Carolina Giraldo
email: carolina.giraldo@ifremer.fr

Bruno Ernande

Pierre Cresson

Dorothee Kopp

Marie Cachera

Morgane Travers-Trolet

Sebastien Lefebvre

Giraldo Carolina

Ernande Bruno

Cresson Pierre

Kopp Dorothee

Cachera Marie

Travers-Trolet Morgane

Depth gradient in the resource use of a fish community from a semi-enclosed sea

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

Introduction

Scientists have long recognized the importance of food web structure and functioning to understand and predict the response of marine ecosystems to environmental change [START_REF] Woodward | Ecological networks in a changing climate[END_REF]. The biotic compartment of marine ecosystems and its trophodynamics respond to variability in the abiotic environment from the individual to the community level [START_REF] Keyl | Environmental variability and fisheries: What can models do?[END_REF]. Consequently, studying how food webs vary along natural gradients from local to regional scales has been recognized as one of the fruitful avenues for future research [START_REF] Ings | Ecological Networks--beyond Food Webs[END_REF]. Such environmentally-induced variability can be particularly significant in semi-enclosed shallow seas that present low inertia to global (e.g., climate change) and/or local variations (e.g., river systems, fisheries) [START_REF] Mackenzie | Impact of 21st Century climate change on the Baltic Sea fish community and fisheries[END_REF][START_REF] Martin | The channel habitat atlas for marine resource management (CHARM): An aid for planning and decision-making in an area under strong anthropogenic pressure[END_REF]). As a result, numerous epi-continental seas (e.g., Baltic Sea, North Sea) are highly dynamic and productive areas fueled by a large number of sources (e.g., freshwater inputs, marine organic matter), characterized by a large number of species and biological interactions, and where commercially important fish are caught every year.

Transfer of energy or matter between benthic and pelagic compartments can be described as a two-way process accounting for the amount of pelagic material that reaches the sea floor (pelagic-benthic coupling) but also for the amount of benthic organic matter that is available and consumed by pelagic species (benthic-pelagic coupling; [START_REF] Gaudron | The comparison of δ13C values of a deposit-and a suspension-feeder bio-indicates benthic vs. pelagic couplings and trophic status in contrasted coastal ecosystems[END_REF].

Studies on pelagic-benthic coupling in polar and temperate regions have successfully identified a close relationship between primary production in the water column and benthic biomass or abundance [START_REF] Grebmeier | Studies of pelagic-benthic coupling extended onto the soviet continental shelf in the Northern Bering and Chukchi Seas[END_REF], Cresson et al. 2014b). Similarly, deep (> 500 m) benthic trophic webs are largely dependent upon sinking organic matter of pelagic origin [START_REF] Iken | Food web structure of the benthic community at the porcupine abyssal plain (NE Atlantic): A stable isotope analysis[END_REF], Cresson et al. 2014a). However, few studies have documented and recognized the importance of benthic production to the whole food web in semi-enclosed shallow marine ecosystems where its contribution is expected to be high due to the proximity between pelagic and benthic species and the virtual absence of physical barriers such as thermoclines. Among the factors influencing benthic-pelagic coupling (e.g., vertical current, primary production, nyctemeral migrations, community composition), depth is one of the main forcing variables [START_REF] Woodland | Benthic-pelagic coupling in a temperate inner continental shelf fish assemblage[END_REF] that can be easily measured and is usually available for all studies. Depth is directly or indirectly related to different physical (e.g., water mixing, sinking time of organic matter) and biological processes (e.g., vertical migration of species) [START_REF] Baustian | Linking the bottom to the top in aquatic ecosystems: mechanisms and stressors of benthic-pelagic coupling[END_REF]) and can be used as a proxy of habitat variation to explore benthic-pelagic coupling and its impact on aquatic food webs' structure [START_REF] Kopp | Reorganization of a marine trophic network along an inshore-offshore gradient due to stronger pelagic-benthic coupling in coastal areas[END_REF].

Energetic links can be studied using fish as integrators of benthic-pelagic coupling (and the other way round pelagic-benthic coupling) by exploring fish trophic interactions and their variation with depth [START_REF] Romero-Romero | Body size-based trophic structure of a deep marine ecosystem[END_REF]. At the community level, fish are characterized by a remarkable diversity of feeding strategies, life-history traits and associated morphologies that are expected to respond differently to environmental variability. Flatfishes (e.g., European plaice), generally characterized as endobenthos-feeders, spend most of their time on the bottom and, for the majority, do not feed by sight [START_REF] Gibson | Flatfishes: Biology and Exploitation[END_REF]. Due to the characteristics of such sedentary species, such as swimming short distances and creating moderate disturbances of the bottom to forage [START_REF] Link | The Feeding Ecology of Flatfish in the Northwest Atlantic[END_REF], we expect their diet to originate from benthos regardless of depth. Benthic round fish (e.g., Gadoids) share a relatively similar diet with endobenthos-feeders (mainly benthic prey). However, differences in their morphological traits and feeding modes (visual predation) should allow them to take greater advantage of available pelagic prey (e.g., copepods) when moving from deep to shallow feeding grounds (Jӧnsson et al. 2013). Small forage fish (e.g., sprat, herring) are generally identified as omnivorous pelagic planktivores [START_REF] Mollmann | Feeding ecology of central Baltic Sea herring and sprat[END_REF]). Yet, studies show that larger specimens of some species (e.g., herring) can also be nectobenthos-feeders preying on small benthic crustaceans and polychaete worms [START_REF] Casini | Feeding Preferences of Herring (Clupea harengus) and Sprat (Sprattus sprattus) in the Southern Baltic Sea[END_REF]). Although the main food sources of this functional group are expected to be of pelagic origin, benthic-derived material may also play a significant role in its diet in shallow environments. Finally, piscivorous round fish (e.g., European seabass) form a broad group that can occupy different habitats (pelagic or demersal) and are among the largest specimens within the fish community. Although they preferentially feed on fish [START_REF] Juanes | Feeding ecology of piscivorous fishes[END_REF] thus occupying higher trophic levels, most piscivores are opportunistic and have flexible diets. Piscivorous round fish are thus likely to consume the most abundant food source regardless of its origin [START_REF] Post | Prey preference by a top predator and the stability of linked food chains[END_REF].

In this study, we used data from the eastern English Channel (EEC), a semi-enclosed sea, as a case study to address the following objectives: 1) identify the main trophic interactions and describe the resulting food web structure at the EEC-scale; 2) explore variation in fish resource partitioning with depth; and 3) compare the general structure of trophic interactions within the fish community at the EEC-scale with other shallow semi-enclosed systems. Based on a previous study [START_REF] Kopp | Reorganization of a marine trophic network along an inshore-offshore gradient due to stronger pelagic-benthic coupling in coastal areas[END_REF] and common knowledge on species' habitat use and feeding strategy, we hypothesize that benthic-pelagic and pelagic-benthic coupling vary with depth as the latter influences fish diets: pelagic-feeding species benefit from benthic subsidies in shallow waters and the contribution of pelagic sources to their diet increases with increasing depth as the benthic-pelagic coupling weakens; similarly, benthicfeeding species benefit from pelagic subsidies in shallow waters and the contribution of benthic sources to their diets increases with increasing depth as pelagic-benthic coupling weakens.

Diet estimates originating from digestive tract (DT) analysis and stable isotope (SI) analysis provide a snapshot of the current prey sources and integrated information about food resource use over several weeks to months, respectively. DTs of the main fish species (in terms of commercial interest and/or ecological dominance) of the EEC have been reported by [START_REF] Cachera | Individual diet variation in a marine fish assemblage: Optimal Foraging Theory, Niche Variation Hypothesis and functional identity[END_REF] who studied sources of fish trophic niche variation at the individual level.

Additionally, SIs from the different compartments of EEC food web, including the main fish species, were reported by [START_REF] Kopp | Reorganization of a marine trophic network along an inshore-offshore gradient due to stronger pelagic-benthic coupling in coastal areas[END_REF] who showed an inshore-offshore gradient in the food web structure and concluded that this gradient was related to a weakening of benthicpelagic and pelagic-benthic couplings towards offshore waters. To further understand the processes underlying this phenomenon, we combined data from DTs and SIs within a Bayesian framework, including depth as an environmental variable [START_REF] Francis | Habitat structure determines resource use by zooplankton in temperate lakes[END_REF][START_REF] Semmens | MixSIAR: A Bayesian stable isotope mixing model for characterizing intrapopulation niche variation[END_REF]. Species of the EEC food web were grouped into functional groups and DTs were used to define the general topology of the food web whereas SIs were used to calculate proportions of the different prey in the diet of the various functional groups. Lastly, for fish functional groups, variation of diet (based on DTs and SIs) along a depth gradient from 7 to 80m was explored.

Materials and Methods

Data

DT and SI data for fish and the majority of the epifaunal invertebrates (n = 799 samples) were collected using a GOV bottom trawl from R. V. "Gwen Drez" during the Channel Ground Fish Survey (October 2009) that covers the whole EEC annually according to a spatially stratified sampling scheme (Le [START_REF] Roy | CGFS2009 cruise, RV Gwen Drez[END_REF]. Additional SI data for zooplankton (n = 16) and for benthic suspension feeders (n = 34) were respectively collected in January 2010 with a WP2 zooplankton net (International Bottom Trawl Survey; Vérin 2010) and in July 2010 using a French dredge (COMOR survey; Foucher and Quinquis 2010). Finally, particulate organic matter was obtained from water samples collected with Niskin bottles during the French sampling program SOMLIT (October 2009 to June 2010). More details about sampling protocols can be found in [START_REF] Kopp | Reorganization of a marine trophic network along an inshore-offshore gradient due to stronger pelagic-benthic coupling in coastal areas[END_REF], [START_REF] Cachera | Individual diet variation in a marine fish assemblage: Optimal Foraging Theory, Niche Variation Hypothesis and functional identity[END_REF] and supplementary tables S1 and S2.

DT data were obtained by identifying prey to the lowest possible taxon in 853 digestive tracts extracted from 16 fish species chosen to represent the main trophic guilds of the EEC fish community (Chelidonichthys cuculus, Chelidonichthys lucerna, Clupea harengus, Dicentrarchus labrax, Gadus morhua, Merlangius merlangus, Mullus surmuletus, Mustelus asterias, Pleuronectes platessa, Raja clavata, Sardina pilchardus, Scomber scombrus Scyliorhinus canicula, Solea solea, Sprattus sprattus, Trachurus trachurus; for more details on digestive tract analysis see [START_REF] Cachera | Individual diet variation in a marine fish assemblage: Optimal Foraging Theory, Niche Variation Hypothesis and functional identity[END_REF].

Nitrogen and carbon SI ratios, denoted by δ 15 N and δ 13 C, respectively, were measured from particulate organic matter, to zooplankton, epifaunal invertebrates, and fish collected from 7 to 80m depth in the EEC. A biplot of the SIs for each fish species arranged in functional groups can be found in Supplementary Figure S1. Specific details on tissue samples analyzed for SI ratios and their preparation can be found in [START_REF] Kopp | Reorganization of a marine trophic network along an inshore-offshore gradient due to stronger pelagic-benthic coupling in coastal areas[END_REF]. Briefly, SI data were normalized to account for lipid content based on C:N ratios [START_REF] Post | Getting to the fat of the matter: models, methods and assumptions for dealing with lipids in stable isotope analyses[END_REF]) and corrected for spatial variation in the isotopic baseline based on SI values of the suspension-feeding bivalve,

Aequipecten opercularis, taken as the baseline. SI values of the isotopic baseline at sampling sites were obtained by kriging interpolation of published data (Jennings and Warr 2003a, b;[START_REF] Barnes | Environmental correlates of large-scale spatial variation in the δ13C of marine animals[END_REF]. For both nitrogen and carbon, the isotopic value of each consumer sample was then corrected by subtracting the kriged baseline value at the sampling location and adding the mean kriged baseline value averaged across the entire area (for all sampling sites:

Food web modeling

The topological food web of the EEC was constructed by aggregating qualitatively the 54 species sampled for SI into functional groups based on their taxonomic classification (e.g. fish, cephalopods, bivalves), feeding strategies (e.g., filter feeders, scavengers), trophic guild (e.g., herbivores, piscivores), habitat use (e.g., pelagic, demersal) and morphology (only for fish, i.e., flatfish, round fish) using DT data reported by [START_REF] Cachera | Individual diet variation in a marine fish assemblage: Optimal Foraging Theory, Niche Variation Hypothesis and functional identity[END_REF] for the main fish species and completed by published data (see Supplementary Table 1 and2

for references).

Aggregation resulted in 15 functional groups from primary producers to piscivorous fish and cephalopods (Table 1). The trophic level (TL) of each species was estimated based on its δ 15 N using the scaled method proposed by [START_REF] Hussey | Rescaling the trophic structure of marine food webs[END_REF] where the trophic enrichment factor (TEF) of the consumer's δ 15 N is dependent on the δ 15 N value of its prey:

𝑇𝐿 𝑐𝑜𝑛𝑠𝑢𝑚𝑒𝑟 = ( 𝑙𝑜𝑔(δ 15 N 𝑙𝑖𝑚 -δ 15 N 𝑏𝑎𝑠𝑒 ) -𝑙𝑜𝑔(δ 15 N 𝑙𝑖𝑚 -δ 15 N 𝑇𝑃 ) 𝑘 ) + 𝑇𝐿 𝑏𝑎𝑠𝑒
where δ 15 N lim is the saturating δ 15 N limit as TL increases, δ 15 N base is the δ 15 N value of the isotopic baseline, δ 15 N TP is the consumer's δ 15 N value at a given TL, and k is a rate constant [START_REF] Hussey | Rescaling the trophic structure of marine food webs[END_REF]. Values for the parameters δ 15 N lim, δ 15 N TP , and k were taken from [START_REF] Hussey | Rescaling the trophic structure of marine food webs[END_REF]. The bivalve A. opercularis was used as isotopic baseline (TL base = 2). Trophic level for each functional group was then calculated as the mean value for all individuals within the group (Table 1).

The topological description of the food web consisted of trophic links between functional groups expressed as a binary matrix. A trophic link was established if at least one species of the consumer group fed on one species within the source group according to DT analysis. The relative contributions of each source to consumers' diets were then estimated using the Bayesian isotope mixing model IsoWeb [START_REF] Kadoya | IsoWeb: A bayesian isotope mixing model for diet analysis of the whole food web[END_REF]. Isotope mixing models are based on the principle that a consumer's isotopic ratios result from the mixing of the isotopic ratios of its food sources according to their relative contributions to its diet after accounting for TEF [START_REF] Post | Using stable isotopes to estimate trophic position: models, methods, and assumptions[END_REF]. While simple analytical mixing models can only estimate contributions of a few food sources to a single consumer's diet at a time, IsoWeb has the advantage of estimating dietary contributions for all consumers in a food web based on SI data and a topological description of the food web given a priori. The model also allows for TEF variation across links assuming that TEFs follow a normal distribution with a mean of 0.8 for carbon and 3.4 for nitrogen [START_REF] Post | Using stable isotopes to estimate trophic position: models, methods, and assumptions[END_REF][START_REF] Saigo | Trophic level, food chain length and omnivory in the Paraná River: a food web model approach in a floodplain river system[END_REF][START_REF] Fukumori | Detritivore diversity promotes a relative contribution rate of detritus to the diet of predators in ponds[END_REF]. The standard deviations of these normal distributions are assumed to follow a half-Cauchy distribution, for both carbon and nitrogen (see details in [START_REF] Kadoya | IsoWeb: A bayesian isotope mixing model for diet analysis of the whole food web[END_REF]. Nitrogen TEF values estimated from Hussey's equation (TL calculations) were close to those proposed in IsoWeb (mean value of 3.1). Hussey's equation provides TEF estimates between prey and predator species pairs, while IsoWeb estimates TEF between functional groups (several species) preventing a direct comparison of the estimates obtained by the two methods. The posterior probability densities of TEFs for carbon and nitrogen from this study are provided in the supplementary Figure 2S.

The IsoWeb model was run with the following parameters: 10 6 chain length, burn-ins 500.000, and thin number 500 for three parallel MCMC chains. Convergence was assessed using the Gelman-Rubin test [START_REF] Gelman | Bayesian Data Analysis[END_REF].

Estimation of predatory fish diet as a function of depth

The resulting EEC-scale food web (referred to as IsoWeb model hereafter) was then used as a template to further explore the effect of depth on food resource use by fish functional groups using the Bayesian isotope mixing model MixSIAR [START_REF] Stock | MixSIAR GUI user manual, version 1.0[END_REF].

Compared The "Planktivorous fish" group was excluded from this analysis due to insufficient sample size (40 individuals) to explore diet variation with depth as a continuous covariate.

Visual inspection of the final isotopic space (referred to as IsoSpace hereafter) and correlation coefficients between prey sources allowed for the determination of when two sources were indistinguishable. For instance, if two sources presented high overlap in their SI ratios, then, likely solutions could involve one of the two sources but not both at the same time [START_REF] Inger | SIAR v4 (Stable Isotope Analysis in R): An Ecologist's Guide[END_REF]. Sources were aggregated a priori only if the absolute value of the coefficient of correlation was higher than 0.5 and if the combined sources had some functional or ecological significance (e.g., "Benthic decapods-omnivores" and "Benthic decapods-carnivores" combined into "Benthic decapods") following suggestions by [START_REF] Phillips | Combining sources in stable isotope mixing models: Alternative Methods[END_REF]. Whenever sources were combined, the model was re-run under the same settings (i.e., same MCMC parameters). Convergence was assessed using the default MixSIAR diagnostic Gelman-Rubin and Geweke tests. For each fish functional group, the plot of the IsoSpace and the mean contribution of sources to the diet as a function of depth are reported. The resulting posterior distributions of sources' contributions to fish functional groups' diets at the EEC-scale (i.e.

without accouting for the influence of depth) and at the minimum and maximum depths are also provided in Supplementary Figure S3 to allow assessing uncertainty in the effect of depth on functional groups' diets.

Results

EEC-scale food web (IsoWeb model)

The EEC encompassed 5 TLs that, apart from the primary producers, ranged from TL 2 for benthic-suspension feeders (mainly bivalves and gastropods) to almost 5 for cephalopods.

Fish TLs varied from 3.17 for planktivorous to 4.4 for demersal piscivorous fish (Table 1).

Benthic-suspension feeders were the main food source for endobenthos-feeding fish (30% of the diet) while copepods represented the main food source (27%) for benthos-feeding fish (Table 2). Almost equal contributions of all sources to the diet of piscivorous fish (both demersal and pelagic) and cephalopods suggest generalist diets for the higher TL species. The combined contribution of benthic sources accounted for 54% of the diet of planktivorous fish while pelagic subsidies (copepods and macro-zooplankton) represented 46%. Likewise, benthic and pelagic sources represented 51% and 49%, respectively, of the diet of piscivorous pelagic fish (Table 2). In contrast, the diet of benthos-feeding, endobenthos-feeding and demersal

piscivorous fish was largely dominated by benthic subsidies (73%, 100% and 100%, respectively; Table 2). Overall, the contribution of benthic subsidies (i.e., benthic OM, suspension feeders, deposit feeders, benthic predators, and benthic decapods) dominated the diet (51 to 100%) of all fish functional groups, even for those that are commonly reported as pelagic (e.g., planktivorous fish) (Table 2).

Fish diet variation with depth (MixSIAR models)

Using the previous model (IsoWeb) as a template (i.e., same topology and TEF factors), changes in diet with depth, described as a continuous variable, were explored for each fish functional group. Benthos-feeding fish (n = 237 individuals) were highly variable in terms of δ 13 C values (δ 13 C range: -15.45‰ to -18.98‰) suggesting that different carbon sources contributed to their diet. An initial MixSIAR model run with the 5 potential sources of this functional group (Figure 1a) showed a large overlap between SI ratios of copepods and deposit feeders as prey (correlation of -0.90). However, these prey groups were not pooled together to keep pelagic and benthic sources separate. As a result, large standard deviations (hereafter referred to as SD) were observed for estimated sources' contributions to diet due to the difficulty of the model to fully distinguish between the two sources. Overall diet (i.e., integrated across all depths) indicated that individuals fed mainly on copepods (36.2 ± 22.7%

of their diet) and deposit feeders (31.3 ± 19.5%) followed by benthic decapods-omnivores prey (13.5 ± 5.3%). Cephalopods and benthic-suspension feeders were minor dietary items representing less than 10% of individuals' diet each (8.5 ± 4.6% and 10.5 ± 8.3% respectively).

Including depth as a continuous covariate of sources' contributions showed that individuals fed primarily on copepods at shallow depths (< 40m) and increased their consumption of deposit feeders with increasing depth (Figure 1b). The contribution of other sources remained low (<15%) and showed little variation with depth. Posterior distributions of sources' contributions (Figure S3, 1 st row) show that, despite some uncertainty in sources' contributions at the global scale, there is a clear discrimination between benthic and pelagic sources as depth increases.

A first model for endobenthos-feeding fish (n = 149 individuals) showed a large overlap between SI values of benthic predators and deposit feeders as sources (correlation of -0.78). They were combined into a common "benthic predators-worms" group leading to a 4source mixing model (Figure 1c). Overall, individuals fed mainly on benthic-suspension feeders (63.8 ± 5.0%) and benthos-feeding fish (23.8 ± 6.4%). The combined source "benthic predators-worms" and benthic decapods-omnivores appeared as minor dietary sources (6.2 ± 6.5% and 6.2 ± 5.4%, respectively). Including depth as a continuous covariate showed that individuals fed mainly on benthic-suspension feeders from shallow waters (7 m) up to 60 m depth. Their contribution decreased substantially in deeper waters (from ~60% at 40 m depth to ~10% at 80 m depth). The contribution of benthos-feeding fish also decreased with increasing depth, whereas the contribution of "benthic predators-worms", that represented a minor source in shallow waters, increased strongly with depth to become the main source in the deepest waters (Figure 1d). Posterior distributions of sources' contributions (Figure S3, 2 nd row) show the clear shift of main sources as depth increases.

Demersal piscivorous fish (n = 133 individuals) were also trophically linked to benthic predators and deposit feeders. Again, the SI ratios of these two sources overlapped largely (correlation of -0.88) and they were thus combined into a "benthic predators-worms" group.

Similarly, the SI values of benthic decapods-omnivores overlapped with those of benthic decapods-predators and these groups were combined into a "benthic decapods" group (correlation of -0.52). The same held for benthos-feeding fish and benthic decapods (correlation of -0.91) but these sources were kept separate because of functional difference, which resulted in large SD values for sources' contributions to diet (Figure 2a). Overall, individuals consumed mainly "benthic predators/worms" (51.2 ± 6.4%) followed by benthosfeeding fish (29.2 ± 12.8%) and "benthic decapods" (19.7 ± 14.9%). Little variation of diet with depth was observed (Figure 2b). "Benthic predators/worms" remained the main food source regardless of the depth, although its contribution decreased slightly (from ~60% to ~40%) with increasing depth, while benthic decapods contribution increased but always remained the lowest one. Posterior distributions of sources' contributions (Figure S3, 3 rd row)

show that besides the relative stability of the main sources, uncertainty in their contributions increases with depth.

Pelagic piscivorous fish (n = 106 individuals) was the group with the highest number of trophic links (7 sources), which increased uncertainty in the mixing models. As for the previous group, benthic decapods species were combined into a single source (correlation of -0.55). Similarly, "planktivorous fish" and "macro-zooplankton" were combined into a "planktivores" group (correlation of -0.55). As a result, 5 potential sources instead of 7 were considered (Figure 2c). Individuals were highly dispersed in terms of δ 15 N values (δ 15 N range: from 11.78‰ to 18.29‰) suggesting feeding at different TLs. A large overlap in SI values was still present between "planktivores" and "benthic decapods" (correlation of -0.85). However, these sources were kept separate to distinguish pelagic and benthic sources. Overall, individuals consumed mainly copepods (37.6 ± 10.2%) followed by "planktivores" (27.21 ± 24.1%) and "benthic decapods" (26.5 ± 16.6%). Benthic-suspension feeders and benthic predators were minor sources representing less than 5% of the diet (3.9 ± 3.8% and 4.9 ± 6.1% respectively). When depth was included as a continuous covariate, individuals appeared to feed mainly on a mixture of copepods and "benthic decapods" in shallow waters (~7m depth), but their contribution progressively decreased with depth while the contribution of "planktivores" increased to dominate diet in the deepest waters (Figure 2d). Posterior distributions of sources' contributions (Figure S3, 4 th row) show that, despite some uncertainty in benthic decapods' contribution at the global scale, there is a clear discrimination between the main food sources, notably of benthc and pelagic origin, along the depth gradient.

Discussion

We present the major trophic pathways towards the main fish species of a semienclosed sea and how they vary with depth. Species are not studied in isolation, but instead are positioned within the food web and grouped into functional groups that take into account several biological (taxonomy, feeding strategy, trophic guild, and morphology) and habitat (pelagic, demersal) characteristics that are frequently used in trophic web studies and modeling approaches. Our results indicate high mixing of pelagic and benthic trophic pathways towards fish in general over a large area (35.000 m 2 ), but also highlight species' ability to forage preferentially on different prey when moving from shallow to deep waters. With increasing depth, benthic-pelagic and pelagic-benthic couplings weaken gradually (and almost linearly)

and species' diet more closely reflects their pelagic or benthic affinity (Figure 3). Some fish functional groups related to the bottom, namely endobenthos-feeding fish and demersal piscivorous fish, rely exclusively on benthic sources indicating some dietary specialization. In contrast, benthos-feeding fish and pelagic piscivorous fish integrate both pelagic and benthic sources as part of their diet depending on depth, suggesting a more opportunistic feeding strategy. More precisely, the benthos-feeding group relies partly on pelagic sources at shallow depth whereas they favor benthic subsidies as depth increases. Inversely, pelagic piscivorous fish feed on benthic sources in shallow waters and focus more and more on pelagic subsidies as depth increases. This conclusion was reached thanks to an original approach combining two Bayesian modeling approach: IsoWEB and MixSIAR.

Modeling considerations

The combined use of two different Bayesian mixing models (IsoWeb [START_REF] Kadoya | IsoWeb: A bayesian isotope mixing model for diet analysis of the whole food web[END_REF]& MixSIAR Semmens et al. 2015), using DT and SI data simultaneously resulted in better estimates than when used independently and compensated for some of each other's' drawbacks. One of the main advantages of IsoWeb is the use of non-isotopic data, generally

Benthic-pelagic and pelagic-benthic couplings in shallow semi-enclosed marine ecosystems

Our results at the scale of the entire EEC (IsoWeb model) indicated equally important dietary contributions of benthic and pelagic subsidies for two functional groups of pelagic fish, namely planktivorous and pelagic piscivorous fish, whereas these groups are usually known to feed primarily on zooplankton and small fishes (e.g., [START_REF] Olaso | Seasonal changes in the north-eastern Atlantic mackerel diet (Scomber scombrus) in the north of Spain (ICES Division VIIIc)[END_REF]. In parallel, benthosfeeding and endobenthos-feeding fish are known to feed on a variety of bottom animals, but preferentially on crustaceans and polychaete worms, respectively, whereas demersal piscivorous fish also forage on bottom-living fish. For these three functional groups, our results are in agreement with previous studies [START_REF] Cohen | Gadiform Fishes of the World (Order Gadiformes): An annotated and illustrated catalogue of cods, hakes, grenadiers, and other gadiform fishes known to date[END_REF]) and confirm that most of their energy is of benthic origin, representing ~73% of their diet (IsoWeb).

Planktivorous fish are important prey for a large number of predators, including larger fish, cephalopods, marine mammals and man (Mackinson and Daskalov 2007). A contribution of benthic sources to their diet suggests that they could be key components of the benthicpelagic coupling by transferring benthic-derived carbon and energy towards higher trophic levels in shallow semi-enclosed seas, which could potentially differentiate the structure and functioning of their food webs from that of deeper open oceanic ecosystems [START_REF] Pikitch | The global contribution of forage fish to marine fisheries and ecosystems[END_REF]). In the same vein, piscivores are characterized by generalist diets due to their mobility.

This includes a wide variety of prey from invertebrates to fish, of both pelagic and benthic origin, thereby contributing to benthic-pelagic coupling [START_REF] Schindler | Food web structure and littoral zone coupling to pelagic trophic cascades[END_REF]. This is exemplified by the large range of δ 15 N values observed in piscivorous pelagic fish that indicates high dietary plasticity and the consumption of prey from different TL.

Numerous studies in large temperate lakes, with similar characteristics as the EEC (e.g., similar depth range, temperature, presence of benthic and pelagic invertebrates and fish) have found patterns comparable to those of this study. Vander Zanden and Vadeboncoeur (2002), using SI and stomach content data on 16 freshwater fish species (from both littoral and pelagic habitats) showed that benthic-derived energy pathways accounted for more than 50% of total fish energy intake and that this pattern is a general feature common to the large lakes of North America (e.g., Lakes Superior, Michigan and Ontario). Similarly, several studies have emphasized the role of predation and nutrient transfer by mobile predators in benthic-pelagic coupling by showing that benthic invertebrates were the major prey source for pelagic fishes [START_REF] Stewart | Dynamics of Consumption and Food Conversion by Lake Michigan Alewives: An Energetics-Modeling Synthesis[END_REF][START_REF] Schindler | Food web structure and littoral zone coupling to pelagic trophic cascades[END_REF][START_REF] Schindler | Habitat coupling in lake ecosystems[END_REF]. Due to the proximity between pelagic and benthic compartments, shallow semi-enclosed seas and large deep lakes (>20.000 km 2 , ~80-100m depth) share similar physical and biological constraints affecting benthic-pelagic coupling. Moreover, it seems clear that in shallow nonstratified waters, the basis of the benthic and pelagic food webs (i.e., primary producers and primary consumers) are not independent and should be studied as inter-dependent and highly connected units [START_REF] Boero | The continuity of living matter and the discontinuities of its constituents: do plankton and benthos really exist?[END_REF], Cresson et al. 2014b) to fully understand processes affecting the structure and dynamics of aquatic trophic networks.

Variation of benthic-pelagic and pelagic-benthic couplings along the depth gradient

The use of depth as a continuous variable partly explained the variation in consumers' isotopic ratios and helped untangling complex interactions in the EEC over a large spatial scale. In shallow waters, pelagic and benthic sources are highly connected and benefit primary and secondary consumers in the two compartments, which are thus difficult to discriminate. It is worth noting that strong links between zooplankton and zoobenthos from both functional (energy fluxes) and structural (life cycle) perspectives [START_REF] Boero | The continuity of living matter and the discontinuities of its constituents: do plankton and benthos really exist?[END_REF], particularly in coastal marine areas, may indicate even higher proportions of benthic-derived energy for benthos-feeding and pelagic piscivorous fish. For example, the proximity of δ 13 C and δ 15 N values of planktivores and benthic decapods rendered their discrimination by the mixing models challenging. This observation highlights the strong connection between compartments, where primary and secondary consumers (i.e., herbivores and omnivores) can have access to both pelagic-and benthic-derived matter. Our results support the hypothesis of a greater consumption of benthic prey by pelagic predators and of pelagic prey by benthic predators in shallow waters. Our results, transposing the initial observations by [START_REF] Kopp | Reorganization of a marine trophic network along an inshore-offshore gradient due to stronger pelagic-benthic coupling in coastal areas[END_REF] in a depth continuum, suggest stronger benthic-pelagic and pelagic-benthic couplings with decreasing depth through plasticity in fish diet, in addition to already known physical processes (e.g., vertically mixed waters) and ecological features (e.g., vertical migration of zooplankton and epibenthic fauna). In semi-enclosed marine ecosystems, depth is correlated with other environmental and oceanographic variables (e.g., distance to shore, distribution of water masses, currents) making it a suitable variable to account for habitat variation and its potential effect on species diet. The gradual, almost linear, change in the strength of benthic-pelagic and pelagic-benthic couplings along the depth gradient (Figure 3) is probably related to the fact that waters are well-mixed in such ecosystems and the resulting absence of physical barrier in the water column. In open oceanic ecosystems, it is likely that the strength of these couplings shifts more abruptly with depth in relation to physical barriers such as thermoclines or transitions between euphotic, dysphotic and aphotic zones. It is also expected that in such ecosystems (e.g., 76% at 75m depth). Unfortunately, the benthic-suspension feeders group included bivalves and gastropods that are present in both shallow and deep waters. Similarly, there is no published evidence of a higher abundance of worms in deeper waters in the EEC that could explain the increase of their contribution with increasing depth and no high resolution data on the spatial distribution of polychaetes worms is available for the area. The absence of a known spatial trend in the abundance of both prey sources prevents from being conclusive about the relationship between endobenthos-feeding fish's diet change and prey spatial distribution.

However, similar diet variability was found in flatfishes from the Bering Sea. In this area, bivalves dominated flatfishes' diet on the inner shelf (shore to 50m), while polychaetes were the main prey on the middle shelf (~50 to 100m). In this case, diet changes were associated with estimated trends in prey abundance [START_REF] Yeung | Habitat and infauna prey availability for flatfishes in the northern Bering Sea[END_REF]. The authors however emphasized the possible mismatch between diet composition and infauna abundance in regions with high prey availability, where endobenthos-feeding fish preferentially feed on polychaetes even when they are not the dominant prey.

Demersal piscivorous fish had direct trophic links with benthic sources only. Small variations with depth were observed, the diet being composed of benthic predators and worms (~40-50%) followed by bottom-living fish (i.e., Benthos-feeder fish, ~31%) and benthic decapods (15-25%). Our results suggest a more generalist diet than the other functional groups with little intra-and inter-specific variability as shown by the weak dispersion of the consumer's SI ratios. Previous studies have however highlighted high spatial and temporal variability in the diet of species from this group (e.g., Atlantic cod, [START_REF] Daan | A Quantitative Analysis of the Food Intake of North Sea Cod, Gadus Morhua[END_REF]) that includes a wide variety of prey. This discrepancy might be explained by the fact that we used SI data that integrate diet over longer periods of time than stomach contents. Spatio-temporal diet variability in highly mobile generalist species such as demersal piscivorous fish may integrate out in terms of SI signature and result in rather similar SI ratios that are unrelated to the sampling depth. Similar patterns have been observed in shelf and lacustrine fish assemblages, where higher TL species integrate coupling of pelagic and benthic pathways, resulting into intermediate δ 13 C values [START_REF] Woodland | Benthic-pelagic coupling in a temperate inner continental shelf fish assemblage[END_REF]Secor 2013, Schindler andScheurell 2002). Although the endobenthos-feeding and demersal piscivorous fish had trophic links with benthic prey only, it is worth noting that our approach accounts only for direct predator-prey interactions and that pelagic organic matter might still fuel benthic fish through indirect trophic links (i.e., contribution of pelagic organic matter to the diet of primary consumers), that would then benefit indirectly from pelagic-benthic coupling.

Concluding remarks

The aim of this study was to provide information on the fundamental structure of the trophic network involving fish functional groups in a semi-enclosed sea and over a large spatial scale.

The use of Bayesian mixing models integrating both DT and SI data indicated high connection between benthic and pelagic compartments. Including depth as an environmental covariate, the results showed that strong benthic-pelagic and pelagic-benthic couplings in shallow waters result in fish benefiting from both benthic-and pelagic-derived matter as part of their diet. As waters become deeper, these couplings weaken and species consume prey (either benthic or pelagic) progressively more according to their habitat preferences. Future studies in epicontinental seas should include environmental variables such as depth as proxies of habitat variation to untangle consumers' resource use and highlight potentially varying energy pathways structuring food webs. Results from this study enhance our comprehension of trophic interactions sustaining the fish community, which is essential for management and informed decision-making. Figure 3: Variation of the proportional benthic contribution (all benthic sources summed) to the diet of fish functional groups (proportional pelagic contribution is simply obtained as 1-benthic contribution).

Figure legends

Blue dashed line: pelagic piscivorous fish; orange dash-dot line: benthos-feeding fish; red continuous line: demersal piscivorous and endobenthos-feeding fish

  δ[corrected] = δ[consumer] -δ[local baseline] + δ[mean baseline] with δ the nitrogen δ 15 N or carbon δ 13 C SI ratio). Any further mentioning of SI data in this paper refers to baselinecorrected SI values.

  DT or stomach contents, to identify main predator-prey relationships that are used to define the general topology of the trophic network. The weaknesses of IsoWeb are the lack of visual outputs to verify that the consumer's SI values lie inside the mixing polygon defined by the sources' positions in the IsoSpace (created by the outer most values of the prey following TEF mean TEFs and SDs calculated for every prey-consumer pair (out of the 10 6 iterations). Values of TEFs used in MixSIAR had therefore narrower variances resulting in narrower SDs in MixSIAR dietary estimates when compared to IsoWeb. Second, prey sources were grouped in MixSIAR models if they overlapped and their grouping had ecological significance (see discussion below). Thus, the number of sources for each predator differed between IsoWeb and MixSIAR models resulting in different dietary estimates.

Figure 1 :

 1 Figure 1: Diet composition of benthos-feeding (a, b) and endobenthos-feeding fish (c, d) as estimated

Figure 2 :

 2 Figure 2: Diet composition of demersal (a, b) and pelagic piscivorous fish (c, d) as estimated by the

Figure 1 Figure 3

 13 Figure 1

  keeping the same trophic links, estimated TEFs, and Bayesian model parameters as the ones previously used by IsoWeb. For every MixSIAR dietary estimate, mean contributions and standard deviations are reported.

to IsoWeb, MixSIAR can incorporate continuous covariates as predictors of sources' relative contributions to consumers' diet and multiplicative error structures. In this study, depth was introduced as a continuous covariate affecting sources' contributions to functional groups' diets. Because the model fits a continuous covariate (here depth) as a linear regressor on sources contributions in Isometric Log-Ratio (ILR) transform-space (see Francis et al. 2011; Egozcue and Pawlowsky-Glahn 2003 for details), it can also extrapolate changes in sources' contributions to diet beyond the limits of the observed distribution of the covariate. As results are presented in the space of sources' relative contributions to consumers' after reciprocal transformation from the ILR transform space, depth effect may look non-linear whereas it is linear in the ILR transform space. MixSIAR models were first run for each functional group

Table 1 : Functional groups of the Eastern English Channel food web. Species composition, reported diet and 718 mean trophic level (TL) based on SI are given for each functional group. Source data and references are 719 available in Supplementary Tables S1 and S2.

 1

	720
	Functional group	Composition	Reported diet	TL
	1. Phytoplankton	POM	-	1
	2. Benthic OM and
		BOM	-	1
	detritus
			Mostly herbivore, although some species are
	3. Copepods	Copepods		2.29 ± 0.32
			omnivorous/carnivorous or parasites
	4. Macro-zooplankton Chaetognaths and fish larvae	Feed on copepods and other zooplankton	3.19 ± 0.03
		Mainly bivalves and gastropods: Aequipecten
	5. Benthic-suspension	opercularis, Glycymeris glycymeris,	Suspension-feeder, deposit-feeeder
				2.01 ± 0.26
	feeders	Laevicardium crassum, Mimachlamys varia,	detrivore, planktivore, bacteria
		Pecten maximus, Crepidula fornicata
		Mostly Nereis sp (this study) and Aponuphis
			Omnivores feeding on phytoplankton,
	6. Deposit feeders	tubicola, Nephtys caeca, Dasybranchus gajolae,		2.72 ± 0.15
			zooplankton, crustaceans and other worms
		Glycera rouxii (Le Loc'h, Hily, and Grall 2008).
	7. Benthic decapods/	Shrimps Palaemon serratus, Processa sp. and	Omnivores feeding on algae, crustaceans,
				3.71 ± 0.79
	omnivores	Hermit crabs Pagurus bernhardus	gastropods and polychaetes
			Carnivores/scavengers feeding on
	8. Benthic decapods /	Shrimp Crangon crangon and crabs Liocarcinus
			echinoderms, crustaceans, marine worms,	3.56 ± 0.39
	predators/scavengers	holsatus, Maja brachydactyla, Necora puber
			molluscs and dead fishes
			Omnivores/scavengers feeding on marine
		Mainly echinoderms Psammechinus miliaris and
	9. Benthic predators		worms, hydroids, small crustaceans,	2.42 ± 0.37
		the gastropod Buccinum undatum
			molluscs, diatoms, macroalgae and detritus
	10. Benthos-feeding	Rays Raja clavata, Gobiidae, Triglidae	Feed on all kinds of bottom animals,
				3.83 ± 0.48
	fish	Trigloporus lastoviza, Chelidonichthys lucerna,	preferably crustaceans

Acknowledgments

The post-doctoral position of C. Giraldo was funded by the regional council "Hauts de France" through the project SIMODYN (grant n° 2014_10396). This study is part of the CPER project MARCO (grant n°2016 regional council "Hauts de France" and Préfecture du Nord Pas de Calais). We thank Paul Auchon for his fruitful ideas regarding this study, and Manuel Rouquette and Mickael Baheux (RHBL Ifremer Boulogne sur Mer) for their valuable help in the laboratory. The authors thank Dr. Tracey Loewen from Fisheries and Oceans Canada for proofreading and editing and 2 anonymous reviewers for their valuable feedback that improved the manuscript.

adjustments; [START_REF] Phillips | Best practices for use of stable isotope mixing models in food-web studies[END_REF] and the lack of a correlation value between prey's SI ratios to determine if sources can be discriminated based on their SI signatures. In this regard, the combined use of IsoWeb with MixSIAR offers several advantages. First, MixSIAR can be built upon IsoWeb calculations (e.g., trophic links, TEF estimates) and provides a simple visual display of the IsoSpace for each node, combining source and TEF uncertainties [START_REF] Semmens | MixSIAR: A Bayesian stable isotope mixing model for characterizing intrapopulation niche variation[END_REF]. Second, MixSIAR provides correlation values between sources that can help in making the decision of aggregating prey groups when appropriate. Finally, MixSIAR can include environmental (e.g., depth) or biological variables as covariates affecting sources' contributions to a consumer's diet.

In some cases, natural variability in SI signatures and TEFs of the different sources reduced the discriminative power of the mixing models and resulted in large standard deviations of sources' contribution estimates (see the full posterior distributions of sources' contributions to fish functional groups, Figure S3). Higher uncertainty was especially observed when the mixing model was unable to distinguish between two sources based on their SI values (e.g., copepods and benthic deposit feeders as sources for benthos-feeding fish considered at the EEC-scale). However, for all groups, major and minor dietary sources and dominance of benthic vs. pelagic sources could be identified at the EEC-scale. Interestingly, variation in fish's SI ratios was partly explained by depth. This resulted in a clearer discrimination between food sources and therefore more certain sources' contribution estimates when depth was included as a covariate (Figure S3: middle and right panels versus left panels), thus ultimately providing more relevant and ecologically significant information. Diet estimates from IsoWeb were slightly different from those provided by MixSIAR, which can be explained by two factors. First, IsoWeb allows variation in TEFs that results in larger SD estimates. MixSIAR models were built upon IsoWeb outputs, therefore using the resulting other parameters than depth need to be considered to fully account for the spatial heterogeneity of benthic-pelagic and pelagic-benthic couplings. For example, at a given depth, varying distance to marginal ice zones in the Barents Sea has been proven to influence pelagic-benthic coupling [START_REF] Tamelander | Trophic relationships and pelagic-benthic coupling during summer in the Barents Sea Marginal Ice Zone, revealed by stable carbon and nitrogen isotope measurements[END_REF]. Similarly, seasonal or inter-annual variations in surface primary and secondary productions modulate the amplitude of primary carbon sources that reaches the bottom and different oceanographic features (e.g., currents, eddies, water column stratification) and ecological features (e.g., structure of the pelagic-food web) can be better proxies than depth to account for variation in the pelagic-benthic coupling [START_REF] Grebmeier | The influence of oceanographic processes on pelagicbenthic coupling in polar regions: A benthic perspective[END_REF]. Finally, the role of species that perform wide diel vertical migrations (fish and zooplankton) in the pelagic-benthic coupling has also been highlighted in the North Atlantic and Mediterranean seas. Most of these species concentrate at the upper and mid continental slope depths (300-500 m) making them accessible to both, benthic and bentho-pelagic feeders, resulting in relatively strong coupling between benthic and pelagic production [START_REF] Tecchio | Trophic dynamics of deep-sea megabenthos are mediated by surface productivity[END_REF][START_REF] Trueman | Trophic interactions of fish communities at midwater depths enhance long-term carbon storage and benthic production on continental slopes[END_REF].

Other sources of dietary variation

Apart from benthic-pelagic and pelagic-benthic couplings, a change in diet with depth is expected due to the mosaic of habitats (i.e., spatial differences in prey abundance or composition due to different sediment types or other abiotic conditions). Endobenthos-feeding fish had no trophic link identified (from previous DT analysis; [START_REF] Cachera | Individual diet variation in a marine fish assemblage: Optimal Foraging Theory, Niche Variation Hypothesis and functional identity[END_REF]) with pelagic prey. Their main prey were all benthic and, due to their sedentary behavior, little variation in diet was expected. However, endobenthos-feeding fish diet varied largely with depth. This pattern could be linked to variation in the abundance of their various benthic prey.

Endobenthos-feeding fish fed mainly on benthic-suspension feeders in shallow waters (e.g., 65.5% of their diet at 25m) and switched to benthic-predators and worms in deeper waters


Joao Pedro Ferreira

Romain Eschalier

Kévin Duarte

Kevin Damman

Finn Gustafsson

Morten Schou

Nicolas Girerd

Renaud Fay

Stéphane Tala

Bertram Pitt

João Pedro Ferreira

Kevin Duarte

; Faiez Zannad

Patrick Rossignol
email: p.rossignol@chru-nancy.fr

Patrick Pr

Rossignol

Reduced Diuretic Dose in Patients Treated With Eplerenone: Data From the EPHESUS Trial

Keywords: myocardial infarction, heart failure, systolic dysfunction, eplerenone, loop diuretics, congestion, treatment effect

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

Introduction

Loop diuretics are used for congestion relief and dose-adaptations are usually a consequence of the clinicians` clinical judgement about the congestive status of the patient 1-3 : those with more severe signs and symptoms of congestion likely receive higher diuretic doses (in a tentative to relieve their congestion) and, as consequence of their worse clinical condition, have worse prognosis [START_REF] Ellison | Diuretic Treatment in Heart Failure[END_REF][START_REF] Domanski | Diuretic use, progressive heart failure, and death in patients in the Studies Of Left Ventricular Dysfunction (SOLVD)[END_REF] .

In EPHESUS (Eplerenone, a Selective Aldosterone Blocker, in Patients with Left Ventricular Dysfunction after Myocardial Infarction) patients were randomized, to either eplerenone or placebo, if they had reduced ejection fraction and signs of heart failure (HF) or diabetes within two weeks after a myocardial infarction (MI) [START_REF] Pitt | The EPHESUS trial: eplerenone in patients with heart failure due to systolic dysfunction complicating acute myocardial infarction. Eplerenone Post-AMI Heart Failure Efficacy and Survival Study[END_REF] . Consequently, many of these patients had HF (90%) and needed diuretics (60%) for congestion relief. In EPHESUS eplerenone had a beneficial effect which translated into reduced hospitalizations and death. One of the possible mechanisms associated with the treatment effect of eplerenone (and spironolactone) is a decrease of congestion by increasing diuresis and natriuresis, induced by a contraction in plasma volume, which we could assess with an indirect estimation based on hemoglobin and hematocrite [START_REF] Duarte | Prognostic Value of Estimated Plasma Volume in Heart Failure[END_REF][START_REF] Rossignol | Eplerenone survival benefits in heart failure patients post-myocardial infarction are independent from its diuretic and potassium-sparing effects. Insights from an EPHESUS (Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study) substudy[END_REF] . Based on these effects, we hypothesized that clinicians would observe patients` clinical improvement which would lead them to reduce loop diuretics.

The aims of the present study are to: 1) characterize patients` according to the dose of loop diuretic they took at baseline; 2) assess the outcome associations of different doses of loop diuretics ;

3) study the effect of eplerenone on the diuretic doses during the follow-up; 4) explore whether the effect of eplerenone could have been modified by the diuretic doses at baseline.

Methods

Study design and population

The aims and methodology of the EPHESUS study have been previously described [START_REF] Montalescot | Early eplerenone treatment in patients with acute ST-elevation myocardial infarction without heart failure: the Randomized Double-Blind Reminder Study[END_REF] . Patients enrolled in EPHESUS had an acute MI complicated by systolic dysfunction (i.e. LV ejection fraction ≤40%) and HF (documented by at least one of the following: presence of pulmonary rales, chest radiography showing pulmonary venous congestion, or the presence of a third heart sound) or diabetes. Indeed, patients with diabetes mellitus were not required to have evidence of HF. Patients entered the study at any point from 3-14 days after MI and were randomly assigned to treatment with eplerenone or placebo in addition to standard medical therapy, which could include angiotensin converting enzyme inhibitors (ACEi) or angiotensin receptor blockers (ARBs), β-blockers, diuretics, aspirin, statins as well as coronary reperfusion therapy. EPHESUS was an event-driven study with a mean follow-up duration of 16 months. The investigation conformed with the principles outlined in the Declaration of Helsinki [START_REF] Rossignol | Determinants and consequences of renal function variations with aldosterone blocker therapy in heart failure patients after myocardial infarction: insights from the Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study[END_REF] . Written informed consent was obtained for all subjects.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Statistical analysis and end-points

Continuous variables are presented as mean ± standard deviation and categorical variables as frequencies (percentages). Hazard ratios are presented with their 95% confidence intervals as HR (95%CI). Loop diuretic doses were converted to furosemide equivalents as follows: furosemide 40 mg = torsemide 10 mg = bumetanide 1 mg = piretanide 10 mg [START_REF] Duarte | Prognostic Value of Estimated Plasma Volume in Heart Failure[END_REF][START_REF] Voors | Tackling Early Heart Failure Deaths and Readmissions by Estimating Congestion[END_REF] ; patients not on loop diuretics at inclusion were coded 0 mg/day. Three groups were then created: patients without loop diuretics (0 mg/day), those with loop diuretics at doses ≤40 mg/day, and those with doses >40 mg/day or intravenous (IV) diuretics. These subgroups were created based on the loop diuretic doses commonly used in clinical practice and also to keep sufficient patient numbers between the loop diuretic categories to perform the analyses, as patients with doses above 40 mg/day represented less than 20% of the population and those with doses above 80 mg/day less than 1%. We assessed the loop diuretic doses at baseline, the mean loop diuretic dose with all available data throughout the follow-up, and the dose at specific time-points (30, 90, 180, 270, 360 days); as EPHESUS had a relatively short follow-up time (median =1.3 years) observations beyond 1-year had few observations. We also performed a sensitivity analysis including only the patients who remained alive during the follow-up.

Comparisons of furosemide equivalent groups at baseline were carried out using Pearson's Chi-2 test and one-way ANOVA test, as required. Cox regression models were performed to assess the associations between loop diuretic doses (continuous and categorical) and the study outcomes, which were: 1) a composite of cardiovascular death or hospitalization for HF; 2) an extended composite of cardiovascular death, HF hospitalization or IV diuretic use; 3) cardiovascular death; and 4) all-cause death. The use of IV diuretics was included in the exploratory extended composite outcome, because it is clinically meaningful by representing an objective intensification of HF therapy, associated with subsequent mortality, has the potential for increasing the number of events and therefore increasing the study power [START_REF] Okumura | Importance of Clinical Worsening of Heart Failure Treated in the Outpatient Setting: Evidence from the Prospective Comparison of ARNI with ACEI to Determine Impact on Global Mortality and Morbidity in Heart Failure Trial (PARADIGM-HF)[END_REF] . The loop diuretic-outcome associations were tested both in unadjusted/crude and adjusted fashion using the baseline diuretic dose, the mean diuretic dose throughout the follow-up, and the time-updated dose. Covariates for adjustment were: age, sex, Killip class, left ventricular ejection fraction, reperfusion therapy, hemoglobin, potassium, sodium, systolic blood pressure, heart rate, estimated glomerular filtration rate, body mass index, history of diabetes, history of hypertension, atrial fibrillation, chronic obstructive pulmonary disease, previous myocardial infarction, previous heart failure hospitalization, peripheral arterial disease. These covariates were selected based on their clinical relevance, prognostic association, and use on previous reports [START_REF] Duarte | Prognostic Value of Estimated Plasma Volume in Heart Failure[END_REF] .

Prespecified interactions (based on the clinical hypothesis) between baseline loop diuretic dose and the study drug were performed. Cox proportional hazards assumptions were tested by plotting the Schoenfeld residuals vs. time without proportional hazards violation. We also performed a landmark analysis to assess the effect of the loop diuretic doses in the first 90 days of the trial and subsequent outcomes (cardiovascular death or heart failure hospitalization) excluding from the analysis the patients who died or were hospitalized in the first 90 days. Linear mixed effects models with repeated measures over time were performed to assess the loop-diuretic dose changes by treatment group allocation (eplerenone vs. placebo). The diuretic doses and the interaction of the treatment by time were specified as fixed effects with random effects at the patient level. All analyses were performed

Results

Characteristics of the study population by baseline loop diuretic dose

A total of 6632 patients were included; at baseline 3352 (50.5%) did not have diuretics, 2195 (33.1%) had diuretic doses between 1 and 40 mg/day, and 1085 (16.4%) had diuretic doses above 40 mg/day. Patients with higher furosemide equivalent doses had worse clinical status; they were older, more often female, had worse Killip class, lower ejection fraction, had coronary reperfusion performed less often, had more often HF symptoms, lower blood pressure, higher heart rate, poorer renal function, had more often diabetes, hypertension, a previous MI, angina, and previous HF hospitalization. Table 1.

Loop diuretic dose and outcome associations

Both higher baseline loop diuretic dose and increase in loop diurectics during the follow-up were associated with worse prognosis for all the studied outcomes. Table 2. However, after adjustment the associations were less marked, suggesting that an important component of confounding occurred i.e. patients with higher loop diuretics are sicker and is likely that their underlying illness is driving the outcome associations and not the diuretic doses themselves. For example, patients treated with furosemide equivalents higher than 40 mg/day had a crude HR (95%CI) of cardiovascular death or HF hospitalization of 4.96 (4.24-5.79), after adjustment for potential confounders the associations decreased to 2.33 (1.95-2.78). Similar findings were present for all the studied outcomes. Table 2 & Figure 1. The associations between the loop diuretic dose and outcomes were linear and are represented in the Supplemental Figure 1, by treatment group and for the outcome of cardiovascular death or heart failure hospitalization. In the Supplemental Figure 2, we also present the association of loop diuretic dose during follow-up in a continuous fashion with the distribution of the loop diuretic doses on the background for the outcome of cardiovascular death or heart failure hospitalization (similar associations were present for cardiovascular and all-cause death). The adjusted landmark analysis taking only the cumulative loop diuretic doses in the first 90 days and subsequent outcomes provided similar associations. Supplemental Table 1.

Eplerenone and loop diuretic dose

Eplerenone treatment was associated with reduced loop diuretic dose prescription throughout the follow-up. Table 3, Figure 2 & Figure 3. The loop diuretic dose reductions with eplerenone were already apparent at 90 days, and became more marked at 180 days and after. For example, at 180 days 14.2% of the patients had furosemide equivalent doses >40 mg/day in the placebo group vs. 11.6% in the eplerenone group, p =0.003. Table 3 & Figure 3. Eplerenone treatment led to a mean furosemide equivalent dose reduction of -2.2 mg/day (-2.9 to -1.6; p<0.001) throughout the follow-up; a reduction that became statistically significant (p <0.05) at day 90 and remained significant throughout the follow-up. Figure 2. These results were similar including only the patients that remained alive during the follow-up. Supplemental Figure 3.

Eplerenone treatment effect

Eplerenone was effective in reducing morbidity and mortality regardless of the baseline loop diuretic dose used: HR (95%CI) for the outcome of cardiovascular death or HF hospitalization =0.83 (0.75-0.92); p for interaction =0.54. Similar findings were observed for all the studied outcomes. Table 4.

Discussion

The present study shows that MI patients with HF or diabetes taking higher diuretic doses had worse clinical features (e.g. older age, worse Killip class, lower blood pressure, worse renal function, and previous hospitalization for HF). Higher diuretic doses were independently associated with worse prognosis with a substantial decrease in the strength of the associations after adjustment (suggesting a reverse causation effect). Patients taking eplerenone had their loop diuretic dose significantly reduced during follow-up without evidence of eplerenone`s treatment effect modification by loop diuretics.

These findings support the utility of eplerenone for reducing congestive signs and symptoms, leading clinicians to reduce the loop diuretic doses in their patients. These findings may help clinicians in better treating their patients and provide insight into the mechanisms of action of eplerenone in patients with the characteristics of those enrolled in EPHESUS.

In a previous report from the EPHESUS trial we have shown that eplerenone decreased body weight and plasma volume, whereas blood protein levels and potassium increased within a month, supporting a eplerenone-induced diuretic effect 8,7. The present report corroborates these findings from the perspective of the clinician i.e. even under blinded treatment clinicians probably noted the decongestion of the patients and reduced the diuretic doses accordingly. This dose reduction became statistically significant at day 90, suggesting that eplerenone may reduce the need for diuretics by improving cardiac function and patient clinical status, rather than having an early diuretic effect.

Particularly, in the high-risk subset treated with furosemide equivalent dose higher than 40 mg/day, patients significantly decreased furosemide doses (below 40 mg/day) after 6 months of treatment with eplerenone, changes in therapy that were sustained over time. This finding is of particular relevance because patients taking higher diuretic doses have higher risk of subsequent events; this is likely not due to the diuretic dose itself but due to the patients` status, with sicker patients needed higher diuretic doses for congestion relief (as observed herein) i.e. reverse causation. In fact, more aggressive uptitration of loop diuretics or more potent loop diuretics (e.g. the ongoing TRANSFORM trial is testing the hypothesis that torasemide may improve outcomes compared to furosemide) might be indicated for achieving an efficient decongestion which is associated with better outcomes [START_REF] Lala | Relief and Recurrence of Congestion During and After Hospitalization for Acute Heart Failure: Insights From Diuretic Optimization Strategy Evaluation in Acute Decompensated Heart Failure (DOSE-AHF) and Cardiorenal Rescue Study in Acute Decompensated Heart Failure (CARESS-HF)[END_REF][START_REF] Voors | Tackling Early Heart Failure Deaths and Readmissions by Estimating Congestion[END_REF] . In the light of the recent DAPA-HF trial results [START_REF] Mcmurray | Dapagliflozin in Patients with Heart Failure and Reduced Ejection Fraction[END_REF] , in which the SGLT-2 inhibitor dapagliflozin (vs. placebo) reduced major cardiovascular events in patients with HF and reduced ejection fraction, 71% of the patients were treated with an MRA, raising the hypothesis that MRAs and SGLT-2 inhibitors may act synergically towards congestion relief. Such hypothesis obviously warrants a dedicated testing aimed at better defining the fine tuning between proximal tubule (SGLT2i), loop of Henle (loop diuretic), collecting duct (MRA) sodium reabsorption inhibition, acknowledging that both SGLT-2 inhibitors and MRA may diplay pleiotrophic beneficial effects in HF beyond their diuretic properties.

Notwithstanding, loop diuretic dose is a marker of disease severity, a marker that was significantly reduced by randomized allocation to eplerenone, a finding that remained similar when including only the patients alive during the follow-up. The reduced need for diuretics in patients randomized to eplerenone may be secondary to the diuretic (supported by the weight loss and protein increase) [START_REF] Rossignol | Eplerenone survival benefits in heart failure patients post-myocardial infarction are independent from its diuretic and potassium-sparing effects. Insights from an EPHESUS (Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study) substudy[END_REF] and potential natriuretic effect of eplerenone [START_REF] Zhang | The specific mineralocorticoid receptor blocker eplerenone attenuates left ventricular remodeling in mice lacking the gene encoding guanylyl cyclase-A[END_REF] . As eplerenone also decreases natriuretic peptides, suggesting a reduction in cardiac filling pressures, it is possible that a hemodynamic effect also contributes for the reduction in the loop diuretic doses [START_REF] Montalescot | Early eplerenone treatment in patients with acute ST-elevation myocardial infarction without heart failure: the Randomized Double-Blind Reminder Study[END_REF] . Since investigators were blinded to therapy, the diuretic dose reductions were performed after careful assessment of changes in patients` signs and symptoms, which improved in patients randomized to eplerenone. Unless contraindicated) all patients with a MI and a LVEF≤40% with HF or diabetes should receive a MRA (such as eplerenone); this treatment can favourably impact patients` outcomes and reduce the need of loop diuretics, which may translate patient`s clinical improvement throughout the trial, even in those who did not have a major adverse cardiovascular event such as hospitalization or death. Loop diuretic dose changes could thus be used as a surrogate for patients` clinical status and possibly as an intermediate end-point in clinical trials of patients with MI and systolic dysfunction. An effect on blood pressure is not likely to play a major role in the clinicians` decision, as eplerenone in this context (MI with systolic dysfunction) did not cause blood pressure drop. At one year, the mean blood pressure had increased by 8/4 mmHg in the placebo group and by 5/3 mmHg in the eplerenone group [START_REF] Pitt | The EPHESUS trial: eplerenone in patients with heart failure due to systolic dysfunction complicating acute myocardial infarction. Eplerenone Post-AMI Heart Failure Efficacy and Survival Study[END_REF] . An observed effect on worsening kidney function, along with alleviated congestive symptoms may also have triggered loop diuretic down-titration; in a previous report, we showed that several features were associated with an early worsening in kidney function (>20% after 1 month), including loop diuretic use at baseline [START_REF] Rossignol | Determinants and consequences of renal function variations with aldosterone blocker therapy in heart failure patients after myocardial infarction: insights from the Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study[END_REF] .

Limitations

Several limitations should be noted in the present analysis. This is a non-prespecified secondary analysis of a randomized controlled trial, therefore these findings should be regarded as hypothesis generating. The diuretic doses were recovered from the patients` case report form filled-in by the site clinicians, as consequence some dose-attribution errors might have occurred; despite the manual drug/dose-checking we performed. The reasons for diuretic dose changes were not captured in the study, which could provide additional granularity in our findings.

Conclusion

In patients with MI, systolic dysfunction and HF or diabetes, higher diuretic doses were independently associated with worse prognosis, but eplerenone treatment led to a loop diuretic dose reduction during follow-up without evidence of treatment effect modification by loop diuretics. These findings suggest that eplerenone may reduce congestion, which enables clinicians to reduce the dose of loop diuretics.

Disclosures

PR reports grants and or honoraria (personal fees) from Ablative Solutions, AstraZeneca, Bayer, Corvidia, CVRx, Fresenius, G3P, Grunenthal, Idorsia, Novartis, NovoNordisk , Relypsa, Servier, Stealth Peptides, Vifor Fresenius Medical Care Renal Pharma, and Cofounder of CardioRenal with FZ.

All the other authors report no relevant conflicts of interest with regards to the present manuscript. Model with treatment (eplerenone or placebo) by baseline diuretic dose (0mg, ≤40mg, >40mg) interaction adjusted on age, sex, Killip class, left ventricular ejection fraction, reperfusion therapy, hemoglobin, potassium, sodium, systolic blood pressure, heart rate, estimated glomerular filtration rate, body mass index, history of diabetes, history of hypertension, atrial fibrillation, chronic obstructive pulmonary disease, previous myocardial infarction, previous heart failure hospitalization, peripheral arterial disease. 3).

using STATA version 15 (

 15 StataCorp. 2017. Stata Statistical Software: Release 15. College Station, TX: StataCorp LLC).

Figure 1 .Figure 2 .

 12 Figure 1. Kaplan-Meier curves for the outcome of CV death or HF hospitalisation by categories of mean loop diuretic dose (furosemide equivalents in mg/day)

Figure 3 .

 3 Figure 3. Loop diuretic doses >40mg/day of furosemide equivalents by treatment allocation

Table 1 .

 1 Patients` characteristics by baseline loop diuretic dose (in mg/day)

	Patients` characteristics	Loop Diur. =0	Loop Diur. ≤40	Loop Diur. >40	p-value
	N.	3352	2195	1085
	Age (years)	61.3 ± 11.5	66.1 ± 10.9	67.7 ± 10.8	<0.001
	Male	2507 (74.8%)	1493 (68.0%)	714 (65.8%)	<0.001
	White race	3024 (90.2%)	1991 (90.7%)	969 (89.3%)	0.003
	Killip I	631 (19.0%)	249 (11.4%)	132 (12.2%)	<0.001
	II	2244 (67.4%)	1427 (65.4%)	606 (56.1%)
	III	380 (11.4%)	434 (19.9%)	281 (26.0%)
	IV	74 (2.2%)	71 (3.3%)	62 (5.7%)
	LVEF (%)	34.2 ± 5.4	32.5 ± 6.3	30.8 ± 6.7	<0.001
	Reperfusion/revasc.	1723 (51.4%)	871 (39.7%)	412 (38.0%)	<0.001
	HF symptoms	2962 (88.5%)	2015 (91.9%)	988 (91.1%)	<0.001
	Hemoglobin (g/dL)	13.4 ± 1.7	13.3 ± 1.7	13.0 ± 1.8	<0.001
	Potassium (mmol/L)	4.3 ± 0.4	4.3 ± 0.5	4.2 ± 0.5	<0.001
	Sodium (mmol/L)	139.6 ± 4.4	139.6 ± 4.3	138.7 ± 4.2	<0.001
	SBP (mmHg)	119.2 ± 16.2	119.4 ± 16.5	117.8 ± 17.4	0.024
	DBP (mmHg)	72.8 ± 10.3	71.7 ± 10.7	70.5 ± 11.6	<0.001
	Heart rate (beats/min)	73.3 ± 10.9	75.3 ± 11.9	77.7 ± 13.1	<0.001
	eGFR (mL/min/1.73m²)	72.8 ± 20.2	65.7 ± 20.3	60.2 ± 20.6	<0.001
	Body mass index (kg/m²)	27.3 ± 4.4	27.4 ± 4.5	27.7 ± 4.9	0.053
	Diabetes	955 (28.5%)	715 (32.6%)	472 (43.5%)	<0.001
	Hypertension	1915 (57.1%)	1407 (64.1%)	685 (63.1%)	<0.001
	Atrial fibrillation	316 (9.4%)	332 (15.1%)	226 (20.8%)	<0.001
	COPD	251 (7.5%)	229 (10.4%)	145 (13.4%)	<0.001
	Previous MI	777 (23.2%)	642 (29.2%)	383 (35.3%)	<0.001
	Angina	1290 (38.5%)	952 (43.4%)	493 (45.4%)	<0.001
	PVD	336 (10.0%)	288 (13.1%)	199 (18.3%)	<0.001
	Previous HFH	148 (4.4%)	190 (8.7%)	174 (16.0%)	<0.001
	ACEi/ARB	2828 (84.4%)	1928 (87.8%)	995 (91.7%)	<0.001
	Beta-blocker	2682 (80.0%)	1585 (72.2%)	694 (64.0%)	<0.001
	Digoxin	271 (8.1%)	430 (19.6%)	303 (27.9%)	<0.001
	Index Q-wave MI	2526 (76.1%)	1502 (69.8%)	675 (65.0%)	<0.001
	Eplerenone allocation	1706 (50.9%)	1076 (49.0%)	537 (49.5%)	0.36
	Legend: LVEF, left ventricular ejection fraction; HFH, heart failure hospitalization; SBP, systolic
	blood pressure; DBP, diastolic blood pressure; ACEi/ARB, angiotensin converting enzyme
	inhibitor/angiotensin receptor blocker; COPD, chronic obstructive pulmonary disease; MI, myocardial
	infarction; PVD, peripheral vascular disease; IV, intravenous; Loop diur., loop diuretic in furosemide
	dose equivalents.

Table 2 .

 2 Loop diuretic dose (in mg/day) and outcome associations , mean diuretic dose per day during follow-up until the event of interest. Legend: CV, cardiovascular; HFH, heart failure hospitalization; IV, intravenous. Model adjusted on age, sex, Killip class, left ventricular ejection fraction, reperfusion therapy, hemoglobin, potassium, sodium, systolic blood pressure, heart rate, estimated glomerular filtration rate, body mass index, history of diabetes, history of hypertension, atrial fibrillation, chronic obstructive pulmonary disease, previous myocardial infarction, previous heart failure hospitalization, peripheral arterial disease.

	End-point	Diuretic	Dose	Events (%) Crude HR (95%CI) Adj. HR (95%CI)	p-value
			0	489 (14.6)	Ref.	Ref.	-
		Baseline	≤40	536 (24.4)	1.78 (1.58-2.01)	1.21 (1.07-1.38)	0.004
			>40	426 (39.3)	3.22 (2.82-3.67)	1.64 (1.42-1.90)	<0.001
			0	237 (10.1)	Ref.	Ref.	-
	CV death/HFH	Follow-up	≤40	720 (23.3)	2.47 (2.13-2.86)	1.63 (1.39-1.90)	<0.001
			>40	494 (41.5)	4.96 (4.24-5.79)	2.33 (1.95-2.78)	<0.001
			0	358 (10.7)	Ref.	Ref.	-
		Time-updated	≤40	575 (26.2)	1.86 (1.58-2.19)	1.22 (1.03-1.45)	0.024
			>40	518 (47.7)	3.94 (3.30-4.69)	1.95 (1.59-2.38)	<0.001
			0	567 (16.9)	Ref.	Ref.	-
		Baseline	≤40	612 (27.9)	1.76 (1.57-1.97)	1.24 (1.10-1.40)	<0.001
			>40	540 (49.8)	3.94 (3.50-4.43)	2.21 (1.94-2.52)	<0.001
	CV		0	388 (15.7)	Ref.	Ref.	-
	death/HFH/IV	Follow-up	≤40	813 (26.8)	1.77 (1.56-1.99)	1.15 (1.01-1.31)	0.029
	diur.		>40	518 (46.0)	3.45 (3.03-3.93)	1.58 (1.36-1.85)	<0.001
			0	446 (13.3)	Ref.	Ref.	-
		Time-updated	≤40	691 (31.5)	1.82 (1.55-2.15)	1.21 (1.01-1.44)	0.035
			>40	582 (53.6)	4.00 (3.34-4.79)	2.03 (1.65-2.49)	<0.001
			0	299 (8.9)	Ref.	Ref.	-
		Baseline	≤40	320 (14.6)	1.69 (1.44-1.98)	1.11 (0.94-1.32)	0.21
			>40	271 (25.0)	3.11 (2.64-3.67)	1.53 (1.27-1.85)	<0.001
			0	166 (7.2)	Ref.	Ref.	-
	CV death	Follow-up	≤40	412 (13.5)	1.92 (1.60-2.29)	1.13 (0.94-1.38)	0.20
			>40	312 (24.2)	3.60 (2.98-4.35)	1.46 (1.18-1.82)	0.001
			0	281 (8.4)	Ref.	Ref.	-
		Time-updated	≤40	320 (14.6)	1.75 (1.49-2.06)	1.13 (0.95-1.34)	0.16
			>40	289 (26.6)	3.93 (3.34-4.63)	1.91 (1.58-2.31)	<0.001
			0	364 (10.9)	Ref.	Ref.	-
		Baseline	≤40	363 (16.5)	1.58 (1.37-1.83)	1.03 (0.89-1.21)	0.66
			>40	305 (28.1)	2.89 (2.48-3.36)	1.43 (1.21-1.69)	<0.001
			0	193 (8.4)	Ref.	Ref.	-
	All-cause death	Follow-up	≤40	480 (15.7)	1.92 (1.63-2.27)	1.16 (0.97-1.38)	0.11
			>40	359 (27.9)	3.58 (3.00-4.26)	1.50 (1.22-1.83)	<0.001
			0	343 (10.2)	Ref.	Ref.	-
		Time-updated	≤40	359 (16.4)	1.61 (1.39-1.87)	1.04 (0.88-1.21)	0.66
			>40	330 (30.4)	3.70 (3.18-4.31)	1.80 (1.51-2.14)	<0.001
	Median (pct 25-75 ) follow-up time =1.3 (1.0-1.7) years.
	Follow-up

Table 3 .

 3 Eplerenone/Placebo treatment and loop diuretic doses (in mg/day) throughout the follow-up

	Time	Diuretic dose N (%) Placebo	N (%) Eplerenone	p-value
		0	1646 (49.7)	1706 (51.4)
	0 (randomization)	≤40	1119 (33.8)	1076 (32.4)	0.36
		>40	548 (16.5)	537 (16.2)
		0	1616 (51.2)	1692 (52.7)
	30d	≤40	1133 (35.9)	1106 (34.4)	0.44
		>40	410 (13.0)	414 (12.9)
		0	1566 (51.1)	1685 (53.9)
	90d	≤40	1085 (35.4)	1052 (33.7)	0.074
		>40	416 (13.6)	389 (12.4)
		0	1544 (51.7)	1672 (54.8)
	180d	≤40	1017 (34.1)	1023 (33.6)	0.003
		>40	425 (14.2)	352 (11.6)
		0	1441 (50.8)	1601 (55.0)
	270d	≤40	984 (34.7)	966 (33.2)	0.001
		>40	413 (14.6)	343 (11.8)
		0	1244 (51.0)	1360 (54.6)
	360d	≤40	841 (34.5)	837 (33.6)	0.005
		>40	353 (14.5)	292 (11.7)

Table 4 .

 4 Treatment effect by baseline diuretic dose interaction 25-75 ) follow-up time =1.3 (1.0-1.7) years. Legend: CV, cardiovascular; HFH, heart failure hospitalization; IV, intravenous.

	Outcome	N. (%)	N. (%)	HR (95%CI)	P-value	Interaction p
		Placebo	Eplerenone
	CV death/HFH	780 (23.5)	671 (20.2)	0.83 (0.75-0.92)	0.001	0.54
	CV death/HFH/IV diur.	904 (27.3)	815 (24.6)	0.88 (0.80-0.97)	0.010	0.24
	CV death	483 (14.6)	407 (12.3)	0.83 (0.72-0.94)	0.005	0.41
	All-cause death	554 (16.7)	478 (14.4)	0.85 (0.75-0.96)	0.007	0.61
	Median (pct

Funding JPF, NG, PR and FZ are supported by a public grant overseen by the French National Research Agency (ANR) as part of the second "Investissements d'Avenir" program FIGHT-HF (reference: ANR-15-RHU-0004) and by the French PIA project "Lorraine Université d'Excellence", reference ANR-15-IDEX-04-LUE and by Contrat de Plan Etat-Lorraine and FEDER Lorraine. Bibliography 1. Ponikowski, P.; Voors, A. A.; Anker, S. D.; Bueno, H.; Cleland, J. G.; Coats, A. J.; Falk, V.; Gonzalez-Juanatey, J. R.; Harjola, V. P.; Jankowska, E. A.; Jessup, M.; Linde, C.; Nihoyannopoulos, P.; Parissis, J. T.; Pieske, B.; Riley, J. P.; Rosano, G. M.; Ruilope, L. M.; Ruschitzka, F.; Rutten, F. H.; van der Meer, P., 2016 ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: The Task Force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC). Developed with the special contribution of the Heart Failure Association (HFA) of the ESC. Eur J Heart Fail 2016.

Supplemental Material

Supplemental Table 1. Landmark analysis considering only the diuretic doses in the first 90 days of the trial and subsequent events (cardiovascular death or heart failure hospitalization) Global mean loop diuretic reduction = -2.3mg (-2.9 to -1.6), p <0.001.