrial dysfunction is associated with the degree of organ failure [START_REF] Vincent | International study of the prevalence and outcomes of infection in intensive care units[END_REF]. Here, sepsis did not affect high-energy phosphate compounds and pHi.

Myocardial ATP content remained unchanged while LVDP decreased. In another model of sepsis using 31P MRS, energetic parameters were also stable despite alterations in cardiac function [START_REF] Boyd | Fluid resuscitation in septic shock: a positive fluid balance and elevated central venous pressure are associated with increased mortality[END_REF][START_REF] Morelli | Microvascular effects of heart rate control with esmolol in patients with septic shock: a pilot study[END_REF], suggesting that energetic metabolism is not limiting cardiac function in sepsis. Conversely, using biochemical analyses, Escames et al. however found a decrease in ATP production in hearts from septic mice [START_REF] Lesur | Gene expression profiles characterize inflammation stages in the acute lung injury in mice[END_REF].

There was a sex-dependent effect on expression of genes involved in the inflammatory response in our septic animals. The lack of difference in TNF, IL-1 beta and IL-6 may reflect the late timepoint of collection of cardiac tissue [START_REF] Matsuishi | Landiolol hydrochloride ameliorates acute lung injury in a rat model of early sepsis through the suppression of elevated levels of pulmonary endothelin-1[END_REF]. However, in line with a previous study, landiolol did not seem to interacting with most inflammatory pathways [START_REF] Lin | NPR3 protects cardiomyocytes from apoptosis through inhibition of cytosolic BRCA1 and TNF-α[END_REF]. Of note, landiolol acts predominantly on ß1-AR. As reported elsewhere (1), the mechanism of ß1-AR activation on inflammatory pathways remains unclear, and at variance with that of ß2-AR activation.

NPR3 has an anti-apoptosis role and protects cardiomyocytes [START_REF] Mitaka | Circulating endothelin-1 concentrations in acute respiratory failure[END_REF]. NPR3 decreased in both sexes after CLP and landiolol could reverse this effect in the septic males only. In our model, we found a different expression of eNOS and pAKT:AKT in males and females. Females have a reduced susceptibility to cardiac ischemic injury with NO generation playing a pivotal protective role [START_REF] Yanagisawa | A novel potent vasoconstrictor peptide produced by vascular endothelial cells[END_REF][START_REF] Lisa | A female way to protect the heart: say NO to calcium[END_REF]. In a model of endotoxemia, the protection of the female heart was associated with the AKT/eNOS pathway [START_REF] Sun | Hypercontractile female hearts exhibit increased S-nitrosylation of the L-type Ca2+ channel alpha1 subunit and reduced ischemia/reperfusion injury[END_REF]. Kimmoun et al. also showed an increase in AKT and eNOS phosphorylation with esmolol in a model of sepsis [START_REF] Kimmoun | β1-Adrenergic Inhibition Improves Cardiac and Vascular Function in Experimental Septic Shock[END_REF]. These pathways may thus be implicated in the different response to beta-blockers according to sex.

Our study has several limitations. The difference between the weight of males and females was significant; males initially weighed 1.4 times the female weight. Landiolol was administered according to the weight of each animal and each variable was adjusted to weight.

The ultra-short half-life of landiolol can have resulted in low tissue concentrations in the isolated heart [START_REF] Iguchi | Development of a highly cardioselective ultra short-acting beta-blocker, ONO-1101[END_REF]. The isolated heart also removes the effects of circulating myocardial depressant factors that are implicated in sepsis-induced myocardial dysfunction so this may not necessarily reflect the in-vivo situation [START_REF] Chen | Gender dimorphism of the cardiac dysfunction in murine sepsis: signalling mechanisms and age-dependency[END_REF]. Another limitation is the choice of the intravenous dose of landiolol, which was infused at a fixed dose. In our study, blood pressure remained constant in males but decreased in females. The dose could be adapted to an individual animal's response, since the pharmacokinetics of ß-blockers depends on gender and age [START_REF] Luzier | Gender-related effects on metoprolol pharmacokinetics and pharmacodynamics in healthy volunteers[END_REF]. Another limitation is that our study did not assess the baseline response to landiolol. Landiolol is hydrolysed to an inactivate metabolite by esterases, which activities are higher in female rats than in male rats [START_REF] Joulin | Cytokine profile of human septic shock serum inducing cardiomyocyte contractile dysfunction[END_REF]. Moreover, the response to general anesthesia may be sex-mediated (47) and hypothermia may affect the cardiac effects of ßblockers (48). Our septic rats did not develop profound hypotension thus the role of norepinephrine was not assessed, requiring future studies. Furthermore, we studied young rats; most patients with sepsis are however postmenopausal so our results may not necessarily reflect responses in older females.

CONCLUSION

Our model confirms that cardiac function was more impaired in septic males compared to their female counterparts. Landiolol ameliorated the cardiac dysfunction in males, particularly in relation to an improvement in diastolic function. Landiolol however produced deleterious effects in females. Phosphorylation of AKT and altered expression of eNOS and NPR3 are potential signaling pathways that merit further investigation in order to better understand sex differences.

Table 3 : Ex vivo assessment using isolated heart rat preparation after 19 hours

 3 . HR: Heart rate; LVDP: Left ventricular developed pressure; RPP: Rate pressure product (LVDP x HR). Data are expressed as means ± standard error of the mean (SEM).* p < 0.05, ** p < 0.01, *** p < 0.001 vs. sham male and vs. sham female respective †p < 0.05, † † †p < 0.001 vs. male of the equivalent group

	Males

The study was funded by Fondation pour la Recherche Médicale (DEA 20150633224), AOP Orphan, Lions Club Marseille Doyen and France Life Imaging (grant ANR-11-INBS-0006).

Supplemental Digital Content Legends

Supplemental Digital Content 1: More details text Supplemental Digital Content 2: Experimental procedure. CLP: cecal ligation and puncture; MRI: magnetic resonance imaging; 31P MRS: 31P magnetic resonance spectroscopy. All rats were equipped with a right jugular catheter before experimental procedure. After surgery, all animals received a normal saline infusion (10 mL/kg) for 30 minutes. Note that expression levels were calculated with only 4 biological replicates for genes annotated [START_REF] Desrois | Effect of isoproterenol on myocardial perfusion, function, energy metabolism and nitric oxide pathway in the rat heart -a longitudinal MR study[END_REF]. CLP: cecal ligation and puncture; IL: interleukin; TNF-a: tumor necrosis factor alpha; Cox-2: cyclooxygenase 2; TREM1: triggering receptor expressed on myeloid cells 1; NR3C1: glucocorticoid receptor, NR3C2: mineralocorticoid receptor, ESR1: estrogen receptor; NPR: natriuretic peptide receptor; EDNRA: endothelin-1 receptor; AT1-AR: angiotensin type 1A receptor; AT2-R: angiotensin type 2 receptor; AGTRAP: angiotensin II receptor associated protein; ADRB1: beta1-adrenoreceptor; PLN: phospholamban; ATP2A2: ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2; RYR2: ryanodine receptor 2; cTNI: cardiac troponin I; eNOS: endothelial nitric oxide synthase; iNOS: inductible nitric oxide synthase. a p < 0.05 vs. sham male and vs. sham female, respectively; b p < 0.05 vs. CLP female; c p < 0.05 vs. male of the equivalent group * p < 0.05, ** p < 0.01, *** p < 0.001 vs. sham male and vs. sham female, respectively § p < 0.05, § § p < 0.01, § § § p < 0.001 vs. CLP male and vs. CLP female, respectively † † † p < 0.001 vs. male of the equivalent group


M Tardif

J.-B Jager

P R Marcoux

K Uchiyamada

E Picard

E Hadji

D Peyrade

Single-cell bacterium identification with a SOI optical microcavity

Single-cell bacterium identification with a SOI optical microcavity M. Tardif 1,2 , J-B Jager 2 , P.R. Marcoux 3 , K. Uchiyamada 4 , E. Picard 2 , E.Hadji 2 , D. Peyrade 1 1 Univ. Grenoble Alpes, CNRS, LTM -Micro & Nanotechnologies for Health, 38054 Grenoble Cedex 9, France ² CEA & Univ. Grenoble Alpes, INAC-SP2M-SINAPS, 38000 Grenoble, France 3 CEA, LETI-DTBS-SBSC-LCMI/LBAM, 38054 Grenoble Cedex 9, France 4 Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8573, Japan Photonic crystals and microcavities can act as on-chip nano-optical tweezers for identification or manipulation of biological objects. So far, virus and bacteria optical trapping has been achieved, their presence in the vicinity of the optical resonator being deduced from a shift of the resonant wavelength. Here we show that bacteria can not only be trapped but identified as well if the trapping time is long enough. A silicon on insulator microcavity with a properly tuned quality factor allows to achieve enhanced trapping times. By combining spatial and temporal observation of bacteria -cavity interaction, the optical identification of three different kinds of bacteria is demonstrated.

Biological objects are strategic components for medical and pharmaceutical development, faster clinical diagnosis and food processing control. The field of photonics has proven to be extremely suitable for the study of these objects. The first tool that allowed their trapping and handling is optical tweezers, 1,2 based on the concentration of a laser beam through a microscope objective.

Unfortunately, this method is limited by the macroscopic scale of the implemented set-up and especially by the damage caused on cells 3,4 and bacteria [5][6][7] during optical manipulation. On the contrary, the evanescent field of onchip optical resonators offers a low light intensity energy that could minimize biological object damage. Therefore, they are more suitable for biomolecules, 8-12 virus 13,[START_REF] Vollmer | Proc. Natl. Acad. Sci. U. S. A[END_REF] and bacteria [START_REF] Van Leest | [END_REF]16 manipulation, at a microscopic scale. For the past 20 years many designs of optical resonators have emerged. From the 1D 17,18 and 2D [START_REF] Notomi | Conf. Proc. -Lasers Electro-Optics Soc. Annu. Meet[END_REF][START_REF] Akahane | [END_REF][21] photonic crystals to toroid, [22][23][24] ring, 25 cylindrical 26 and microspherical [START_REF] Gorodetsky | [END_REF]28 resonators, extremely narrow resonances and quality factors as high as 10 9 have been reached. With their integration in optofluidic devices, these structures allow micro and nano objects manipulation [29][30][31][32][33][34][35] and biological object detection. 37,38 More specifically, at the cell scale, despite the low index contrast with liquid medium, it has been proven that bacteria trapping is achievable on H-L types PhCs microcavities. [START_REF] Van Leest | [END_REF] For these structures, the trap stiffness was quantified and the confined Brownian motion of the bacteria has revealed power dependence to the light power excitation. [START_REF] Van Leest | [END_REF] In this direction, Lin et al. have demonstrated that this optical trap do not affect Escherichia coli viability by studying its photothermal damage. Thus, according to the theory, designed lineic microcavities appear to be suitable tools to manipulate bacteria.

Most of literature work has been focused on detection where high quality factor structures are required to precisely detect the shift of the resonance wavelength due to refractive index variation near the resonator 12,36,37 (Figure 1(a)). What we propose here is the use of a broader resonance, adapted for single bacterium analysis and label-free identification (Figure 1(b)). To achieve this condition, the resonance must be wide enough to have an overlap between the resonance without bacterium and the resonance when the bacterium interacts with the field in the cavity (redshift).

Starting from this statement, we investigate an optical method based on microcavities to perform single-cell characterization on bacteria. The analysis of the cell Brownian motion video in the trap combine with the optical structure transmission signal allow a fine characterization of the cell behavior. Moreover, by analysis the fluctuations of the transmitted signal over time during bacteria trapping, we expect to observe variations between bacteria phenotypes.

A 1D microcavity resonator is made on SOI substrate (250 nm / 2 µm / 700 µm) by combining electron beam lithography and reactive-ion etching. The two mirrors are designed for mode adaptation within the cavity 38 (Figure 2(a)) in order to get a quality factor of 4000. In this study we used the same optical structure for all the experiments. By injecting the resonance wavelength in this structure (laser at λres = 1.5 µm, injected power Pinj = 10 mW, and linewidth Lw = 10 -3 pm), an intense and confined electromagnetic field is built in the cavity. The evanescent part of this field above the cavity creates optical gradient forces able to trap and manipulate colloidal objects.

Above the optical structures, a PDMS fluidic system leads swimming objects to the evanescent field. It consists of a 100µm thick PDMS film with molded µ-fluidic channel in which we place a drop of water/bacteria solution. Then it is covered by a 150 µm thick layer of glass. This optofluidic chip is shown in Figure 2(b). A polarizationmaintaining fiber enables the optical coupling of the light from a tunable laser to the microcavity. Two microscope objectives are placed on-top/in-line with the chip and connected to detection devices, which allows spatial (images) and temporal (signal) characterization of the trapping. Images of the cavity are created by a microscope connected to a CCD camera, with a time resolution of 1/24s. To track bacterium trajectories we detect their position on every recorded image for a relevant duration (few seconds), using tracking macro of Fiji (image processing package of ImageJ). 39 This analysis is shown in Figure 2(b-SPATIAL).

The signal transmitted by the optical structure is detected by a photodiode, increased by a trans-impedance amplifier and displayed on an oscilloscope. The oscillations observed Figure 2(b-TEMPORAL) are due to the shift of the resonance wavelength of the optical structure in presence of bacterium above the cavity. Indeed, the resonance wavelength depends not only on the optical index of the cavity, but also on the index of the environment. When the bacterium interacts with the electromagnetic field in the cavity, the environmental index ranges from nwater = 1.33 (bacterium outside the cavity, only water above it) to 1.368 < nbacterium < 1.413 (bacterium exactly above the cavity). 40 This phenomenon is shown in Figure 1: it induces a less than 1 nm shift of the resonance wavelength from the grey to the blue curve.

As we keep injecting the same wavelength during all trapping experiments (resonance at nwater), the signal will oscillate between the two intensity values IMIN and IMAX while the bacterium oscillate between two positions: trapped above and untrapped around the cavity. By this method, we are able to record the position of the trapped object and the fluctuation of the transmitted signal simultaneously.

The objects under study are the following bacteria, shown Figure 2(c): Staphylococcus epidermidis diplococcus, Escherichia coli and Bacillus subtilis aggregate. They were selected for their size, close to the previously studied polystyrene beads 30,41,42 and the differences in morphology and Gram-type between them (one Gram+ bacillus, one Gram-bacillus and one Gram+ coccus). An E. coli cell and a diplococcus of S. epidermidis have equivalent size and shape: oval shape and size of 1 µm x 2 µm, compared to a more elongated shape of The Brownian motion of each type of studied bacterium and the effect of trapping on its trajectory is shown in Figure 3. We characterize the effect of the trap by a parameter that we call "confinement factor" which consists in the ratio of the rectangular area covered by bacteria position (L x l) without and with trapping. The trapping is demonstrated by the reduction of the displacement area (confinement factor between 3.2 and 57) and its major effect is a transversal confinement (area reduced by a factor ranging from 2.8 to 15.5, in comparison to 1.1 to 6 for lateral confinement). In this microcavity, previous studies have evidenced that the evanescent field is spread over two maxima of electromagnetic field. 42 We observe in Figure 3(b) that S. epidermidis and E. coli interact with one maximum of the field at a time (S. epidermidis stays on one while E. coli keep switching from one maxima to another). This is allowed by their reduced size compared to the size order of the field maxima of the cavity. The flagella (E. coli and B. subtilis) seem to induce a larger displacement in the trap and the ability to interact with several maxima of the field. This behavior is consistent with the increased motility of the flagellated bacteria and with the shape of the electromagnetic field in the microcavity.

In Table 1, more details about confinement are shown. It highlights a stronger effect of the trap on S. epidermidis and E. coli, than on B. subtilis (confinement factor are 57, 30.6 and 3.2 respectively). This table also outlines a more effective confinement along transversal axis than along lateral one. For S. epidermidis lateral and transversal confinement are of the same order of magnitude, which can be linked to the spherical shape of the bacterium. On the other hand, for E. coli and B. subtilis the transversal confinement prevails. It is consistent with the behavior of the bacteria in the trap: they align on the structure, with their longer dimension parallel to the cavity. From this we can infer that the lateral and transversal contributions provide information on the shape of the object and its symmetry. The size and shape of B. subtilis induce only transversal confinement (lateral confinement of 1.1), which explains the low effectiveness of the trap on that bacterium (confinement factor of 3.2) in accordance with experimental results (trapping duration under 10 minutes against more than 1 hour for S. epidermidis and E. coli).

Finally, each type of the three investigated bacteria species owns a very distinct spatial signature, which is linked to their size, shape (symmetry) and flagella.

In parallel, monitoring the optical signal transmitted by the cavity (Figure 4) enables an accurate detection of the trapping effectiveness: high frequency oscillations and intensity drop of about 20% (Figure 4(a)).

On Figure 4(b) the temporal variation of the optical signal transmitted by the microcavity during the trapping of the three kinds of bacteria is shown. It is compared to the signal detected in the same conditions without trapping. Because the amplitude is the most relevant criteria to discriminate these curves, we calculate the histogram function of the transmitted signal (Figure 4(c)). Its characteristic elements are the full width at half maximum (FWHM) and the probability to have each intensity value. E. coli is characterized by a FWHM at least twice larger than the two other bacteria. It can be linked to its large lateral displacement in the trap (Figure 3(b, blue curve)). On the contrary, S. epidermidis trapping signal has a very low FWHM which highlights the unique position of equilibrium of the bacterium (high probability for one intensity value), matching with one field maximum. Finally, the optical signal transmitted by the adapted SOI microcavity is self-consistent for bacteria discrimination in this example. We highlighted here that amplitude is the most relevant criteria to differentiate the optical signal transmitted by the microcavity, in which the substantial part of the information is located below 15 Hz. It provides information on the maximum and minimum intensity values of the signal shown in Figure 1 and Figure 4. On Figure 5, these Imax and Imin values are represented on the resonance peak of the microcavity, at the injected wavelength.

If we consider that the quality factor remains constant during the trapping (low change of optical index when the microcavity environment is water or bacteria), it allows to calculate the redshift of the resonance wavelength between the two extreme positions of the bacteria in the trap (from the trap center (Imin) to its edge (Imax)). This redshift is characteristic of bacterium phenotype, as represented Figure 5(b1): its value ranges from 0.20 nm for S. epidermidis to 0.30 nm for E. coli. Each colored peak represents the resonant mode available according to the type of trapped bacterium. By projecting these curves on the resonance peak of the optical trapping structure we are able to assign to every type of bacterium a specific area of the peak (Figure 5(b2)) and discriminate them.

Finally, the two methods of characterization (spatial and temporal) are consistent with each other: the displacement of E. coli on the two evanescent field maxima can be linked to the large amplitude of its trapping signal and the higher value of the FWHM of its histogram function. S. epidermidis oscillates close to its balance position (one field maximum), matching to one transmitted intensity value of highest probability.

In this paper we show that planktonic single cell bacterium can by trapped by the evanescent field of a properly tuned microcavity. Both spatial (Brownian and partially-trapped motions) and temporal (optical transmission measurement) methods are relevant to study finely the bacterium behavior within the trap.

The spatial signature demonstrates that morphological properties of bacteria (size, shape and presence of flagella) can be identified thanks to the so-called confinement factor parameter. The temporal signature through the analysis of the fluctuations of the optical transmitted signal over time during bacteria trapping allows us to identify bacteria phenotypes.

Compared to the spatial characterization, the temporal method requires only cavity transmission measurements. Thus, this promising label-free technic that can be performed in a much more compact way, with a smaller footprint, in a device scheme might be suitable for a reduce cost fast analysis device.

In the future we plan to extend this study to a larger number of bacteria, with different shapes, sizes and overall properties.

FIG. 1 .

 1 FIG. 1. Redshift of the microcavity resonance wavelength in presence of a bacterium above it. (a) High quality factor structure, ideal for detection of the target object, no trapping of bacteria. (b) Intermediate quality factor structure, ideal for bacteria trapping and analysis.

FIG. 2 .FIG. 3 .FIG. 4 .FIG. 5 . 1 ) 2 )

 234512 FIG. 2. (a) SEM image of the single microcavity used for all the experiments. (b) Trapping Set-up: fluidic system on a 1D PhC, light injected in the optical structure with a polarized fiber and recorded on structure output (TEMPORAL). Second trapping analysis way by microscopy column above the cavity (SPATIAL). The measurements related to E. coli are shown in blue and those associated with S. epidermidis are shown in orange, (c) SEM images of the under study bacteria: (1) diplococcus of Staphylococcus epidermidis, (2) Escherichia coli and (3) aggregate of Bacillus subtilis.

Table 1 .

 1 Numerical analysis of spatial behavior of trapped and nontrapped bacteria. The transversal direction is parallel to the x axis and the lateral one is parallel to the y axis.

	1 µm x 7 µm
	for B. subtilis (aggregate). E. coli and B. subtilis have
	flagella, a molecular motor they use to propel themselves
	through an aqueous medium. Bacterial strains (E.

coli ATCC 117775, S. epidermidis ATCC 14990, B. subtilis ATCC 11774) were obtained from KwikStik lyophilized reference strains (Microbiologics, St. Cloud, MN).

Acknowledgement

The authors would like to express their gratitude to Pascal GENTILE and Christophe PIN for imaging and preparation of bacteria. A thank you is also extended to Julien CORDEIRO for his blender images and to Patricia LAURENT for the bacteria samples preparation. J.B. J. thanks the technical group of the "Plateforme Technologique Amont" of Grenoble for their help in nanofabrication.