Yet much experimental and theoretical work over the past 40 years has explored the events during photosynthesis subsequent to absorption of light from intense, ultrashort laser pulses15. Here, we use single photons to excite under ambient conditions the light-harvesting 2 (LH2) complex of the purple bacterium Rhodobacter sphaeroides, comprising B800 and B850 rings that contain 9 and 18 bacteriochlorophyll molecules, respectively. Excitation of the B800 ring leads to electronic energy transfer to the B850 ring in approximately 0.7 ps, followed by rapid B850-to-B850 energy transfer on an approximately 100-fs timescale and light emission at 850–875 nm (refs. 19). Using a heralded single-photon source21 along with coincidence counting, we establish time correlation functions for B800 excitation and B850 fluorescence emission and demonstrate that both events involve single photons. We also find that the probability distribution of the number of heralds per detected fluorescence photon supports the view that a single photon can upon absorption drive the subsequent energy transfer and fluorescence emission and hence, by extension, the primary charge separation of photosynthesis. An analytical stochastic model and a Monte Carlo numerical model capture the data, further confirming that absorption of single photons is correlated with emission of single photons in a natural light-harvesting complex.Photosynthesis is generally assumed to be initiated by a single photon Using a heralded single-photon source along with coincidence counting, we establish time correlation functions for B800 excitation and B850 fluorescence emission and demonstrate that both events involve single photons. One of the photons, the herald, was directly detected by a single-photon counting module, Detector 1, and signifies the presence of the other photon, which is focused by a home-built microscope into a solution of LH2 complexes under ambient conditions  based on a heralded single-photon source19). After confirming the fluorescence spectra, we switched the excitation source to heralded single photons for PCQLS measurements correlating heralds of incident photons with fluorescent photons (Supplementary Information Section Rp = 3.47 × 106 pairs per second as generated at the source (corresponding to an average photon pair number np = Rp/Rr = 0.0458 generated per pump laser pulse) , yielding an overall heralded fluorescence channel efficiency ehf = Rhf/Rh = 1.244 × 10−4 that encapsulates all experimental inefficiencies from the photon pair source to Detector 2, including the internal electronic dynamics within the LH2 complexes. For all these measured count rates, the contributions from dark counts of the detectors were very small and can be safely ignored (Supplementary Table 19) is much shorter than the laser repetition period 1/Rr = 13.2 ns, we then identified all corresponding pairs of herald and heralded fluorescent photons together with their relative time delay τ  = I(0) × exp(−τ/τ0) after reconvolution with the instrument response function determined by the crosscorrelation between heralds and heralded incident residue . Contributions to this fluorescence resulting from other states, such as long-lived triplets or charge transfer states, are excluded by quantum yield and timescale analysis, as has been discussed in the context of single-molecule studies of LH2 fluorescence22 (t = 0), conditioned on herald detection using a standard three-detector measurement24 (t = 0) value, with ‘t = 0’ defined by the gate window, can then be obtained as24g(2)(t = 0) = (NH × NC)/(N2 × N3), where NH is the herald count, NC is the coincidence count between Detector 2 and Detector 3, and N2 (N3) is the count at Detector 2 (Detector 3). The measured conditional g(2)(t = 0) values of both heralded incident and heralded fluorescent photons are found to be much lower than the single-photon threshold of 0.5 (refs. 25), which unambiguously confirms both their single-photon nature and the very small probability of having two photons in either channel (t = 0) value, namely g(2)(t = 0) = 2np/(1 + np), following a simple model  M, , with np = Rp/Rr  being the average number of photon pairs produced by each pump laser pulse, resulting from the fact that the generated photon pair can be described by a squeezed vacuum state26. A detailed analysis of the relationship of ehf to np is given in Supplementary Information Section We also studied the dependence of the probability distribution on the probability weight of multi-photon components in the heralded incident single photons by lowering the rate of incident photons by up to a factor of four , of the distribution at the number, N, of heralds per each heralded fluorescent photon can then be shown to be  for N ≥ 1, as expected from a single-photon fluorescence subsequent to a single-photon absorption. A second key feature is that for very small heralded fluorescence efficiencies ehf << 1, the geometric distribution in equation (P(N) ∝ exp(−Nehf), which agrees well with fits of the experimental distributions , is not exactly equal to zero due to the small finite probability of two fluorescent photons conditioned on the same herald, included in our counting scheme (t = 0) value of the heralded fluorescence in Fig. P(N) from equation (ehf. The plots show excellent agreement between the experiments and the analytical stochastic model, which further supports our experimental inference of single-photon transitions in absorption and a single-photon absorption and emission cycle.The most important feature of equation  is that equation  reduces eme Fig. . These aPN =  for N ≥ In addition to the analytical model, we also carried out numerical Monte Carlo calculations to simulate the experimental results with individual stochastic trajectories using the experimental parameters (Supplementary Information Section Rhodobacter sphaeroides under ambient conditions that photosynthesis is initiated by absorption of a single photon and then proceeds on the basis of the associated single quantum of energy. Our results are particularly relevant for in vivo conditions because natural sunlight has a very low flux, about 60 photons per nm2 per second for a 20-nm bandwidth at 808 nm at the full power of the sun1, similar to the single-photon flux used in our experiments.We emphasize that this PCQLS method is distinct from the conventional time-correlated single photon counting measurements, which use classical laser pulses to excite the sample and thus, cannot provide information on the correlations between individual incident and fluorescent single photons. By allowing us to time, count and correlate individual incident photons and individual fluorescent photons, PCQLS has made it possible to directly show on an ensemble of LH2 pigment–protein complexes from purple bacteria 27 could in principle allow greater spatial resolution of the pathways followed by individual excitations. We anticipate further advances from exploiting the new spectroscopic variables available from quantum light-based studies29. For example, entanglement in the photon pair could be exploited to improve spectral and temporal resolution and to break the classical Fourier transform limit31. Comparing photosynthetic light harvesting driven with laser, single-photon32 and thermal light33, respectively, could clarify the differences between in vitro and in vivo studies. Measuring the photon statistics of frequency-resolved fluorescence could provide new insights into the role of quantum coherence in excitonic energy transfer35. In short, we anticipate that similar to the way in which single-molecule experiments opened new avenues of investigation, PCQLS will prove a valuable and unique tool for exploring the spatio–temporal dynamics of individual excitations in a broad range of complex biological, chemical and physical systems and thereby gaining insight into their behaviour.The PCQLS approach should enable new experiments capable of tracking single-excitation pathways in photosynthesis and other photo-driven processes in complex systems. Extending PCQLS to a pump–probe setup and applying it to photosynthetic complexes could reveal the excitonic dynamics after initial absorption, with femtosecond-timing resolution and/or spatial–temporal information on the interplay between initial light absorption and subsequent energy transfer. Spectrally resolving the fluorescent photons from complexes with clearly defined energy funnelsAny methods, additional references, Nature Portfolio reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at 10.1038/s41586-023-06121-5.Supplementary InformationSupplementary Code 1The script HFProbDist_Rept.m calls the function HFProbDist.m multiple times and writes data into files.Supplementary Code 2The function HFProbDist.m uses a numerical Monte Carlo model to simulate the probability distributions of the number of heralds per heralded fluorescent photon.


The quantum beating between two exciton fine-structure splitting (FSS) levels enables coherent ultrafast optical control of the excitonic degree of freedom. From the anomalous temperature dependence, we identify and fully parametrize all the regimes of exciton spin depolarization, finding that approaching room temperature, it is dominated by a motional narrowing process governed by the exciton multilevel coherence. Importantly, our results present an unambiguous full physical picture of the complex interplay of the underlying spin decoherence mechanisms. These intrinsic exciton FSS states in perovskite NCs present fresh opportunities for spin-based photonic quantum technologies.Coherent optical manipulation of exciton states provides a fascinating approach for quantum gating and ultrafast switching. However, their coherence time for incumbent semiconductors is highly susceptible to thermal decoherence and inhomogeneous broadening effects. Here, we uncover zero-field exciton quantum beating and anomalous temperature dependence of the exciton spin lifetimes in CsPbBr 3 nanocrystals.Lead halide perovskites host bright triplet excitons which have applications in optospintronic devices. Here the authors observe quantum coherence between exciton sublevels without magnetic field and clarify the mechanisms of exciton spin relaxation in ensembles of CsPbBr For instance, in single quantum dots (QDs), the orthogonal photoluminescence (PL) doublets originating from the superposition of spin up and down states, can serve as a basis set for quantum computing and optical gating5. Such coherent optical control can be achieved by leveraging the exciton fine-structure splitting (FSS) in confinement-enhanced semiconductor QDs7, valley excitons in transition metal dichalcogenides (TMDs)8, and the exciton complexes in low dimensional structures11. Nevertheless, strong decoherence is usually triggered by increasing temperature or intense excitation because of the enhanced exciton-phonon and/or exciton-exciton Coulomb interaction14. Furthermore, the small energy splitting between two interacting FSS levels is highly prone to thermal energy and inhomogeneous broadening effects. This typically limits the observation of such exciton coherence to extremely low temperatures and at the single-particle (nanostructure) level. Therefore, for practical optospintronic applications, it is imperative to search for new semiconductor systems that could transcend these limitations.Semiconducting nanostructures are promising platforms for logic operations and optical modulation with controllable carrier and exciton states15 and rich optical spin physics23. Interestingly, recent single-particle PL measurements reveal the FSS of exciton states in CsPbX3 NCs because of the strong spin-orbit coupling (SOC) and shape anisotropy in the absence of strong quantum confinement28. In CsPbBr3 NCs, the Rashba effect flips the dark singlet and bright triplet states yielding fast radiative recombination of excitons25, upon which coherent single-photon emission has also been evidenced in single CsPbBr3 NCs29. The existence of exciton FSS in the family of facile processable, defect tolerant, bandgap-tunable colloidal perovskite NCs, provides a highly promising system for coherent optical manipulation of the excitonic degree of freedom. However, these non-resonant steady-state PL studies do not reveal the coherence dynamics. There is also a lack of time-resolved PL studies on exciton coherence in perovskites probably because of the fast exciton recombination and resolution limits of the technique. Presently, the spin decoherence mechanisms are under intense debate. Strong SOC induced spin relaxation has been empirically suggested to originate from the Elliott-Yafet (EY) mechanism31 in CH3NH3PbI3 thin films16 and CsPbI3 NCs32; while it is the D’yakonov-Perel’ (DP) mechanism33 for CsPbBr3 thin films34 and layered perovskites35; and the polaron mediated Maialle-Silva-Sham (MSS) mechanism36 in CsPbBr3 quantum wells37. These mechanisms could possibly be modified by the FSS due to the exchange interaction.Lead halide perovskites (LHPs) and their nanostructures are intriguing candidates for functional devices exhibiting outstanding optoelectronic propertiesHerein, we explicate the coherent dynamics of the exciton FSS states in colloidal perovskite NC ensembles using circularly polarized transient absorption (CTA) spectroscopy. Upon optical orientation, the exciton multilevel coherence remains preserved and manifests as an oscillatory spin dynamics signature at low temperatures, which originates from the quantum beating between two FSS states. Very interestingly, these coherent dynamics are relatively unaffected by the inhomogeneous broadening caused by the NC size distribution. While at high temperatures with strong interactions with phonons, we find EY process to dominate the spin decoherence, at lower temperatures a motional narrowing process controls the spin dynamics. Such motional narrowing is observed to evolve through two regimes (strong and weak scattering) producing an anomalous temperature dependence of exciton spin lifetime, which we clearly observe experimentally and parametrise through Monte Carlo simulations. Our results provide a comprehensive understanding of the exciton physics in perovskite NCs and suggest possible optical control of the exciton states that could be employed for quantum information science.J-states, consisting of an upper Je = 3/2 and lower Je = 1/2 for the electron states in the conduction band while leaving the valance band almost unaffected (Jh = 1/2)38. Importantly, the total angular momentum J, as a consequence of SOC, is a mixture of up and down spins rather than pure spin states16. In the excitonic description, the total angular momentum J = Je + Jh of the exciton remains conserved while being threefold degenerate due to interaction with the center of mass (CoM) momentum of the exciton25. Taking asymmetry into account, the band edge J = 1 triplet exciton state then splits into sublevels with three orthogonal linear dipoles Πx, Πy, and Πz, where Πz is assumed to be optically inactive, being along the observing z-axis25 as shown in Fig. 3 NCs exhibiting an orthorhombic phase below room temperature (RT). As shown in Fig. 39. As a complementary measurement to the time-resolved Faraday/Kerr rotation, the time evolution of the net exciton spin polarization Sz, can be obtained by subtracting the kinetics obtained from two counter-circular probes following photoexcitation with the same circularly polarized pump  populates the lowest J = 1 state with excitons possessing angular momentum of | +1〉 (|−1〉). Upon a σ+ (σ−) probe, the blocked | +1〉 (|−1〉) transition due to phase-space filling and screening effects results in bleaching at the exciton resonance (X). In addition, the counter-circular configuration coherently generates | +1〉|−1〉 biexcitons, which are expected to be absent within the co-circular configuration because of the selection rule40. However, biexciton induced absorption is observed below bandgap with a red-shifted energy of ~2.39 eV for both circular probes as shown in Fig. 3 NCs with the optical orientation of the excitons. The Πx and Πy linear dipoles, because of the superposition of the spin up and down excitons, are simultaneously excited by the phase-sensitive circular pump. Consequently, the cross-relaxation process is modulated periodically by the coherent superposition of Πx and Πy states, i.e., quantum beating between the two states. Figure σ+ and σ− probe following σ+ pump, and picosecond optical oscillations are clearly resolved for the NC ensemble. Taking the exciton FSS into consideration -handed circularly polarized pump 28 and arsenide-based QDs (tens of μeV)42 measured by single-particle PL. In addition, the energy gap dependence of the splitting energy is also consistent with the literature reports43, where enhanced splitting appears in the smaller NCs as shown by shorter oscillation periods in Fig. x and Πy dipoles because of the random NC orientations. Hence the beating between the two coupled levels can be resolved. A single-particle level experiment may clarify the dipole alignments, which would allow the enhancing and erasing of the quantum beating according to the polarization distribution. Moreover, the independence of oscillating frequency on the excitation density  and biexciton (XX) resonances, and at the above bandgap photoinduced absorption (PIA) peak as seen from the mes Fig. . We noteity Fig.  indicateτs, i.e., the envelope of the oscillatory dynamics. To understand the exciton spin relaxation process, we examine the temperature dependences of Sz in these CsPbBr3 NCs. Figure τs can be obtained, as summarized in Fig. 3 polycrystalline thin film (PTF) samples are also inspected  the spin undergoes full rotating cycles between scattering producing damped spin oscillation. This limit is usually found at low temperatures. On the other hand, in the strong scattering limit i.e., the spin cannot rotate full cycle between scattering events) the motional narrowing process leads to an exponential time decay of the spin, yet with the spin-relaxation time τs having an inverse relationship45 with τp thatτp and τs.The motional narrowing process is an alternative spin dissipation mechanism, where spin can coherently precess along an axis with its direction dependent on the quasiparticle’s crystal momentum. The phenomenon of motional narrowing refers to the linewidth decrease of a resonant frequency due to the motion in an inhomogeneous system. Motional narrowing of spins has been demonstrated in nuclear magnetic resonance (NMR) and spin relaxation in III-V semiconductors. As a result, the motion narrows down the dephasing linewidth by interrupting the spin precession. There are two distinct characteristics of this precessional process. In the quasi-collision-free limit , which simultaneously account for both spin relaxation channels. At the highest temperatures the spin relaxation for all NC sizes is dominated by the EY process. Interestingly all the NC sizes display the same EY lifetimes . In fact, we obtain the best fit assuming size-independent EY lifetimes with a 27.The spin precession is assumed to be around in-plane momentum-dependent vectors of effective magnetic fields originating from the FSS energy splitting, which is changed in direction by the spin-preserving, momentum-randomizing scatterings. We assume a momentum-independent strength of the effective magnetic field with a constant spin precession frequency Ω. Yet the momentum scattering rate ion Fig. , verifyi3 NCs can be divided into three regimes with different dominant mechanisms: 1) the EY mechanism where the scattering with LO phonons depolarizes spins; 2) the strong-scattered motional narrowing mechanism where the momentum scattering preserves spin polarization; and 3) the quasi-scattering-free motional narrowing of spins which yields oscillatory spin dynamics. These as-described exciton spin relaxation processes are schematically shown in the insets of Fig. 50, and the latter for the exchange-interaction-induced exciton spin relaxation51 in 2D structures like monolayer MoSe212 and quantum wells36. In perovskite NCs, an effective magnetic field originating from the FSS can be naturally established. Such intrinsic field must be momentum-dependent and in-plane as mentioned previously, which can be understood in terms of the Zeeman effect where the field strength can be deduced from the splitting energy. Consequently, CoM motion in momentum space randomizes the spin precession, which determines the relaxation process. Specifically, at rather high temperatures, the enhanced scattering yields varying directions of Ω and hence is expected to prolong the spin lifetime, which corresponds to the strong scattering regime in the motional narrowing process. However, in a quasi-collision-free regime (below ~100 K), the excitons are “localized” in momentum space and the spin dynamics are oscillatory because of the quantum beating between two FSS levels with splitting energy of Δ, at a frequency of Ω = Δ/ℏ. Such coherence between the FSS levels provides an upper limit for the spin dephasing rate.From high to low temperature, the exciton spin relaxation in CsPbBr3 nanocrystals, which will evoke further theoretical and experimental investigations of the exciton physics in perovskite nanostructures. In addition, we demonstrate that ultrafast spectroscopy allows estimating the exciton fine-structure splitting in ensembles of colloidal perovskite nanocrystals at zero magnetic field, even though that the exciton multi-levels are not spectrally resolved. However, such measurements can only provide average splitting because of the random dipole distribution and nanocrystal size distribution. Furthermore, the quantum beats may be smoothed with increasing temperature due to the motional narrowing process. On the other hand, single-particle PL measurements allow direct observation of the FSS levels with high energy resolution. Importantly, these FSS states are promising for quantum information science. For example, the two eigenstates Πx and Πy, and the biexciton state can be engineered as a set of basis for logic devices5. The coexistence of the EY process and FSS-induced motional narrowing of exciton spin paves the way for spin engineering in perovskite nanostructures.In conclusion, our results provide a thorough understanding of the exciton coherence and spin relaxation in colloidal CsPbBr3 NC samples are approximately 5 nm (NC5), 6 nm (NC6), 8 nm (NC8) and 12 nm (NC12) in size. NC5 was synthesized by hot-injection according to a reported method with slight modifications52. Briefly, a Cs-Oleate stock solution was prepared by loading 0.815 g of Cs2CO3, 2.5 mL of oleic acid and 40 mL of octadecene into a round bottom flask and degassed under vacuum at 100 °C for 30 min. Thereafter, the solution was kept under nitrogen flow and the temperature was raised and kept at 120 °C prior to injection. In a separate round bottom flask, 75 mg of PbBr2, 183.8 mg of ZnBr2 along with 2 mL of oleic acid, 2 mL of oleylamine and 5 mL of octadecene were loaded and degassed under vacuum at 100 °C for 30 min. Then, the temperature was raised and kept at 170 °C until all salts dissolved. Subsequently, 0.5 mL of the prepared Cs-Oleate stock solution was swiftly injected and the reaction was quenched with an ice bath after 15 s. To separate the NCs, ethyl acetate was added to the crude solution in a volume ratio of 2:1 and the mixture became turbid. Then, the mixture was centrifuged at a Relative Centrifugal Force (RCF) of 11,627 × g for 10 min. The supernatant was discarded, and the precipitate was redispersed in anhydrous toluene. Subsequently, the redispersed NCs were centrifuged at a RCF of 1860 × g for 10 min and the precipitate was discarded. The supernatant containing NC5 was kept in the refrigerator for further use. Centrifuge rotor radius is 10.4 cm (Thermofisher FiberliteTM F15-8x50cy).Our CsPbBr53. Briefly, a 0.1 M Cs-Octanoate stock solution was prepared by dissolving Cs2CO3 in octanoic acid. Separately, a PbBr2 stock solution was prepared by dissolving 225 mg of PbBr2, 2.9 g of trioctylphosphine oxide along with 0.2 M of octylphosphonic acid in 5 mL of anhydrous toluene. A 0.05 M solution of dimethyldidodecylammonium bromide (DDAB) was prepared by dissolving DDAB in anhydrous toluene. To prepare the NCs, 0.5 mL of the PbBr2 stock solution was loaded into a vial under vigorous stirring. 55 μL of Cs-Octanoate stock solution was then swiftly injected. After the desired growth period , 155 μL of DDAB solution was injected and the solution was left to stir for further 5 min. Thereafter, multiple centrifugations and redispersion steps were performed according to the published method. The resulting NCs were kept in the refrigerator for further use.The synthesis of NC6, NC8, and NC12 was performed according to a previously reported method under room temperature ambient conditions2 with a stoichiometric molar ratio of 1:1 are dissolved in dimethyl sulfoxide (DMSO) with a concentration of 0.2 M to form a precursor, which is then used for film deposition. Specifically, 20 μL of the precursor is dropped onto a 10 mm × 10 mm glass substrate for spin-coating with a speed of 4,000 r.p.m. followed by an annealing process at 100 °C for 10 min. All procedures are done in a nitrogen-filled glove box, and the as-deposited thin-film is of ~40 nm thick. For the NC solids, pre-synthesized colloidal CsPbBr3 NCs are directly used for spin-coating with a speed of 500–800 r.p.m., and the resultant thickness is tens of nanometers.For the polycrystalline thin-film, CsBr and PbBrTM transient absorption spectrometer from Ultrafast Systems LLC powered by an ~800 nm Libra™ Ti: Sapphire laser  from Coherent Inc. Briefly, the output from the laser system is split into two beams with one directed into an optical parametric amplifier (Coherent OPeRa SOLOTM) for energy-tunable pump and the other delayed one coupled onto a sapphire crystal for white light probe. The circularly polarized light was generated by achromatic quarter wave-plates and linear polarizers.The measurements were conducted using a commercial Helios54. Each exciton is represented by a 3-dimensional spin vector, which precesses about an effective in-plane magnetic field arising from the exciton-fine structure splitting, with a precession frequency Ω and a direction which lays on the sample’s plane and depends on the center-of-mass momentum of the exciton. Apart from precession, the excitons can undergo two processes: a momentum randomizing motional narrowing scattering with a rate of τEY.We simulate the net relaxation of the exciton spin polarization as the result of two competing scattering events which result in (i) motional narrowing and (ii) spin randomization, primarily attributable to the Elliott-Yafet (EY) mechanismτEY are fitted to NC12 and then used for all samples and extrapolated at lower temperatures. Further details on the Monte Carlo simulations are collected in Supplementary Note The spin dynamics are calculated using Monte Carlo over an ensemble of 2,000 excitons, and the parameters are extracted by best fitting with experimental results, performed using Powell minimization. While Ω and Supplementary InformationPeer Review File


Dietary patterns (DPs) during pregnancy have been well researched. However, little is known about maternal diet after pregnancy. The aim of the study was to explore maternal DPs longitudinally, examine trajectories over 12 years after pregnancy and identify associated factors.Of 14,541 pregnant women enrolled in the Avon Longitudinal Study of Parents and Children (ALSPAC) complete dietary information was available for 5336 women. Principal components analysis (PCA) was used to derive DPs. DP scores at each time point were used to create DP trajectories using group-based trajectory modelling (GBTM). Multinomial logistic regression assessed the association with maternal factors.A total of six distinct DPs were identified over time with different numbers of DPs at each time point. The “healthy” and “processed” DPs persisted over the 12-year post-pregnancy. Three trajectories of “healthy” and “processed” DPs were identified from GBTM. Half the women were on the moderately healthy DP trajectory with 37% on the lower trajectory and 9% on the higher healthy DP trajectory. 59% of women were on the lower processed DP trajectory with 38% on the moderate trajectory and 3.3% on the higher processed DP trajectory. Low educational attainment, low social class and smoking in pregnancy were independently associated with being on a less favourable DP trajectory over the 12 years.Health professionals should provide support on smoking cessation along with healthy eating advice during ante-natal counselling. Continued support on eating healthily after pregnancy would be beneficial for mothers and families.The online version contains supplementary material available at 10.1007/s00394-023-03185-x. Diet is an important modifiable risk behaviour that has significant links with a range of non-communicable diseases (NCDs) and related health conditions, including obesity ; type 2 Much research has focussed on diet during pregnancy and its effects on offspring’s health –13, howeChanges in dietary habits among women from before to during pregnancy have been systematically examined . SuggestIn the review of Lee et al., there were two studies that had analysed DPs longitudinally from pregnancy to 5 years post-pregnancy , 21 and In the general population, some studies have investigated DPs longitudinally examining their stability over time , 26, andAvon Longitudinal Study of Parents and Children (ALSPAC) is a population-based, prospective, longitudinal British cohort of pregnant women, their partners and offspring established in the 1990s to investigate the development of health and potential diseases from the time of pregnancy in the women to the adulthood of offspring . PregnanFood frequency questionnaires (FFQ) were used to collect dietary data from the women at 32 weeks of pregnancy and then at 4, 8 and 12 years post-pregnancy . Full deThe data on frequency of consumption were numerically transformed into times consumed per week, to give a quantitative meaning to the frequency categories, as follows: (i) 0; (ii) 0.5; (iii) 2; (iv) 5.5 (v) 10 times per week. Daily foods were converted to times per week and recoded accordingly to keep similar quantitative values. All variables used in the analysis were standardised by subtracting the mean and dividing by the standard deviation for each variable . The queMaternal dietary patterns at 32 weeks of gestation and at 4 years of child’s age have been previously identified in this cohort using principal component analysis (PCA) with varimax rotation. Detail of the identified dietary patterns has been described elsewhere , 42. In Covariates were selected based on previous relevant studies; they were obtained from self-completed postal questionnaires sent during pregnancy –44. Thes2). Pre-pregnancy BMI was categorised according to the World Health Organization as “underweight (< 18.5 kg/m2)”, “normal (18.5 and < 25 kg/m2)”, “overweight (> 25.0 and < 30 kg/m2),” and “obesity (≥ 30 kg/m2)” [Mothers were asked to report alcohol consumption before their current pregnancy by questionnaire at 18 weeks of gestation as: never, < 1 glass/week, at least 1 glass/week, 1–2 glasses/day, at least 3–9 glasses/day and at least 10 glasses/day. The total glasses/week or day were grouped  . Women w kg/m2)” .Using the DP scores constructed at each time point, group-based trajectory modelling (GBTM) was applied to evaluate trajectories of DPs . This meA censored normal distribution appropriate to continuous data was used to generate representative trajectory curves that best fit the distinct trajectory groups for the overall study population . It cannThe distribution of maternal characteristics (collected during pregnancy) were presented as frequencies and percentages and the distribution of categorical variables were compared using chi-squared tests. Multinomial logistic regression was used to examine the relationship between these mutually adjusted characteristics and membership of the dietary trajectories. The likelihood of a woman following a specific DP trajectory in relation to maternal characteristics, was expressed using relative risk ratios (RRR). The characteristics investigated were maternal age, education, employment status, marital status, social class, ethnicity, parity, pre-pregnancy BMI, smoking and alcohol behaviours. All statistical analyses were carried out using Stata/SE .A total of 12,191 women had dietary data at 32 weeks of gestation, 9598 women at 4 years, 7822 women at 8 years and 6932 women at 12 years post-pregnancy. Complete data at all four time points was available for 5336 women. Table Three dietary patterns were chosen to best describe the DPs of mothers at 8 and 12 years post-pregnancy compared to 5 and 4 DPs extracted in pregnancy and 4 years post-pregnancy, respectively , 42. MorThe correlations between the DP scores at each time point are summarized in Table n = 1971, 37.4%) had an initially negative DP score with a stable progression which remained negative across time ; women in Group 2  had a moderate initial DP score with a slow decrease thereafter ; and, women in Group 3  had the highest DP score and increased over the time . Similarly, women were allocated to one of three trajectory groups for the processed DP: women in Group 1  had a negative DP score initially, with a slight increase thereafter, but it remained negative across the time points (named as “lower processed DP trajectory”); women in Group 2  had an initial moderate score with a slight increase and stable thereafter (named “moderately processed DP trajectory”); however, women in Group 3  had the highest processed DP score initially and rapidly increased over the time (labelled as “higher processed DP trajectory”). The distribution of maternal characteristics in women following the “healthy” and “processed” DP trajectories are presented in the Supplementary file Tables S4 and S5.Figure The associations between maternal baseline characteristics and DP trajectories are presented in Tables This study models group-based trajectories of longitudinal dietary patterns among British women from pregnancy across multiple timepoints post-pregnancy. Women’s dietary intakes were collected prospectively in ALSPAC four times from pregnancy to 12 years post-pregnancy. Six different dietary patterns were derived using PCA analysis over the four time points. A “healthy” and a “processed” DP were persistent at each time and used to explore the trajectories of the women’s diets across time. Three trajectories were identified for each of the two patterns.For the “healthy” DP trajectories, the majority (53.4%) of women followed the “moderately healthy DP trajectory”. This implies that this group of women have a moderate score on the healthy DP at each time point. A further 37.4% of women followed a “lower healthy DP trajectory” where they had a lower score on the healthy DP at each time point. The remaining women (9.2%) were on a “higher healthy trajectory”, representing higher scores on the healthy DP across time. This is the smallest group and we can speculate that these women should have a better health profile than the group who had followed the “lower healthy DP trajectory” throughout the period of interest. Further research will be necessary to confirm this. For the “processed” trajectory, most women followed the “lower processed DP trajectory” (58.6%)”. These women had a lower score on the processed DP throughout. The “moderately processed DP trajectory” (38.1%) was the second largest trajectory group with women having moderate scores on the “processed” DP at all time points. While the “higher processed DP trajectory” was followed by only 3.3% of the women, this trajectory implies the consumption of a highly processed diet at each time point. Further research is necessary to understand the health effects on women who were following the “higher processed DP trajectory” over time.Of the remaining DPs in pregnancy “confectionary” and “vegetarian” were not derived after 4 years post-pregnancy and the “traditional” DP only occurred in pregnancy. A new DP emerged at 8 years post-pregnancy and continued at 12 years it was named “high meat”. These findings suggest that some DPs emerged in this cohort were not necessarily stable over time. A shift toward a more meat-based diet among women supports the global view of nutritional transition towards animal-based diets which may be linked to increased risk of nutrition related NCDs –56. FurtOur study found that women were more likely to have consistently low or moderate scores on a DP across time than consistently high scores on that pattern. Only a small number of women who started with a high “healthy” DP score, remained high over time, thus having a “healthy” dietary pattern throughout. This was also true for the “processed” DP trajectories. Women with medium or low educational attainment were more likely to follow the “lower healthy” and “higher processed” DP trajectories in this cohort. This level of stability in dietary trajectories and association with the educational attainment of women, was similar to another British study the Southampton Women’s Survey (SWS) that examined longitudinal changes in dietary quality trajectories in women from pre-pregnancy to 8–9 years post-pregnancy . The staA limited number of studies have investigated changes in DPs among adult populations using group-based trajectory modelling over long time periods and diverse dietary trajectories have emerged , 57–59. Compared to women following the “higher healthy DP trajectory”, maternal characteristics in pregnancy did not differ in those following the “moderately healthy DP trajectory”. However, women following the “lower healthy DP trajectory” were more likely to have smoked heavily in pregnancy. A similar difference occurred between women following the “higher processed DP trajectory” compared those following the “lower processed DP trajectory”. These results suggest that smoking in pregnancy was associated with having a less favourable DP at all time points. These findings are consistent with previous studies showing an association between smoking and unhealthy dietary intake among women , 61–64. There were social class differences between women following the different DP trajectories. Women who belonged to the lower social class had a higher chance of following the “lower healthy DP trajectory” compared to following either the higher or moderately healthy DP trajectories. Furthermore, women with a low or middle social class background were more likely to follow the “moderately” or “higher processed DP trajectory” rather than following the “lower processed DP trajectory”. People with lower social status are likely to have less disposable income and evidence suggests that lower income groups tend to fall into lower quintiles for food expenditure . AnotherThe major strength of this study is its prospective cohort design, over 12 years of follow-up (from pregnancy to post-pregnancy) and complete dietary information from more than 5000 women. The ALSPAC cohort provided a unique opportunity to explore longitudinal dietary patterns and dietary trajectories because of its large sample size and long follow-up. The use of dietary patterns at 4 time points from pregnancy allowed the assessment of dietary stability over time and the development of dietary trajectories among British women. Additionally, GBMT modelling proved to be an important technique when classifying the dietary patterns into interpretable trajectories. A wide range of potential confounders was available to the study.Nevertheless, this study has some limitations that need to be recognised. Firstly, data on dietary intake was self-reported and hence may be susceptible to measurement bias and inaccuracies. However, the use of FFQs is well-accepted for dietary assessment in observational nutritional science . SecondlFollowing pregnancy, continuing with a healthy and optimal diet during post-pregnancy is important to support maternal health and wellbeing and for the child to learn healthy eating practices. In our study, maternal DPs were found to have some variability from pregnancy to later time points, with some evidence of a move towards higher consumption of a meat-based diet. However, within the persistent “healthy” and “processed” DPs, dietary trajectories were relatively stable over the 12 years post-pregnancy. Our findings add to the evidence for strengthening and improving maternal care during pregnancy and for continued counselling to mothers regarding healthy eating and smoking cessation while bringing up their children.Our findings suggest that it is advisable that health professionals target smoking cessation at the same time as promoting a healthy diet in antenatal care. If resources allowed healthy eating and ante-smoking education that started well before pregnancy may prove even more beneficial to family health. Also, follow up support for healthy eating after the birth of the child which is likely to benefit the mother and her family. To increase effectiveness, introducing a triage system to prioritise socially and economically disadvantaged women with risk behaviours would be beneficial for promoting life course healthy eating among women.Supplementary file1 (DOCX 64 KB)Below is the link to the electronic supplementary material.


However, the exercise-induced changes in K+ intra-to-extracellular gradient is by itself insufficiently large to be a major cause for the force decrease during fatigue unless combined to other ion gradient changes such as for Na+. Whilst several studies described K+-induced force depression at high extracellular [K+] ([K+]e), others reported that small increases in [K+]e induced potentiation during submaximal activation frequencies, a finding that has mostly been ignored. There is evidence for decreased Cl− ClC-1 channel activity at muscle activity onset, which may limit K+-induced force depression, and large increases in ClC-1 channel activity during metabolic stress that may enhance K+ induced force depression. The ATP-sensitive K+ channel (KATP channel) is also activated during metabolic stress to lower sarcolemmal excitability. Taking into account all these findings, we propose a revised concept in which K+ has two physiological roles: (1) K+-induced potentiation and (2) K+-induced force depression. During low-moderate intensity muscle contractions, the K+-induced force depression associated with increased [K+]e is prevented by concomitant decreased ClC-1 channel activity, allowing K+-induced potentiation of sub-maximal tetanic contractions to dominate, thereby optimizing muscle performance. When ATP demand exceeds supply, creating metabolic stress, both KATP and ClC-1 channels are activated. KATP channels contribute to force reductions by lowering sarcolemmal generation of action potentials, whilst ClC-1 channel enhances the force-depressing effects of K+, thereby triggering fatigue. The ultimate function of these changes is to preserve the remaining ATP to prevent damaging ATP depletion.Perturbations in K In brief, the K+ loss from muscle fibers occurs over several APs due to the K+ efflux associated with the AP repolarization phase, resulting in an increased extracellular [K+] ([K+]e) and lowered intracellular [K+] ([K+]i). As the [K+] gradient across the sarcolemma decreases, resting EM depolarizes (for details see next section entitled “+ channels (NaV channels) become inactivated, reducing AP amplitude and slowing AP propagation along the sarcolemma and into t-tubules. For this review, any physiological conditions leading to either a reduction in AP amplitude or a complete loss in the capacity of the sarcolemma to generate an AP compared to AP measured in unfatigued and normal physiological conditions is considered a decrease in membrane excitability. As membrane excitability is reduced due to the K+-induced depolarization, less Ca2+ is released by sarcoplasmic reticulum (SR) resulting in reduced muscular force or work, i.e. fatigue.Muscle fatigue is defined as a transient decrease in the capacity of skeletal muscles to generate force or do work when repetitively activated. Fenn and colleagues in the 1930’s documented Kcle Fenn . A very a et al. . Althougrce Fenn . Later o+ shifts are still considered an important factor in the mechanism of muscle fatigue. However, studies have demonstrated that: (1) by itself, the lower intra-to-extracellular K+ gradient, and hence depolarization, may in many cases not be sufficient to explain the contraction-induced decrease in membrane excitability and force; (2) moderate increases in [K+]e actually potentiates sub-tetanic force; and (3) changes in Cl− conductance (GCl) may be crucial in determining when K+ can either potentiate or depress force. A first objective is to review how perturbations in K+, Na+ and Cl− gradients across the muscle membrane during muscle activity affect membrane excitability and force and to discuss whether the changes in gradients are sufficient to contribute to the decrease in membrane excitability and force as originally conceptualized. A second objective is to discuss the long-ignored phenomenon of K+-induced force potentiation and consider its physiological role at the onset of moderate exercise. A third objective is to discuss the link between metabolism and membrane excitability. The fourth and final objective is to propose an evolved concept integrating the dual roles of K+  during muscle activity and fatigue and how they are modulated or controlled by Na+, Cl− ClC-1 channels, KATP channels and Na+,K+-ATPase .Over the years, this concept gained tremendous support and KM is a diffusion potential that is created when ions cross the sarcolemma through their channels. Thus, the sarcolemma must be permeable to the ions, which must have a concentration gradient across the sarcolemma to contribute to EM. In resting skeletal muscle, [K+]i, measured in different species and muscles, is approximately 160 mM (ranging between 90 and 190 mM), whilst the interstitial [K+] ([K+]int), measured mostly in human muscles, is typically just over 4 mM  or in Na+ free saline solutions  and resting EM are the same  was measured under several conditions involving changes in [Cl−]e and [K+]e either with or without a constant [K+]e [Cl−]e product; in all cases the changes in [Cl−]i were as predicted for passive Cl− distribution across the sarcolemma  its electrogenic nature and (ii) the maintenance of the [Na+] and [K+] gradients.Overall, resting EM reaches a threshold, defined as the EM at which NaV channels start to open resulting in a subsequent increase in Na+ conductance (GNa) allowing large Na+ influx that rapidly depolarizes the sarcolemma from −70 mV to + 30 mV. The repolarization back to −70 mV depends on (i) NaV channel fast inactivation to stop the depolarization and (ii) the activation of voltage-sensitive K+ channels (KV) that increases GK and K+ efflux, allowing EM to return to its original resting level  in amphibian muscles and 5 to 9-times greater than GK in mammalian muscles , a ClC-1 channel blocker, result in more negative AP threshold; this suggests that the Cl− influx provide an outward current counteracting any depolarizing stimulus to trigger an AP, at least in t-tubules .ClC-1 channels allow for Cl+, K+ and Cl− fluxes during APs are changes in both intra- and extracellular Na+, K+ and Cl− concentrations, which then affect resting EM, membrane excitability and consequently force. To fully understand these processes, we must first look at the magnitude of these ionic changes during muscle activity and fatigue.A major consequence of the Na+]int are 4–7 mM greater than increases in venous plasma [K+]  from Table +]int, from human studies, and [K+]i, from mostly animal studies, are in resting muscles respectively 4.3 ± 0.1 and 134.1 ± 6.8 mM, giving rise to a Nernst potential for K+ (EK) of -92 mV. After fatigue, the values are respectively 10.1 ± 0.9 mM, 108.1 ± 5.9 mM and -63 mV. This implies that mean EK decreases by 29 mV; mostly from the increase in [K+]int  compared to the decrease in [K+]i . More importantly, the 29 mV decrease in EK is almost twice as large as the 16 mV resting EM depolarization. This is further supported by one study in which resting EM, [K+]int and [K+]i were concomitantly measured reporting a 24 mV decrease in EK versus a 12 mV resting EM depolarization . Studies describing large resting EM depolarization and force decrease also reported decreases in AP overshoot, from as small as 2 mV to as large as 43 mV (Table +] gradient and (ii) a depolarization-induced NaV channel inactivation.The above studies for which there was no force loss and depolarization with muscle activity Table  bottom, mV Table , top. Tw+] gradient reduces the Na+ current during the depolarization phase leading to smaller AP overshoot. For most experiments presented in Table +] gradient decreased by 1.1 to 1.8-fold and for a minority of studies (4 out of 19 measures) the decrease exceeded twofold. Increasing [Na+]i  stopping the depolarization phase so that the subsequent K+ efflux can repolarize the membrane and (ii) allowing the unidirectional AP propagation. Studies on steady state fast inactivation reported that 10% of NaV channels are fast inactivated when resting EM is on average −90 mV (range between studies from −105 to −70 mV); a value increasing to 50% at −67 mV (range from −87 to −50 mV) and with all NaV channels fast inactivated at −40 mV (range from −60 to −20 mV)  and with all NaV1.4 channels being slow inactivated at −10 mV (from −20 mV to 0 mV). Other studies using skeletal muscle fibers reported more hyperpolarized steady state slow inactivation; i.e., 10% decrease at −107 mV (range from −120 to −90 mV), 50% decrease at −90 mV (range from −110 to −70 mV) and no current at −60 mV (range −80 to −50 mV)  muscles, AP amplitude decreased by 50% at resting EM of −70 and −60 mV, respectively  and the prolonged depolarization (K+-induced depolarization). Mean overshoots were quite similar between the two measurements for resting EM from −90 to −67 mV. At resting EM less negative than −67 mV, overshoot depression was greater with the K+-induced than the microelectrode-induced depolarization. Despite a stable resting EM 10–15 min after the microelectrode penetration, overshoot continued to decrease, reaching values that were even less negative than those measured at elevated [K+]e. For example, at resting EM of −50 mV, mean AP overshoot was −8 mV after 5 min of depolarization further decreasing to −40 mV for the 10–15 min period during which resting EM remained at −50 mV. The most likely mechanism responsible for the further decrease in overshoot while resting EM remained is slow inactivation that can take up to 20 min to reach steady state , defined as the [Ca2+]i during contraction, as a major mechanism by which force decreases during fatigue  despite significant resting EM depolarization and lower overshoot, until a [K+]e is reached above which force declines abruptly. For twitch and sub-maximal tetanic contractions, small increases in [K+]e actually potentiate force, while at higher [K+]e, twitch and sub-maximal tetanic force are decreased. In this section, we discuss the K+-induced force depression on tetanic force and how it is modulated by changes in [Na+] gradient, GCl and NKA activity, then followed by a subsequent section discussing K+-induced potentiation.Decreases in active [Ca+]e is raised from 4 to 9 mM and is completely abolished at 12 mM [K+]  and if the critical [K+]e (measured in mouse and rat muscles) are representative of the situation in human, mouse and rat muscles, then 12 mM [K+]int at 37 °C is expected to reduce tetanic force by 50% in fatigue resistant muscles such as the soleus, but having little effect in fatigable muscles such as the EDL. Human muscle has a mixed fiber type composition, so the expected effects would be intermediate between these two different types of muscles. Overall, these results do not strongly support the concept that perturbations in K+  per se are not a major mechanism for the decrease in force during fatigue at least in muscles with low fatigue resistance. Further studies are necessary to determine [K+]int in animal models as well as the critical [K+]e in human muscles. However, as discussed below, one cannot exclude K+ as a potential factor in muscle fatigue without looking at an interaction with Na+, Cl− and NKA.The next question is whether the changes in [Kt et al.  demonstr+] ([Na+]e) compared to control conditions  reduced peak force by 10% and 30%, respectively  and remained constant when [Na+]e was reduced from 147 to 85 mM (1.7-fold in the [Na+] gradient); concomitant changes of both gradients resulted in a 50% force reduction  reduced force by 3%,while concomitant changes in [K+]e and [Na+]e resulted in a force depression of 40%, more than threefold greater than a calculated additive effect of 12%  to 20–25% of the initial values measured at 4 mM  was then lowered from 7.4 to 6.8 by raising CO2 in the gas phase from 5 to 24%, in order to reduce GCl, as Cl− ClC-1 channels are pH-sensitive . Mimicking the reduction in GCl by lowering [Cl−]e as well as by exposing soleus to 9-AC at pHe 7.4 had the same effect on force and M-wave as the low pHe. The authors concluded that a partial decrease in GCl is the mechanism by which acidic pHe caused an increase in tetanic force and M-wave area during the K+ -induced depolarization.In one study, an increase of [K+]i in order to alter t-tubular resting EM  from 7.1 to 6.6 shifted the force-[K+]i relationship toward lower [K+]i, i.e., more depolarized t-tubules. A similar shift was not observed when i) Cl− was removed from the bathing solution and ii) when contractions were elicited via an activation of the voltage sensor . The authors concluded that greater t-tubular depolarization was necessary to induce force loss in the presence of Cl− (or GCl) in acidic than in normal pHi.The above conclusion was further supported by another study in which mechanically skinned fibers with intact t-tubules were exposed to various [K−]e was decreased stepwise from 127 to 0 mM. Despite greater depolarization, the rate at which force decreased became slower when [Cl−]e was lowered from 127 to 60 mM (to lower GCl) and then became faster from 60 to 0 mM Cl−. The authors concluded that any decrease in GCl worsens the K+-induced depolarization whereas small decrease in GCl improves membrane excitability and tetanic force while large decrease in GCl worsens the K+-induced decrease in membrane excitability and force by mechanisms explain below.In a third study  for greater K+-induced depolarization and (ii) greater GNa:GCl ratio. If small decrease in GCl improves AP amplitude and force at raised [K+]e, then one can suggest that the increase in AP amplitude due to greater GNa:GCl ratio largely overcomes the expected lower AP amplitude due to the greater K+-induced depolarization. Third, there is an optimum decrease in GCl for which the extent of the depressive effects of any depolarizations on excitability and force is at its lowest. As shown by de Paoli et al.  is 12–20 mV in EDL, soleus and diaphragm muscles  to catecholamines/agonists results in a hyperpolarization  phosphorylation of SR Ca2+ release channels, known as the ryanodine receptors (RyR1) and (ii) in some muscles, such as diaphragm and amphibian muscle but not mammalian limb muscles, a phosphorylation of the t-tubular voltage sensor/Ca2+ CaV1.1 channels  in fast twitch dominant rat EDL muscles. Similarly, the K+-induced twitch potentiation was more pronounced in EDL than in soleus muscles of guinea pigs   or by a low-frequency train, both of which fall on the steep portion of the Ca2+-tension relationship. In contrast, there is no potentiation during fully fused contractions, which lie on the plateau of the Ca2+-tension relationship.In a recent report, Overgaard et al.  re-addre2+ transients during activation, is however, still not elucidated. Possibly the small increase in resting [Ca2+]i that is observed following [K+]e elevation could be involved in the pathway  and the Cl− ClC-1 channel, being an ion pump and two ion channels that affect membrane excitability.So far, we discussed how changes in ion gradients across the muscle membrane during fatigue affects its excitability. As discussed in the section entitled “2+ ATPases and the Na+,K+-ATPases (NKA), which respectively consume approximately 50–60%, 40–50% and 5–10% of the energy during exercise  coupling are either directly (ATPases) or indirectly (ion channels) dependent on muscle energy status. In contracting skeletal muscle fibers, the three main ATP consuming processes are the myosin ATPases, the SR Ca+, Na+ and Cl− discussed above, there are numerous other factors affecting muscle excitability and none has been more agreed on than metabolically derived factors affecting NKA and ion channel  activity. It is now well established that NKA and KATP channels are primarily fueled by glycolytically-derived ATP with direct effect of muscle glycogen on muscle excitability  utilize different local pools of glycogen, clearly demonstrating compartmentalized glycogen metabolism and emphasize that spatially distinct pools of glycogen are used for separated energy requiring processes  led to reduction or increase in glycolysis, respectively, while the oxidative phosphorylation remained constant  inactivation  mouse model. The authors concluded that 1 Hz stimulation resulted in an activation of KATP channels. Exposing skeletal muscles to ischemia or chemical metabolic inhibition causes large KATP channel activity . Together, these results strongly suggests that not only are KATP channels activated during fatigue but that they are crucial at protecting muscles from deleterious exhaustion of [ATP].Electrically stimulating mouse tibialis muscle at 1 Hz resulted in sarcolemmal hyperpolarization by 10 mV, reduction in AP overshoot by 14 mV, as well as reduction in SR CaATP channels are activated during fatigue is still not well understood. Firstly, the decreases in bulk [ATP] during or at the end of a fatigue bout range between 10 and 50%  pharmacologically exposing wild type muscles to glibenclamide, a KATP channel blocker, and (ii) genetically using a knockout model in which the Kir6.2 subunit is no longer expressed; i.e., muscles from Kir6.2−/− mice. Notably, in all the studies described here, the effects of abolishing KATP channel activity were quantitatively the same for the pharmacological and genetical approaches. Furthermore, glibenclamide had no effect in Kir6.2−/− skeletal muscles  The decreases in active [Ca2+]i and tetanic force were faster and the final extent of the decrease greater. (2) The increases in unstimulated [Ca2+]i and force, measured between contractions, were greater. In some cases the increase in unstimulated [Ca2+]i was so large that unattached single fibers supercontracted from an elongated fiber to a very small square structure. (3) Resting EM of several fibers depolarized to greater extent from a mean −80 mV to −30 mV (compared to just −60 mV for control conditions). Large depolarizations during fatigue were also reported in rat EDL exposed to glibenclamide  or to 1 µM verapamil, a CaV1.1 channel blocker at a concentration that had no effect on the pre-fatigue tetanic contraction of Kir6.2−/− FDB. Both lower [Ca2+]e and verapamil significantly reduced during fatigue the rate at which tetanic force decreased and the extent of the increase in unstimulated force, while it fully restored the capacity to recover force following fatigue in KATP channel deficient muscle. Based on these results, the authors suggested that the contractile impairment in regard to tetanic force during fatigue and recovery in KATP channel deficient FDB were in part due to some cellular damages possibly caused by the high unstimulated [Ca2+]i.Experiments were then repeated using smaller muscle preparations, i.e., FDB muscle bundles and single fibers  At the end of the fatigue bout, there was a net loss of total adenylates (ATP + ADP + AMP contents). (3) Compared to wild type, Kir6.2−/− FDB had greater glucose uptake, similar glycogen mobilization and greater glucose oxidation during the first min that resulted in a 3.5-fold greater ATP production. (4) However, during the remaining 2 min of the fatigue period glycogen was no longer mobilized and oxidative phosphorylation stopped. The decrease in ATP production and the large increases in unstimulated [Ca2+]i and force between contractions increases the ATP demand by Ca2+ and myosin ATPases, which most likely were the cause for the lack of increase in ATP content in Kir6.2−/− FDB that occurred in wild type FDB during the final two min of the fatigue bout.A last series of experiments demonstrated several metabolic dysfunctions in Kir6.2ATP channels are activated during fatigue. They contribute to the reduction of AP amplitude, which then lowers SR Ca2+ release and force to preserve ATP as it reduces the ATP demand by Ca2+ ATPase and myosin ATPase. The lack of KATP channel activity during fatigue causes: (i) fiber damage and (ii) major excitability, contractility and metabolic dysfunctions suggesting that the channel is crucial in terms of myoprotection. That is, once activated, the channel prevents massive sarcolemmal depolarization and the subsequent large increases in unstimulated [Ca2+]i and force. The apparent faster fatigue rate and lower force recovery in KATP channel deficient muscles, using either glibenclamide in wild type muscles or Kir6.2−/− muscles, is most likely because of (i) NaV channel inactivation associated with the large depolarization as well as (ii) fiber damage associated with greater increase in unstimulated [Ca2+]i and ATP depletion.In summary, KModulation of the K+-induced force depression by changes in GCl”, changes in ClC-1 channel activity or GCl significantly alter how changes in [K+]e affect membrane excitability and force in resting, unfatigued muscles. For these observations to be of any physiological significance, one then expects changes in GCl during muscle activity and fatigue. In fact, there is evidence for the activation of ClC-1 channels during metabolic stress such as metabolic inhibition and fatigue  potentiates twitch and sub-tetanic force, (ii) augments blood flow by triggering blood vessel vasodilation  stimulation frequency and iii) muscle physiological/metabolic state as regulators of NKA activity, GCl (ClC-1 channels) and GK (KATP channels) during exercise and fatigue.The documented contrasting effects of Ks Renaud . Renaud s Renaud  emphasizPaterson . Then, wy Renaud . Three fin-vivo stimulating frequencies directly affect increases in [K+]e. Considering the range of known activation frequencies  that modulate the K+ effect. Nevertheless, it should be noted that during non-fatiguing moderate exercise, where activation frequencies are sub-maximum and increased [K+]e are low/moderate, it seems reasonable to consider K+ to be a primarily positive regulator of contractile performance in muscles.The first two factors are interrelated as +]int that can reach 10–12 mM as observed even in moderate 30 W knee extension exercise  increases in NKA activity due to increases in [Na+]i, activation by catecholamines and CGRP, and iv) decreases in GCl as ClC-1 channels close following phosphorylation by PKC . The expected high [K+]int- and [Na+]i-induced loss of membrane excitability and thus force as discussed above is counteracted by the increased in NKA activity and closure of ClC-1 Cl− channels. This occurs because under those conditions the critical [K+]e that causes force depression is shifted to higher [K+]e, which also shifts the critical resting EM to less negative potential. Furthermore, sub-maximal tetanic forces are potentiated by the increased [K+]int as well as by catecholamines increasing SR Ca2+ release. The duration of this condition  lasts as long as there is no or minimal metabolic stress becoming shorter as the muscular activity becomes more intense.At the onset of any muscle activity, one expects that ATP demands are met by adequate ATP production so that there is no metabolic stress related to an energy deficit. Four major events occur under this metabolic/energy condition Fig. A; (i) laCl and GK as ClC-1 and KATP channels are activated in part by the decrease in ATP concentration allowing the ATP dissociation from the channels and in part by intracellular signaling pathway(s) that has yet to be identified  move the action potential threshold toward less negative EM and (ii) counteract the Na+ inward current during the depolarization reducing action potential amplitude. Although the effect of an increase in GCl on membrane excitability and force generation has not been studied directly due to the lack of a ClC-1 channel opener, one can expect that the increased GCl lowers the critical [K+]e switching the K+ effect from potentiation to depression.When muscle ATP does not meet the demand, especially in restricted areas of the muscle fiber, the ATP depletion triggers phase 2. Phase 2 involves large increases in Gied Fig. B. The inATP channel deficient muscles, as discussed above in the section entitled “KATP channels: another physiological function for K+during fatigue”. Finally, as recently reviewed, after intense muscle activity plasma [K+] falls rapidly and can decline to below pre-exercise levels indicating ongoing elevated muscle NKA activity.  during repetitive AP firing that have been reported in rat and mouse EDL also occur in human skeletal muscles  the increase in muscle [K+]e during fatigue not being high enough by itself to induce force depression during fatigue, or actually declining during continued exercise and ii) the fact that for submaximal tetanic contraction, small increases in [K+]e actually potentiate muscle force. This review emphasizes that K+ exerts dual roles. A first role is one of force potentiation during low-to-moderate exercise intensities, as part of several mechanisms that optimize muscle contraction. A second role is one of force depression that occurs during a metabolic stress that restricts ATP availability. At the onset of muscle activity and during moderate muscle activity, the K+-induced potentiation predominates while the K+-induced depression is prevented, primarily because of NKA activation and a reduced GCl as ClC-1 channels close. The K+-induced force depression in muscle occurs when metabolic stress/energy deficit occurs and leads to the activation of ClC-1 and KATP channels, which reduces sarcolemmal excitability and thus force. The K+-induced force depression is further enhanced by the synergistic depressive effect of reduced Na+ and K+ gradients.KCl in phase 1 involves ClC-1 channel phosphorylation by PKC, the mechanism by which metabolic stress activates both ClC-1 and KATP channels remains to be elucidated. Although ATP modulates the activity of both channels, where decreases in ATP result in greater channel activity, it is more than likely that intracellular signaling pathways are implicated. For example, the adenosine A1-receptor activates KATP channels under patch clamp conditions (Barrett-Jolley et al. ATP channels during metabolic stress (Yoshida et al. Although the decrease in G


Fourteen  elite or world class squash player’s fluid balance, sweat [Na+] and hydration practices were calculated throughout a training session in moderate environmental conditions . Rehydration practices were also quantified post-session until the players’ next training session, with some training the same day and some training the following day. Players had a mean fluid balance of −1.22 ± 1.22% throughout the session. Players had a mean sweat rate of 1.11 ± 0.56 L·h−1, with there being a significant difference between male and female players (p < 0.05), and a mean sweat (Na+) of 46 ± 12 mmol·L−1. Players training the following day were able to replace fluid and sodium losses, whereas players training again on the same day were not. These data suggest the variability in players hydration demands and highlight the need to individualise hydration strategies, as well as training prescription, to ensure players with high hydration demands have ample time to optimally rehydrate.Elite squash players are reported to train indoors at high volumes and intensities throughout a microcycle. This may increase hydration demands, with hypohydration potentially impairing many key performance indicators which characterise elite squash performance. Consequently, the main aim of this study was to quantify the sweat rates and sweat [Na Consequently, an individual’s hydration strategy is personalised, depending on the individual, their sweat rate, and their sweat [Na+].Accordingly, it is important that squash players have an appropriate hydration strategy to maintain their physical and cognitive performance on court. Developing an optimal hydration strategy is not a one-size-fits-all approach. An athlete’s sweat rate is the main determinant of their hydration strategy , with swetention . Na+ conexercise . Like sw, (2017)  reportin+] losses and hydration practices of athletes in a variety of racket sports, such as tennis [+] or hydration practices of elite squash players during a training session. Calculating the fluid balance, sweat [Na+] and hydration practices of elite squash players during a training session would provide players, coaches, and practitioners with information to optimise players’ hydration practices throughout training.Previous research has quantified the fluid balance, sweat  of elite squash players throughout a training session. A secondary aim was to investigate players’ hydration practices during the training session  to determine whether these were optimal in relation to a players individualised sweat rate and sweat Na+ losses. A final aim of the study was to determine players’ pre-conceived sweat rate, sweat [Na+], and cramping frequency, and to quantify the relationship between these, players’ sweat rates, and players’ sweat [Na+].Therefore, the primary aim of this study was to quantify the sweat rates and sweat [Na+] quantified post-session. Players completed a food and drink diary immediately after the session until the time of their next training session, in order to quantify their rehydration practices. Data was collected during a training session with an England Squash senior squad in March 2021. This training session was the first of a training microcycle. The training session was consistent with training that would normally be undertaken at an England Squash senior squad and consisted of players’ habitual warm up, squash-specific routines and conditioned games. The training session lasted 90 min (from 10:00 to 11:30). Player’s pre-exercise hydration status was assessed before the session through urine osmolarity; players’ sweat rate, fluid and food intake measured during the session; and players’ sweat  collection, participants were asked three questions regarding their perceived sweat rate, frequency of cramps and perceived sweat [Na+]. The questions (and answers) were as follows: (1) “How would you classify your sweat rate?” ; (2) “How often do you suffer from muscle cramps?” ; (3) “Do you think you lose a lot of Na+ in your sweat?” . Before answering question three, participants were asked to consider whether they felt sweat irritating their eyes. These questions were asked to quantify whether players’ preconceptions regarding their sweat rate and sweat Na+ losses were correct, and whether players altered their hydration strategy accordingly . These questions have been shown to provide sufficient reproducibility in identifying relationships between participants sweat rate (ICC = 1.0), frequency of cramps (ICC = 0.9), sweat [Na+] perceptions (ICC = 0.7), and quantifiable variables   and their self-reported cramping frequency, (3) players’ sweat [Na+] and their self-reported sweat [Na+]. Pearson’s Correlation Coefficient and Spearman’s Rank-Order Correlation were interpreted according to accepted thresholds  . Players mean sweat [Na+] was 46 ± 12 mmol·L−1. Players had a mean sweat Na+ rate of 934 ± 248 mg·L·h−1, equating to a mean total Na+ loss of 1598 ± 1023 mg throughout the training session.−1; females = 0.78 ± 0.45 L·h−1; p = 0.01; d = 1.53; CI = 0.16–1.22). There were no significant differences in sweat [Na+] .Male players had a significantly greater sweat rate in comparison to female players  and post-session Na+ intake  in the following day training session group and the same day training session group. All seven players in the following day training session group replaced 1.5 L of fluid for every kilogram of body mass lost throughout training, with five replacing the amount of Na+ lost during the session. Only two out of the seven players in the same day training session group managed to replace 1.5 L of fluid for every kilogram of body mass lost throughout the training session, with only one player managing to replace the amount of Na+ lost.+ intake . There wr = 0.51; p = 0.62), a very weak positive association between perceived incidence of cramps (Q2) and sweat [Na+] , and a very weak negative association between perceived sweat [Na+] (Q3) and sweat [Na+] .There was a moderate positive association between players perceived sweat rate (Q1) and sweat rate (+] of elite squash players during a training session. The secondary aims were to (1) quantify the differences in sweat rates and sweat [Na+] between male and female elite squash players; (2) calculate players hydration practices during the training session to determine whether these were optimal in relation to players’ sweat rate and sweat Na+ losses; (3) to establish whether players who have two training sessions on the same day, have sufficient time to appropriately rehydrate in comparison to players who are next training the following day; (4) to determine players perceived sweat rate, sweat [Na+] and cramping frequency and to quantify the relationship between these and players’ sweat rates and sweat [Na+].The primary aim of this study was to quantify the fluid balance, sweat rates and sweat  of 46 ± 12 mmol·L−1; (4) males had a significantly greater sweat rate than females; (5) players in the same day training session group were not able to replace fluid and sweat Na+ losses, whereas players in the following day training session group were able to do so; (6) there was a moderate positive, but non-significant association between players perceived sweat rate and their sweat rate.The main findings were (1) elite squash players had a mean fluid balance of −1.22 ± 1.22% throughout the session; (2) elite squash players had a mean sweat rate of 1.11 ± 0.56 L·h−1)  of 46 ± 12 mmol·L−1. This is comparable to data collected by Ranchordas et al., (2017) [+] of 44 ± 13 mmol·L−1 across a variety of different sports , utilising the same technique (pilocarpine iontophoresis). The present data equated to a mean sweat Na+ rate of 934 ± 248 mg·L·h−1, and a mean total Na+ loss of 1598 ± 1023 mg throughout the training session. Na+ can be easily consumed in the diet through adding NaCl  to meals or sport hydration/electrolyte tablets to water . Consequently, players should aim to consume approximately 1000 mg of Na+ per hour of squash training to replace Na+ losses. Elite squash players had a mean sweat , ranging from 34 mmol·L−1 to 70 mmol·L−1. Therefore, while this recommendation of 1000 mg of Na+ per hour of squash training may be advisable for players with moderate sweat [Na+], players with a high sweat [Na+] may require a more aggressive strategy to replace sweat Na+ losses. Variability in sweat [Na+] may be due to heat acclimation, through an increased Na+ ion reabsorption capacity of the eccrine sweat gland  (p = 0.53). This is consistent with Barnes et al., (2019) [+ balance is equivocal [+] are likely to be due to interindividual and intraindividual differences  and their sweat [Na+]. This suggests that players could not predict how much Na+ they lost in their sweat, and contrasts with findings from Ranchordas et al., (2017) [+] and self-reported [Na+] . [Na+] in sweat may be more difficult to predict, as there is no visual stimulus, unlike sweat rates, aside from Na+ residue on apparel, which is only visible in darker coloured clothing. Players were shown to perceive a very weak negative association between their perceived sweat . This is consistent with findings from Ranchordas et al., (2017) . Pilocarpine iontophoresis is a regional sweat collection technique, with the present study utilising a player’s forearm to quantify sweat [Na+]. Taylor et al., (2013) [+], with regional sweat [Na+] techniques also shown to overvalue sweat [Na+] in comparison to whole body sweat [Na+]  technique proposed by Shirreffs & Maughan (1997) [+]. However, these regression equations are derived from sweat-patch tests, which may compromise the validity of their results [+] composition between pharmacologically induced sweat  and thermally induced sweat  are equivocal, with some studies reporting pharmacologically induced to have greater sweat [Na+] and some reporting similar sweat [Na+]   (whole bn (1997)  is not pn (1997) ,139 has  results . The loc results ,140. Exe results ,142. Desat [Na+] ,144. ConFinally, another limitation of this study is that we did not quantify female players’ hormonal status to better understand how this may influence their sweat rate. Female sex hormones may influence female players’ sweat rates see .−1 and sweat [Na+] of 40 mmol·L−1, losing 3.17 L of fluid and 2589 mg of Na+ throughout the session. This contrasts with player 5, who had a sweat rate of 0.42 L·h−1 and sweat [Na+] of 41 mmol·L−1, losing a total fluid and Na+ loss of 0.63 L and 530 mg respectively. Consequently, the hydration strategies employed with each player may be different, even though the training loads may be the same. Player 5 had a low hydration demand, and fluid and Na+ losses can be accounted for throughout or immediately post-session. This is unlike player 1, who had a high hydration demand, with it being unlikely to replace sweat and Na+ losses during the session, requiring an aggressive rehydration strategy post-session, as well as consideration of the proximity of the next training session undertaken.This study highlights that it is possible to collect hydration-related data in elite squash players and conveys a framework to determine them. It also displays the variability in hydration demands across players and emphasises the importance of individualised hydration strategies, rather than a one-size-fits-all approach. For example, player 1 had a sweat rate of 2.12 L·h


The effects of α and ß adrenergic receptor modulation on the risk of developing heart failure (HF) remains uncertain due to a lack of randomized controlled trials. This study aimed to estimate the effects of α and ß adrenergic receptors modulation on the risk of HF and to provide proof of principle for genetic target validation studies in HF.Genetic variants within the cis regions encoding the adrenergic receptors α1A, α2B, ß1, and ß2 associated with blood pressure in a 757,601-participant genome-wide association study (GWAS) were selected as instruments to perform a drug target Mendelian randomization study. Effects of these variants on HF risk were derived from the HERMES GWAS .P = 0.001) and 0.95 . Conversely, lower α2B activity was associated with increased HF risk: OR 1.09 . No evidence of an effect of lower ß2 activity on HF risk was found: OR 0.99 . Complementary analyses showed that these effects were consistent with those on left ventricular dimensions and acted independently of any potential effect on coronary artery disease.Lower α1A or ß1 activity was associated with reduced HF risk: odds ratio (OR) 0.83 (95% CI 0.74–0.93, This study provides genetic evidence that α1A or ß1 receptor inhibition will likely decrease HF risk, while lower α2B activity may increase this risk. Genetic variant analysis can assist with drug development for HF prevention. HoweverSeveral adaptive changes in HF are mediated by an over-activation of the sympathetic nervous system as evidenced in HF with reduced or mildly-reduced left ventricular ejection fraction (LVEF) , 4. The Many traditional candidate gene studies have assessed the effect of adrenoreceptor modulation on HF, but their results are controversial or inconclusive . On the We aimed to decipher the role of the various adrenoreceptors in HF by using a drug target MR to estimate the effects of their inhibition on the risk of developing HF and the left ventricular (LV) dimensions, and to provide proof of principle for genetic target validation studies in HF to prioritize novel therapeutic approaches. For targets causally related to HF, we also studied their effect on the risk of coronary artery disease (CAD) to gain further insight into the mechanisms involved.2.2.1.Drug target validation MR studies follow the same principle as classical MRs that assess the causal relationship between an exposure  and an outcome , but restricted the genetic variants selection to the cis-region of the gene encoding the drug target of interest (exposure) to build the genetic instrument rather than selecting them from across the genome  9)..9).We used relevant downstream traits: blood pressure (BP) and heart rate (the main cardiovascular biomarkers affected by sympathetic nervous system modulation), as proxies for receptor activity, to select the cis-variants and weight their effect  9). As . As 9). 2.2.2.2.1.A list and a description of the GWAS summary statistics used are provided in Expression quantitative trait loci (eQTL) were obtained from the Genotype-Tissue Expression (GTEx) portal (release version 8) that includes 15,201 RNA-sequencing samples from 54 non-diseased tissues of 838 postmortem donors  . As 49 tProtein quantitative trait loci (pQTL) data were obtained from a cohort of 3,301 participants of European descent from the INTERVAL study that includes about 50,000 healthy participants nested within a randomized trial of varying blood donation intervals . The rel2.2.2.The Heart Failure (HF) risk GWAS comprising 40,805 HF cases and 542,362 controls was derived from a GWAS meta-analysis of HF of the HERMES consortium of European ancestry, which includes 68,157 HF cases and 949,888 controls . All stun = 16,920), LVESV , LVEF , and LV mass  (Genetic association estimates for the LV dimensions were obtained from GWAS of cardiac magnetic resonance imaging (MRI)-derived LV measurements drawn from the UK Biobank: Left ventricular mass (LV mass), Left ventricular end-diastolic volume (LVEDV), Left ventricular end-systolic volume (LVESV), and Left ventricular ejection fraction (LVEF) in a total of 16,923 European individuals with a maximum sample size of LVEDV  .Estimates for CAD risk were obtained from the CARDIoGRAMplusC4D  Consortium's 1,000 Genomes–based transethnic meta-analysis of 60,801 case subjects and 123,504 control subjects . The maj2.3.To build the genetic instrument, the gene encoding regions of the nine adrenergic receptors, ADRA1A (α1A), ADRA1B (α1B), ADRA1D (α1D), ADRA2A (α2A), ADRA2B (α2B), ADRA2C (α2C), ADRB1 (ß1), ADRB2 (ß2) and ADRB3 (ß3), as well as their promoter and cis-enhancer regions were first selected . This tain silico functional predictions, and distance from the canonical transcript start site (We verified that the identified SNPs were specific to the genes encoding the adrenergic receptors when located in intergenic regions by using the genetic.opentargets.org database where we checked that the SNPs had a high Variants to Genes (V2G) score affiliated to the gene encoding the corresponding adrenergic receptor. The V2G score is a single aggregated score for variant-gene prediction obtained by combining eQTLs and pQTLs, chromatin interaction and conformation datasets, art site .We then identified the genetic variants of these regions as single nucleotide polymorphisms (SNPs) that are in both exposure  and outcome GWAS and checked their specific relationship to the corresponding adrenergic receptor.P ≤ 5 × 10–8), with a Minor Allele Frequency (MAF) >0.01, and clumped to a linkage disequilibrium (LD) threshold of r2 < 0.1 using the 1000G European reference panel to ensure their independence.SNPs were then selected based on association with diastolic BP, systolic BP, or heart rate at genome-wide significance (P ≤ 1 × 10−4) or for LD clumping (r2 < 0.6).When only one or two independent SNPs were found for a given gene using these criteria, we selected additional SNPs to perform MR analyses using the previously defined criteria, but with different thresholds either for their association with diastolic BP  were harmonized to match coded effect alleles consistently.Indeed, for each SNP we need to ensure that the measured effects on the exposure and the outcome correspond to the same effect alleles. It's worth noting that discrepancies might arise when comparing SNPs across different GWAS, necessitating a harmonization process before any subsequent analyses can be conducted. The methodology for SNP harmonization closely follows the approach outlined in the work by Hemani et al. .To enhance the quality of our harmonization process, certain SNPs are excluded from consideration. Specifically, we exclude palindromic SNPs, which are characterized by having the same possible alleles on both the forward and reverse strands. Additionally, SNPs with major allele frequencies (MAF) approximating 50% (MAF > 0.42) are also omitted. This exclusion criteria is implemented to mitigate potential ambiguities in the subsequent analyses , 25.r2 between the SNPs described in the literature and the selected SNPs. In the end, we did not find any supplementary independent genetic variants that were significantly associated with the diastolic BP, systolic BP, or HR exposures.We also searched for additional published SNPs, in particular for ADRA1A and ADRA2B –34 to en2.3.1.p = 0.05 significance threshold was used to examine the association between a genetic variant and a phenotype.The genetic variants selected for the MR analysis were characterized with regard to their metabolic profile using the type 2 diabetes Knowledge Portal that enables the analysis of 325 cardio-metabolic traits in 281 datasets. A 2.4.A two-sample MR study design with an approach relevant to drug target validation was used , 35.When a single variant was available, the MR analyses were conducted using the Wald estimator, which is a causal estimate obtained for a single genetic variant by dividing its gene-outcome association by its gene-exposure association. For multiple variants, the fixed-effect “Inverse Variance Weighted” (IVW) method for correlated variants was employed.R2 > 0.1 or above) and variants from the same genetic region are used, they tend to still be in LD. It is therefore necessary to take in account their correlation. Furthermore, the adoption of a fixed-effects model assumes that all genetic variants are targeting the same causal effect parameter. Such assumption is reasonable when all the genetic variants are in the same gene region and then are likely to affect the risk factor in the same way.This approach combines Wald ratio together in fixed effect meta-analysis, where the weight of each ratio is the inverse of the variance of the SNP-outcome association. Each instrumenting SNP is treated as an independent “study”, and the Wald ratios estimated for each SNP are meta-analysed under a fixed effects model. The fixed-effect “IVW” method for correlated variant is a specific parametrization of the generalized least squares technique that accounts for pairwise LD between variants at each locus using the 1000G European reference panel . Since tOdds ratios (OR) were derived from the corresponding MR estimate for each adrenergic receptor and are given for a 1 mmHg decrease in BP or 1 beat per min (bpm) decrease in heart rate. For the LV dimensions, the MR results are expressed as effect size (β) and are given for a 1 mmHg decrease for BP or a 1 bpm decrease for heart rate, with a unit that depends on the LV dimension considered. For the MR analyses using eQTLs, the results are expressed as effect size weighted by the expression level of the gene encoding the corresponding adrenergic receptor.P ≤ 5 × 10–8) and with a LD threshold of r2 < 0.1 were also performed.Sensitivity analyses were employed to assess the validity of these findings. They included diagnostic tests for horizontal pleiotropy (Cochran's Q statistic and MREgger test) and SNP outliers . If horizontal pleiotropy was detected, then the MR Egger method would have been employed to conduct the MR analysis. When, for a given gene, additional SNPs have been selected using alternate thresholds, supplementary MR analyses using only the SNPs at genome-wide significance  . MR esti2.5.For targets having a causal relationship with HF, we performed a mediation analysis to determine whether their impact on HF was mediated partially or not by an effect on the risk of CAD.First, we performed MR analyses using CAD risk GWAS as outcome and the SNPs selected for the different exposure GWAS (BP and heart rate) to check whether these adrenergic receptors also influenced CAD risk. Then, to test whether the predicted effect on HF risk was mediated by the effect on CAD risk, we performed MR analyses using the previously obtained HF risk GWAS adjusted for CAD risk using Multi-trait Conditional and Joint Analysis (mtCOJO) as outcome . The mtC3.The overall design and flow of the study are displayed in 3.1.Among the nine sympathetic nervous system receptors, at least one SNP associated at genome-wide significance with BP or heart rate was identified in the cis region of genes encoding α1A (diastolic BP), α2B (diastolic BP and heart rate), ß1 and ß2 (diastolic and systolic BP) , S3. WhiAll selected SNPs were used in all further MR analyses except those using LV dimensions as outcomes, as fewer α1A, ß1 and ß2 SNPs were in common between the BP and LV dimensions GWAS .3.2.The main analysis was performed with the genetic instruments weighted by diastolic BP as proxy for target activity since it was not possible to identify variants significantly associated with systolic BP and/or heart rate across all 4 genes. The OR derived from the corresponding MR estimate for each adrenergic receptor are given for a 1 mmHg decrease in BP.3.2.1.P = 0.001) and 0.95  respectively (P = 3 × 10−7). No evidence was found for an effect of ß2 activity in HF risk: OR 0.99 .A lower α1A or ß1 activity was associated with a lower risk of developing HF: OR 0.83  and a higher LVEF . No evidence was found for an effect of ß1 on LVEDV or LV mass , LVEDV , lower LVEF  and higher LV mass  (A lower α2B activity was associated with higher LVESV ( × 10−5) .3.2.3.P = 0.26). A lower ß1 or α2B activity was associated with a lower CAD risk: OR 0.95 , and OR 0.95 , respectively . Furthermore, our results are consistent with recent studies that suggest a lack of detrimental or beneficial effect of long-term ß2 modulation on LV volumes and function and the risk of HF  were consistent with the anticipated effects of target modulation. Finally, we searched for previously reported associations between the variants and the target in the literature. We then ensured the validity of our genetic instrument.The robustness of our findings was ensured by the consistency with the sensitivity analyses that included supplemental analyses which used when possible additional exposures  or variants modulating gene expression (α1A and ß2).However, while drug-target MRs are powerful tools to assess the presence and direction of the effect of the modulation of a potential target on a disease, their results may not be directly translated to the clinic due to the differences between genetic and pharmacological perturbation of a target including drug pharmacokinetics and duration of the intervention (lifelong for genetic). Nevertheless, our study gives relevant insights on the potential beneficial or harmful effects of the modulation of the adrenergic receptors on HF risk.4.2.This drug target MR suggests that the inhibition of several adrenoreceptors may be preventive , neutral (ß2) or detrimental (α2B) on the risk of developing HF. Furthermore, drug target MR can be considered a useful tool to identify and validate candidate targets in HF. This will help focusing on the most promising strategies that can lead to patient benefit, accelerate drug development, and limit studies of potentially non-efficacious drugs.