Reducing the number of influenza A virus (IAV) infected pigs at weaning is critical to minimize IAV spread to other farms. Sow vaccination is a common measure to reduce influenza levels at weaning. However, the impact of maternally-derived antibodies on IAV infection dynamics in growing pigs is poorly understood. We evaluated the effect of maternally-derived antibodies at weaning on IAV prevalence at weaning, time of influenza infection, number of weeks that pigs tested IAV positive, and estimated quantity of IAV in nursery pigs. We evaluated 301 pigs within 10 cohorts for their influenza serological  test) and virological  status. Nasal swabs were collected weekly and pigs were bled 3 times throughout the nursery period. There was significant variability in influenza seroprevalence, HI titers and influenza prevalence after weaning. Increase in influenza seroprevalence at weaning was associated with low influenza prevalence at weaning and delayed time to IAV infection throughout the nursery. Piglets with IAV HI titers of 40 or higher at weaning were also less likely to test IAV positive at weaning, took longer to become infected, tested IAV RT-PCR positive for fewer weeks, and had higher IAV RT-PCR cycle threshold values compared to piglets with HI titers less than 40. Our findings suggest that sow vaccination or infection status that results in high levels of IAV strain-specific maternally-derived antibodies may help to reduce IAV circulation in both suckling and nursery pigs. Mycoplasma hyopneumoniae [Influenza A virus (IAV) is a primary cause of acute respiratory disease in pigs and it is also part of the porcine respiratory disease complex (PRDC), which includes other pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus 2 (PCV2), and eumoniae . IAV infeumoniae , decreaseumoniae  and redueumoniae . IAV in Influenza is widespread in US pigs and breed-to-wean (BTW) pig farms play a central role in the spread of IAV across geographical regions –9. SucklSow vaccination is the main measure to control influenza in BTW farms –35. Sow In addition, pigs have complex IAV infection dynamics, such as recurrent infections, multiple waves of infection, co-circulation of genetically distinct viruses and frequent reassortment events observed in growing-finishing pigs –49. HoweThis study was carried out after the protocol was approved by the Institutional Animal Care and Use Committee (Protocol Number: 1510-33054A) and the Institutional Biosafety Committee (Protocol Number: 1508-32918H) of the University of Minnesota. The participating producers provided written consent to conduct the study in their farms. All samples were collected by trained veterinarians and researchers. Pigs were raised indoors in conventional mechanically ventilated nursery barns with climate control. Pigs had ad libitum access to fresh food and water and farmers monitored the health of the pigs twice daily to detect and treat any sick pig. Pigs were cared according to recommendations by the herd veterinarian which included antipyretic and antibiotic treatments on as needed basis.Mycoplasma hyopneumoniae, PCV2 and PRRSV .Piglets (n = 301) were identified at weaning and tested during the growing period in the nursery which included piglets from 3 to 10 weeks of age. Five batches of weaned piglets were placed into 2 separate nursery farms. Pigs in each batch were divided into 2 rooms for a total of 10 cohorts. Approximately 30 pigs within each cohort were selected and pigs were placed in a randomly selected pen within each room. Each nursery farm was a single-barn site with two rooms with all-in/all-out flow by site . Piglets were vaccinated at weaning against M. hyopneumoniae and PCV2. Additionally, incoming replacement females (gilts) were vaccinated twice with an inactivated IAV commercial vaccine .Piglets originated from a single air-filtered 3,200 sow farm located in Minnesota with a history of testing IAV positive by RT-PCR in piglets prior to weaning for at least the last 2 months before the study began. The sow farm was located within 10 miles from the nursery farms and known to be negative for wild-type PRRSV strains but positive for ® MLV vaccine, Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO) as part of their biannual vaccination against PRRSV. During 8 weeks within the study period (December 2015 to January 2016), sows were also vaccinated with 2 commercial IAV inactivated vaccines  with cohorts 3a, 3b, 4a and 4b originating from vaccinated sows. There was no sow influenza vaccination before and after the described 8-week period during the study timeframe. Cohort details are explained in The study was conducted from November 2015 to April 2016. During that time, sows were vaccinated against PRRSV using a modified-live vaccine  and stored at -80°C until testing. Swabs were tested by a real-time RT-PCR targeting the IAV matrix gene .TM—96 viral RNA extraction kit  following manufacturer’s instructions using an automatized robotic extraction equipment: MagMaxTM–Express 96 Deep well magnetic particle processor . RT-PCR test reagents were from AgPath-ID one-step RT-PCR kit  and reactions were run in a 7500 Fast RT-PCR system . Thermal cycles for IAV detection were 10 min at 45°C, 10 min at 95°C, 45 cycles of 1 sec at 94°C and 30 sec at 60°C within a 25μL of total volume [Briefly, sample viral RNA was extracted using a MagMaxAdditionally, two nasal swab samples with the lowest ct values in each cohort from weeks when IAV prevalence was the highest were selected for virus isolation in Madin-Darby Canine Kidney (MDCK) cells  and wholHA gene consensus sequences were annotated for completeness, functionality and subtype classification using the influenza virus sequence annotation tool (FLAN) . CompletHA gene sequences were aligned and translated into amino acid sequences to further analyze the amino acid similarities between viruses from the BTW farm, the studied cohorts and the IAV commercial vaccines used in sows. Alignment, amino acid translation and sequence comparisons were done using the ClustalW algorithm, Neighbor Joining method and a Jukes-Cantor substitution model in Geneious software . H1 antiFinally, blood samples were left at room temperature for serum separation and then refrigerated overnight. Samples were centrifuged for 10 min at 1500 rpm and 4°C. Sera then were stored at -20°C until testing. Sera were tested by hemagglutination inhibition (HI) assay using a representative and dominant IAV isolate (H1 delta 2) among all cohorts and BTW farm. Briefly, sera were pretreated with receptor destroying enzyme (RDE) and incubated at 37°C for 14 hours overnight. Incubated sera were hemadsorpted by adding a 20% turkey red blood cell solution, centrifuged, and stored at -20°C until titration. Two-fold serial dilutions from 1:10 to 1:640 were tested for each serum sample using an antigen solution containing 16 hemagglutination units (HAU) in 50uL and a 0.5% turkey red blood cell solution. Positive and negative controls were used in each plate to confirm the results obtained in plate. Plates were read manually by tilting them and visualizing inhibition of agglutination (presence of a mat of red blood cells in the bottom of the well) . AdditioIndividual HI titer data was tabulated and classified as HI-positive or -negative using a reciprocal HI titer of 40 as the cutoff point (≥40 were considered positive). RT-PCR data were tabulated and classified as positive or negative based on a cycle threshold (ct) cutoff point of 37.5. Then, the percentage of IAV RT-PCR and HI serum antibody positive pigs over time in the nursery was calculated and values reported as prevalence and seroprevalence, respectively. Prevalence and seroprevalence data were summarized by week for each cohort.Time to IAV infection was calculated by counting the number of weeks from weaning until a pig tested IAV RT-PCR positive. The number of weeks that a given pig tested IAV RT-PCR positive during the growing period in the nursery was calculated by counting the total number of weeks that a pig tested IAV RT-PCR positive for the entire nursery period. Because the real-time RT-PCR used in this study is semi-quantitative, the estimated quantity of IAV virus was approximated from the RT-PCR ct values, with the lowest ct values having the highest estimated virus quantity and ct values of >37.5 having estimated virus quantity approaching zero. For a given pig, the lowest ct value during the entire study was chosen for the median calculations by cohort. Median values of time to IAV infection, number of weeks that a pig tested IAV RT-PCR positive and lowest ct values were calculated for each cohort and for each reciprocal HI titer group using the dplyr package in R 3.4.2 statistical software .® statistical software  [® statistical software  [To test the association between IAV seroprevalence of the cohort and HI titer at weaning with prevalence at weaning, time to IAV infection, number of weeks that pigs tested IAV RT-PCR positive and lowest ct values during the nursery period, a Spearman non-parametric correlation test was used in STATA 12ion, TX) . Differeary, NC) . Models A total of 301 piglets from 10 cohorts were tested from weaning to the end of the growing period in the nursery. Overall, there were 885 serum samples of which 28% had a reciprocal HI titer of 40 or higher. Moreover, 348 of 2176 (16%) nasal swabs were IAV RT-PCR positive. In total, 185 of 301 pigs (62%) tested IAV RT-PCR positive at least once during the study .We obtained 20 IAV HA gene sequences including 19 from the cohorts and 1 from the BTW farm prior to the start of the study. We characterized the HA gene sequences as H1 delta 2 (1B.2.1) viruses having 99.4% nucleotide and 99.3% amino acid similarity among themselves. The H1 delta 2 HA gene sequences obtained during the study shared 94.3% and 94.8% amino acid similarity to the H1 delta 2 commercial vaccine strains used in the BTW farm and there were no amino acid changes in the H1 antigenic sites .There was appreciable variability in prevalence and seroprevalence between cohorts over time . IAV preIAV NP ELISA results summarized as percentage of ELISA positive pigs over time in each cohort are also shown in Per cohort, high seroprevalence at weaning was significantly associated with lower IAV prevalence at weaning  and it took a longer time, more than 6 weeks, to become IAV infected after weaning. The number of weeks that pigs tested IAV positive, and lowest ct values during the growing period in the nursery were not significantly associated with seroprevalence at weaning although, there was a numerical trend that reflected limited circulation of IAV after weaning. HI titer group at weaning was significantly associated with infection dynamics after weaning as detailed in Individual HI titer at weaning was significantly associated with the IAV RT-PCR positive status during the growing period in the nursery as shown in Control of influenza in BTW farms is needed to minimize the spread of IAV across pig production systems and regions given that piglets infected at weaning covertly transmit IAV to other farms. Sow vaccination is the most common IAV control measure although the impact of MDA on infection after weaning is poorly understood. Our study showed that high levels of strain-specific MDA at weaning can decrease prevalence of IAV at weaning and more importantly, decrease the overall IAV circulation during the growing period in the nursery. Reducing IAV circulation at weaning should help decrease IAV transmission between farms.Our study supports previous experimental work that demonstrated high MDA measured by strain-specific HI titers reduced significantly the transmission of IAV in weaned pigs . It alsoOur study was also similar to other studies that reported variable levels of IAV circulation and seroconversion in growing pigs and had one or more peaks of infection after weaning , 42–47. Sow vaccination and MDA can protect sows and piglets from IAV clinical disease , 69, 71.Our sequencing approach demonstrated the circulation of one predominant strain (H1 delta 2) in the 10 cohorts and in the BTW farm. We found no differences in the H1 antigenic sites between the delta 2 H1viruses detected in our study and the vaccine strains. Our results indicated that the HI antibodies at weaning affected IAV infection dynamics in the nursery. We considered that pigs originating from vaccinated sows had higher levels of antibodies able to cross-react with the farm specific delta 2 H1 strain and that these were, at least in part, responsible for the decrease in IAV infection. Although almost all pigs tested ELISA IAV NP positive at weaning, NP antibodies did not appear to be protective against IAV infection in the cohorts with high infection at weaning. This may be an additional indication that HI strain-specific antibodies were not present to protect pigs from IAV infection against the circulating H1 farm strain prior or at weaning, especially in the first 4 cohorts originating from non-vaccinated sows. Finally, we cannot fully discard the possibility that sow vaccination may have boosted IAV natural infection before vaccination.Understanding IAV immunity is critical when trying to optimize the vaccination strategies currently used in swine production systems. There are still gaps in our understanding of the quality and quantity of antibodies being generated after IAV vaccination and/or natural infection. This is particularly important in pigs where we have several strains cocirculating in the same population. The complex evolution and landscape of IAV in pigs highlights the need for better vaccines and comprehensive vaccination strategies , 31, 80.In conclusion, we found that high levels of strain-specific hemagglutination inhibition antibodies at weaning decreased IAV infection at weaning, delayed time to become influenza-positive, decreased the number of weeks that pigs tested influenza-positive during the nursery stage, and decreased the overall estimated virus quantity during the infection period. Our results suggest that IAV infection and circulation in nursery pigs may be decreased by using adequate influenza sow vaccination protocols.S1 Table(XLSX)Click here for additional data file.


Higher δ18Ow values during the Dark Age Cold Period (1550 to 1250 years BP) and the Little Ice Age (700 to 200 years BP) are suggestive of reduced ISM rainfall, whereas lower δ18Ow values during the Medieval Warm Period (1200 to 800 years BP) and the major portion of the Roman Warm Period (1950 to 1550 years BP) indicate a wetter ISM. This variability in ISM rainfall appears to be modulated by the Atlantic Multi-decadal Oscillation (AMO) via changes in large-scale thermal contrast between the Asian land mass and the Indian Ocean, a relationship that is also identifiable in the observational data of the last century. Therefore, we suggest that inter-hemispheric scale interactions between such extra tropical forcing mechanisms and global warming are likely to be influential in determining future trends in ISM rainfall.Indian Summer Monsoon (ISM) rainfall has a direct effect on the livelihoods of two billion people in the Indian-subcontinent. Yet, our understanding of the drivers of multi-decadal variability of the ISM is far from being complete. In this context, large-scale forcing of ISM rainfall variability with multi-decadal resolution over the last two millennia is investigated using new records of sea surface salinity (δ Despite the strong bearing and societal relevance of the monsoon, reliable projections of ISM rainfall remain a challenge2, mainly because of the complex dynamics and the myriad of interactions with other tropical and extra-tropical processes across various timescales3.The Indian summer monsoon (ISM) is a large-scale coupled land-ocean-atmosphere phenomenon that plays a dominant role in transporting water vapour to the Indian subcontinent4, the Indian Ocean Dipole5 and Tibetan snow cover6. Additionally, intraseasonal active or break events on short timescales, presumably associated with internal dynamics of the monsoon system itself, have large impacts on the interannual variability of monsoon rainfall8. Example includes the famous break of July 2002, where rainfall was reduced by 50%9. These large year-to-year variations in rainfall associated with internal and external interactions pose a considerable challenge for Indian monsoon forecasting10 and for attributing monsoon variability to large-scale forcings. This is particularly true for multi-decadal and longer time scales given that observational data only covers few cycles and paleoclimate records span longer time scales which fill this gap.On a year to year basis, the ISM exhibits considerable complex and time-varying interactions with the El Niño/Southern Oscillation (ENSO)11. Global climate model experiments tend to suggest an intensification of the ISM in response to global warming partly because of the larger water-holding capacity of a warmer atmosphere, resulting in an estimated 10% increase by the end of the 21st century12; yet, there are large inter-model discrepancies . This is in striking contrast with the recent observational record, which indicates an overall decrease of the ISM rainfall during the period 1950–201215, followed by a weak revival between 2012–201416. Decadal-scale internal climate variability associated with ENSO and the Atlantic Multi-decadal Oscillation (AMO) can also play an important role in explaining the observed variations in NH monsoon precipitation during the last century17 and possibly beyond19. Thus a number of drivers, can contribute to modulating ISM rainfall, including internal variability of the climate system as well as interactions with natural and anthropogenic factors, either of global  or regional  nature22.There is currently a considerable debate on the impact of different forcing agents versus the role of internal variability in determining past and future long-term trends of the ISM23. The relatively coherent response of the various monsoons organised into the NH monsoon system to natural and anthropogenic forcing suggests that monsoon rainfall is sensitive to common hemispheric and/or planetary scale controls. Implicit in this view is the hypothesis that large-scale forcings may override regional-scale factors both natural and anthropogenic in determining future NH monsoon rainfall trends24. Conversely, several studies also suggest a varying response of the individual monsoon systems to the same forcing due to internal feedback dynamics25. Therefore, a better understanding of the large-scale controls of past decadal-scale variability of the monsoon is key to achieve more robust projections of its future changes. However, observations are spatially and temporally limited, mostly to the 20th century, which only allow identifying few long-term fluctuations17. Further extending these records back in time using paleoclimate reconstructions is essential to reduce current uncertainties, to identify its local and global scale forcing mechanisms, and to quantify the background monsoon variability on top of which shorter timescale fluctuations as well as longer-term anthropogenic-induced changes are superimposed24. Here we present a new record of ISM rainfall and SST variability from the Northern Indian Ocean spanning the last 2,000 years at multi-decadal resolution and investigate the large-scale forcing mechanisms of ISM rainfall.More integrated studies of the wider Northern Hemisphere (NH) summer monsoon suggest coherent changes of monsoon precipitation and circulation at hemispheric scales3 from major (Ganges-Bramhaputra and Irrawaddy) and minor  rivers fed by the ISM monsoon precipitation and glacier melt water25. Thus the catchment of these rivers cover a large swath of the summer monsoon rainfall regions of North, Northeast and Central India. Secondly, the BoB directly receives heavy precipitation particularly in the northern parts. Precipitation plus runoff exceeds evaporation on an annual cycle. Fresh water from these sources is mixed into the upper layers of the BoB interacting with the surface circulation. After the summer monsoon some of the strongest lowering of seasonal salinity occurs along the Coromandel Coast in the northern and western BoB  from the calcite shells of planktonic foraminifera Globigerinoides ruber (hereafter G. ruber) to derive δ18Owater (δ18Ow) to reconstruct ISM rainfall variability in the BoB at muti-decadal to centennial time scales . δ18Ow exploits the higher O16/O18 isotopic ratios of precipitation associated with rainfall as opposed to sea water which has lower O16/O18 ratios after appropriate corrections. On land, δ18O of precipitation exhibits spatial variability and shows a negative relationship with precipitation which could vary over the monsoon season24. However, the effects of such spatial and temporal variability are minimised in our study as the BoB receives rainfall from a large area of the Indian monsoon fed region and salinity anomalies persist over the monsoon and post monsoon periods. Also, the δ18Ow and salinity show a strong positive relationship (r2 = 0.89 with a slope of 0.15) in the BoB28. Therefore, δ18Ow, at least in a qualitative sense, reflects salinity variations related to monsoon rainfall at the core site. Calcite tests of G. ruber are used for reconstructions because G. ruber fluxes from sediment trap time series from the BoB suggest that they record average surface conditions in the BoB29. Previous studies have successfully used G. ruber to reconstruct ISM rainfall variability at millennial scale in the BoB since the last glacial period31. High sedimentation rates (0.2 to 0.6 cmyr−1) in Core MD161/17 allow us to reconstruct ISM rainfall variability at multi-decadal temporal resolution for the first time with robust chronology derived from 11 radiocarbon dates of mixed planktonic foraminifera species  and δies Fig. .18Ow record shows a large variability (1.1‰) over the last 2000 years, with high and low δ18Ow values representing decrease and increase of ISM rainfall respectively ; RWP) are associated with negative δ18Ow anomalies, suggesting higher ISM rainfall. During Dark Age Cold Period (DACP) (1550 to 1300 year BP), high positive δ18Ow anomalies are indicative of weaker ISM rainfall. The Medieval Warm Period (MWP) from 1200 to 800 years BP is characterized by negative δ18Ow anomalies compared to the preceding period (i.e. 2000 to 1300 years BP), which reveals that BoB experienced highest ISM rainfall and strong freshening during this MWP over the last 2000 years. In contrast, during the Little Ice Age (LIA), positive δ18Ow anomalies indicate a decrease in ISM rainfall from 700 to 200 years BP. Our ISM rainfall record from the BoB shows similarity with the speleothem record from the Sahiya Cave  and Mg/Ca based SST . Cross correlation analyses of these two time series records also showed a significant negative correlation with zero-lag suggesting a strong temporal coupling between BoB SST and ISM rainfall over the last 2000 years at this core location  and NHT show significant positive correlation at zero year lag  evaporation and cloud cover lead to summertime cooling of the Indian Peninsula as well as of the north-equatorial Indian Ocean, with additional contribution from increased upwelling driven by the intense monsoon winds over its western sector1. As the Himalayas acts as a physical barrier for the southerly moisture-laden flow, precipitation and associated surface cooling are confined largely to the Indian Subcontinent, while the Tibetan Plateau is mainly dry and warm35. This maintains the meridional temperature gradient sustaining monsoon circulation and rainfall during the summer monsoon season36. Our reconstructions shown in Fig. 1: salinity variations in the BoB reflecting monsoon rainfall and freshwater flux and2 lowering of the surface SSTs of the northern Indian Ocean during the monsoon period representing evaporative cooling associated with intensified monsoon winds and cloud cover. The inverse relationship  fluctuations at these time scales. Figure 37. Further afield, a strong positive association between ISM rainfall and SST anomalies in the North Atlantic Ocean and surface temperature over western Eurasia is evident in Fig. 38. Large basin-wide sea surface temperature anomalies are also identifiable across the Pacific, with cooling along the equatorial central-eastern Pacific, and an extensive warming in the extra tropics stretching from Japan to the western coast of the US. The tropical anomalies are somewhat reminiscent of a La Niña-like cooling in the eastern equatorial Pacific. However, the pattern also features substantial differences in two important and fundamental characteristics of the actual La Niña sea surface temperatures anomalies or of its longer-lived manifestation, the Pacific Decadal Oscillation (PDO)39. First, it does not display zonal opposite-sign SST anomalies over the western equatorial Pacific, and second, the anomalies do not extend over the northeastern Pacific. We will return to this point later. Overall, this corroborates the assembled paleo-reconstructions presented in Fig. We further explore the link between large-scale temperature changes and ISM variability at multi-decadal scale by means of observational data during 1901–2012. To make a direct comparison with the multi-decadal proxy records we remove interannual and short time scale variability in the observational data by low-pass filtering. This isolates the decadal or longer time-scale components of the signal from higher-frequency variability which is known to be largely influenced by internal monsoon dynamics41 via its link with Atlantic Thermohaline Circulation (THC) variations and associated inter-hemispheric heat transport fluctuations43. The AMO has also been suggested to play a major role in modulating the 20th century multi-decadal variations of Indian and African (Sahel) summer rainfall, as well as the tropical Atlantic atmospheric circulation . Figure 46. The regional circulation anomalies associated with the southwesterlies in the northern Indian Ocean are part of a hemispheric-wide response pattern to AMO variability featuring enhanced easterly trade winds across the northern equatorial Pacific and enhanced Walker cell north of the equator, leading to larger ascent (precipitation) over Indo-china and the Maritime Continent and subsequent changes in the meridional direction . These patterns, including the Indo-Pacific sea surface temperature anomalies, bear remarkable similarity to those described in previous studies47 and related to variations in the Atlantic oceanic circulation. Figure 48). This analysis, albeit not exhaustive, is suggestive of an AMO-ISM teleconnection with signatures of both an atmospheric pathway via modulation of the upper-tropospheric temperature across Eurasia (e.g.50), and one that also involves atmosphere-ocean feedbacks in the Indo-Pacific region (e.g.51). Overall, this is indicative of an AMO imprint on the global interhemispheric temperature gradient with a positive phase of the AMO associated with a warmer NH than the SH  is poor in both observational data Fig.  and palath century shedding light on distal teleconnections underpinning multi-decadal ISM variations. It is interesting to note that Fig. 17 Fig. In summary, we suggest that multi-decadal ISM rainfall variability during the last 2000 years was modulated by AMO related fluctuations in NH temperatures whereby warmer (cooler) Asian landmasses along with a cooler (warmer) Indian Ocean set up stronger (weaker) surface and tropospheric meridional temperature gradients strengthening (weakening) monsoon circulation and rainfall. Therefore, the reconstructed temporal relationships between ISM rainfall, BoB SST and NHT over the last 2000 years allow to extend and strengthen the findings based on the rather limited observational record for the 2017  was analyzed for δ18O by reacting with 100% orthoposphoric acid at a constant 75 °C in a Kiel Carbonate III preparation device and evolved CO2 gas was analyzed by using Thermo Electron delta plus advantage stable isotope ratio mass spectrometer. Cleaning protocol of G. ruber for Mg/Ca analyses were followed by Barker et al.63, in addition a reductive cleaning step was used according to Martin and Lea64. Mg/Ca ratios were measured by using an axial viewing Varian 720 ICP-OES . Mg/Ca values were then used to estimate SSTs using the equation Mg/Ca = 0.449exp(0.09*T)65. δ18Osw were computed by applying the following equation of Bemis et al.66: δ18Osw = 0.27 ((T − 16.5 + 4.8*d18O)/4.8). The derived δ18Osw estimates were corrected for continental ice volume using Shackleton’s data set67 and presented here as δ18Ow. SST and δ18Ow anomalies are computed by subtracting the mean values of all data points within two millennia from the SST and δ18Ow values respectively.Core MD161/17 was collected at a water depth of 790 m from the KGB. Age model was based on linear interpolation between 11 AMS radiocarbon dates which covers a time span of 2 kyr (Supplementary Table Supplementary file.


Tumour necrosis factor alpha (TNF-α) is a pleiotropic cytokine with both injurious and protective functions, which are thought to diverge at the level of its two cell surface receptors, TNFR1 and TNFR2. In the setting of acute injury, selective inhibition of TNFR1 is predicted to attenuate the cell death and inflammation associated with TNF-α, while sparing or potentiating the protective effects of TNFR2 signalling. We developed a potent and selective antagonist of TNFR1 (GSK1995057) using a novel domain antibody (dAb) therapeutic and assessed its efficacy in vitro, in vivo and in a clinical trial involving healthy human subjects.We investigated the in vitro effects of GSK1995057 on human pulmonary microvascular endothelial cells (HMVEC-L) and then assessed the effects of pretreatment with nebulised GSK1995057 in a non-human primate model of acute lung injury. We then tested translation to humans by investigating the effects of a single nebulised dose of GSK1995057 in healthy humans (n=37) in a randomised controlled clinical trial in which subjects were subsequently exposed to inhaled endotoxin.Selective inhibition of TNFR1 signalling potently inhibited cytokine and neutrophil adhesion molecule expression in activated HMVEC-L monolayers in vitro , and also significantly attenuated inflammation and signs of lung injury in non-human primates . In a randomised, placebo-controlled trial of nebulised GSK1995057 in 37 healthy humans challenged with a low dose of inhaled endotoxin, treatment with GSK1995057 attenuated pulmonary neutrophilia, inflammatory cytokine release  and signs of endothelial injury (P<0.05) in bronchoalveolar lavage and serum samples.These data support the potential for pulmonary delivery of a selective TNFR1 dAb as a novel therapeutic approach for the prevention of acute respiratory distress syndrome.ClinicalTrials.gov NCT01587807. Does selectively targeting tumour necrosis factor receptor 1 (TNFR1) signalling with a novel domain antibody (dAb) reduce pulmonary inflammation?A dAb selective to the TNFR1 reduced pulmonary inflammation in both non-human primate and human pulmonary endotoxin challenge models when dosed prophylactically. Data from mechanistic studies in human cells with this dAb support the importance of TNFR1 signalling in mediating endothelial–neutrophil interactions associated with the development of lung injury.This investigation provides original data to support the utility of a novel TNFR1-targeting dAb as a potential therapy in patients with acute respiratory distress syndrome and supports further clinical investigation in patients.Acute respiratory distress syndrome (ARDS) affects approximately 190 000 patients per annum in the USA, and is associated with mortality rates of up to 40%.Recently, it has become evident that the pleiotropic effects of TNF-α diverge at the level of its two cellular receptors, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2).H) chain TNFR1 dAb antagonist (GSK1995057) administered directly to the lungs of cynomolgus monkeys and humans, and explore the underlying mechanism of action using in vitro human tissue assays.dAbs represent the smallest functional antigen-binding units of human antibodies (10–13 kDa), are derived from single variable regions of either heavy or light chain sequences of human IgG and are sufficiently stable to allow nebulisation directly to the lungs.10.1136/thoraxjnl-2017-210305.supp1Supplementary file 1H framework sequences and ‘scrambled’/non-sense hypervariable domains.The assessment of permeability of human pulmonary microvascular endothelial cell (HMVEC-L) monolayers was undertaken using an electrical cell impedance sensor technique. Once electrical resistance achieved steady state, cells were incubated for 1 hour in the presence of GSK1995057 or dummy dAb (10 nM) or vehicle control for 1 hour prior to stimulation with TNF-α (10 ng/mL). Neutrophil transmigration assays were performed using neutrophils isolated from healthy volunteers. Neutrophil transmigration through confluent HMVEC-L incubated with GSK1995057 or dummy dAb (10 nM) or vehicle control for 1 hour prior to stimulation with TNF-α (10 ng/mL) was measured using CytoSelect Leukocyte Transmigration assay (Cambridge Biosciences) and endothelial ligand cell surface expression was assessed using HMVEC-L after 4 hours of treatment. Detailed descriptions of assay conditions are contained in the online The objective of this study was to investigate the dose–response relationship of single doses of GSK1995057 given as a pretreatment in a lipopolysaccharide (LPS)-induced model of pulmonary inflammation in sedated young adult cynomolgus monkeys. The primary endpoint was neutrophil counts in bronchoalveolar lavage fluid (BALF) at 6 and 24 hours after dosing.Animals were housed according to the USDA Animal Welfare Act  and as described in the Guide for the Care and Use of Laboratory Animals.Escherichia coli serotype 055:B5 (4 mL of 100 µg/mL) was administered via aerosolisation (DeVilbiss Ultraneb-99 ultrasonic nebuliser) over 5 min. Blood and bronchoalveolar lavage (BAL) samples were collected at baseline , 6 and 24 hours after LPS challenge. Detailed descriptions of the techniques used for bronchoscopy and bronchoalveolar lavage are contained within the online A description of the methodology for administration of anaesthesia and LPS is present in the online Cynomolgus monkey BAL samples were analysed by Myriad RBM using their Multi-Analyte Platform (MAP) technology on the Human MAPv1.6 panel of 89 biomarkers, 78 of which are confirmed to be cynomolgus monkey cross-reactive.All studies were conducted in accordance with the GSK Policy on Care, Welfare, and Treatment of Laboratory Animals, and were reviewed by the Institutional Animal Care and Use Committee at Charles River Laboratories.Healthy subjects were recruited by advertising. Screening consisted of a history and physical examination, blood investigations, ECG and spirometry  and the primary endpoint of the trial was BALF neutrophil count with BALF and plasma cytokine, chemokine, epithelial and endothelial biomarkers as secondary endpoints. The dose of GSK1995057 and timing for BAL was derived from data obtained from the dose-finding study in cynomolgus monkeys also presented in this manuscript. A detailed description of the study design, administration of the study drug, bronchoscopy, BAL and sample collection are contained within the online Subjects in part 2 of the trial were randomised to receive a single nebulised dose (26 mg) of GSK1995057 1 hour prior to receiving a nebulised challenge of 50 µg of The measurement of biomarkers in BALF and serum samples from the clinical trial participants were tested under contract by Myriad RBM  using their proprietary multiplex Luminex immunoassay platform; the human inflammation multiplex MAP (iMAP). Biomarkers of interest not included on this panel were measured using commercial ELISAs (surfactant protein-D (SP-D) and Club cell secretory protein (CC16) ELISAs from BioVendor), following manufacturers’ recommendations under contract by Quotient BioResearch . Changes from baseline in free and total TNFR1 were evaluated at varying time points up to 48 hours after the start of nebulisation using an ECLIA on the MSD platform and were conducted by GSK.The study was undertaken in accordance with the Declaration of Helsinki and ICH GCP. Written informed consent was obtained from all subjects prior to enrolment in the study.Analysis of non-clinical data from in vitro and in vivo studies with GSK1995057 was undertaken using analysis of variance on appropriately transformed data, with the exception of neutrophil migration data  and soluThe clinical trial was designed to recruit enough subjects to provide data from 36 subjects available for analysis . While the sample size was based on feasibility, subject numbers were determined to ensure at least 80% power to detect a 65% decrease in BALF neutrophil counts based on mean effect sizes observed in monkey LPS challenge studies. An interim sample size re-estimation (blinded to investigator site team) was undertaken when BAL neutrophil counts from 16 subjects (eight in each study arm) were available for statistical analysis. This confirmed the estimates used to assess the statistical power of the planned sample size, and therefore no change to the sample size was made. Distributional assumptions were assessed by visual inspection of residual plots. Normality was examined by normal probability plots, while homogeneity of variance was assessed by plotting the residuals against the predicted values for the model. Two sample t-tests were used to evaluate the effects of GSK1995057 on biomarkers from BALF samples. Mixed model repeated measures using random effects was used to evaluate the effects of GSK1995057 on biomarkers from serum samples using SAS V.9.3. Baseline values of the biomarker were included as covariates in the analysis of biomarkers from serum samples. Data are shown as geometric mean and SE.To investigate the hypothesis that TNFR1 mediates neutrophilic inflammation in the lung, we assessed the impact of selective TNFR1 antagonism using GSK1995057 on neutrophil transmigration and endothelial permeability in a human in vitro cell system. Using the CytoSelect Leukocyte Transmigration assay, we observed that pretreatment of HMVEC-L monolayers with GSK1995057 significantly reduced the transendothelial migration of neutrophils towards the neutrophil chemoattractant IL-8 ; P<0.05.We investigated further the effects of a single aerosolised dose of GSK1995057 on pulmonary inflammation in a non-human primate model of acute lung injury. Twenty-five cynomolgus monkeys received either nebulised vehicle control, nebulised GSK1995057  or an instillation of fluticasone propionate  via the intratracheal route, 1 hour prior to nebulised LPS (400 µg) challenge. Nebulised GSK1995057 was well tolerated in monkeys and no adverse effects associated with dosing were observed (data not shown). The primary endpoint was the number of neutrophils in the BALF at 6 and 24 hours after LPS exposure. BALF neutrophil counts were markedly suppressed at 6 hours for all three doses of GSK1995057, and at 24 hours for the higher dose levels  13 and 14 hours after administration of LPS and GSK1995057, respectively, which was considered consistent with the expected clinical effects of LPS challenge. The BALF neutrophil data are presented both with and without this biological outlier. GSK1995057 had no effect on the numbers of macrophages or lymphocytes within the BALF at 6 hours (data not shown).Pretreatment with GSK1995057 was associated with a reduction in the post-LPS BALF neutrophil count compared with placebo . This reIn keeping with our in vitro and animal model studies, GSK1995057 significantly reduced the BALF concentrations of IL-1β, IL-6 and IL-8 following LPS administration (P<0.01 in all cases; Here we demonstrate that GSK1995057 reduced pulmonary inflammation in non-human primate and human models of ARDS, and confirm a mechanistic link between TNFR1 signalling and neutrophil/endothelial interactions. The observation that GSK1995057 significantly blunted neutrophil migration through endothelial monolayers in vitro suggests an important role for TNFR1 in mediating neutrophil–endothelial interactions, possibly by regulating the cell surface expression of endothelial cell adhesion molecules E-selectin, ICAM and VCAM. In patients with ARDS, neutrophil infiltration into the lungs is further augmented by a compromised alveolar-capillary barrier brought about by the action of proinflammatory mediators.In some patients with ARDS, it may be sufficient to deliver short-acting therapeutics directly to the lungs; however, factors influencing the pulmonary delivery and disposition of antibodies or antibody fragments are complex,The current study has limitations. While BAL protein increased 24 hours after LPS challenge in monkeys, and GSK1995057 pretreatment attenuated this response, a similar GSK1995057 treatment effect was not apparent in human BAL taken 6 hours after LPS challenge in the clinical trial. BAL protein levels were not significantly increased in humans at 6 hours after LPS challenge, suggesting limited alveolar-capillary leak in response to pulmonary LPS challenge at this early time point. We were unable to assess the contribution of TNFR2 signalling in these studies, primarily due to a paucity of specific biomarkers associated with the TNFR2 pathway. Although we were unable to include a non-selective/pan-TNF-α inhibitor antibody as a comparator in our clinical trial, the observed effects of GSK1995057 on neutrophilic infiltration appear similar to the effects of the long-acting anti-TNF-α antibody adalimumab in the same model.In summary, our data suggest that selective antagonism of TNFR1 using an inhaled dAb may offer therapeutic benefit in patients with ARDS, a common and devastating condition that currently has no effective disease-modifying therapy. While the clinical effectiveness of this approach in established disease is yet to be tested, a phase IIa clinical trial of a TNFR1-targeting dAb in patients is currently under way.


Experiments performed in immobilized and freely swimming larvae and in adult zebrafish brains expressing the genetically encoded calcium indicator GCaMP5G demonstrate, for the first time, the fundamental ability to directly track neural dynamics using optoacoustics while overcoming the longstanding penetration barrier of optical imaging in scattering brains. The newly developed platform thus offers unprecedented capabilities for functional whole-brain observations of fast calcium dynamics; in combination with optoacoustics' well-established capacity for resolving vascular hemodynamics, it could open new vistas in the study of neural activity and neurovascular coupling in health and disease.Non-invasive observation of spatiotemporal activity of large neural populations distributed over entire brains is a longstanding goal of neuroscience. We developed a volumetric multispectral optoacoustic tomography platform for imaging neural activation deep in scattering brains. It can record 100 volumetric frames per second across scalable fields of view ranging between 50 and 1000 mm Recently, these efforts have led to whole-brain activity imaging in transparent organisms using single3 and multiphoton4 light-sheet and light-field5 microscopy. However, these approaches are unable to tackle deep neural activity in intact scattering brains where current state-of-the-art optical imaging strategies based on rapidly scanning multiphoton microscopy are limited to volumes below 1 mm3 because of limited penetration depth and field of view (FOV).Neuronal activation occurs concurrently or in a highly coordinated manner in different areas across the nervous system, reflecting functional interconnection between specialized neuronal subcircuits. Imaging neuronal activation with high temporal and spatial resolution over an entire intact brain, including deep and normally inaccessible areas, could thus play a critical role in the attempt to decipher the fundamental operating principles underlying neural circuit activity. Major efforts are underway to advance the ability to optically image the activity of large, distributed neural populations6, can potentially provide functional neural imaging beyond the limit of current optical imaging technologies. To date, functional optoacoustic brain imaging mainly focused on probing hemodynamics and blood oxygenation variations9, slow and delayed processes that only indirectly reflect neural activation. In contrast, modern approaches toward imaging neural activation are largely based on measuring calcium dynamics, which more directly correlate with neural activity. Calcium dynamics are conventionally tracked using fluorescent sensors, which change their fluorescence output because of a change in their extinction coefficient, fluorescence quantum yield or both as a function of intracellular calcium concentrations11. Recent advances in the field of genetically encoded calcium indicators (GECIs) have provided a variety of genetically modified reporter animal models with calcium indicators in specific neuronal structures14, which in some cases can give rise to signals down to the level of single action potentials15. The new generation of GECIs has several key advantages over organic indicators—they can be expressed over very large volumes and entire brains using the generation of transgenic animals or viral expression; they allow repeated imaging in the same region and animal over multiple days and even up to years; they can be targeted for expression to specific cell populations and even in specific cellular organelles, allowing for flexible, diverse and precise associated methodologies not nearly possible with the traditional organic staining methods.Conversely, optoacoustic imaging, an extremely powerful approach with demonstrated capacity for centimeter-scale penetration into highly scattering tissues12. To test this hypothesis, we selected the zebrafish reporter line HuC:GCAMP5G that exhibits expression of the calcium sensor in a large fraction of neurons to obtain high-resolution optoacoustic measurements from larvae and isolated adult brains.We hypothesized that some members of the GCaMP family of fluorescent GECIs could provide a suitable calcium-dependent optoacoustic contrast because of their strong calcium-dependent extinction changes−2. The maximal volumetric imaging frame rate of the system is determined by the 100 Hz pulse repetition rate of the laser. The optoacoustic detection system was designed to render real-time volumetric ) tomographic capacity with unprecedented spatiotemporal resolutions at depths beyond penetration limits of modern optical microscopes (~0.5 mm in highly scattering brains16). In general, spatial resolution in optoacoustic tomography scales with the available bandwidth of the ultrasound detection system, which, as opposed to light microscopy, is not affected by light scattering in tissues. To enable scalability in imaging brains of different sizes, three different implementations of custom-built piezocomposite matrix arrays  are considered herein. The systems offer real-time imaging with scalable FOV of 50–1000 mm3 and respective spatial resolution varying between 35 and 200 μm. Under FONT1 we refer to the previously reported optoacoustic imaging system17, employing 256-element spherical ultrasound array with 4 MHz central frequency and 90o solid angular coverage, which was shown to enable imaging with 200 μm resolution over a 1000 mm3 volumetric FOV. Higher spatial resolution is enabled herein with the newly developed FONT2 and FONT3 systems consisting of 512-element spherical matrix arrays (π (140o) and ~2π (180o), which allows for an excellent tomographic coverage and high detection sensitivity within the effective FOV of each system. This major performance improvement over previous state-of-the-art implementations17 enables efficient imaging of brains at different scales, from the larval zebrafish nervous system with volumes below 1 mm3 up to the whole adult mouse brain occupying ~420 mm3. The space between the active transducer surface and the imaged samples was filled with an agar gel medium to guarantee acoustic coupling and convenient placement of the samples. The parallel digital acquisition (DAQ) platform was further custom-developed to enable simultaneous sampling of the 512 ultrasound channels and online data transmission over 1 Gb Ethernet connection to the host PC equipped with a high-end graphic processing unit  for real-time data processing. The developed digital acquisitions operate at sampling frequencies of 40 megasamples per second (FONT2) and 125 megasamples per second (FONT3).To enable tomographic imaging of whole scattering brains of different sizes, we developed a new generation of functional optoacoustic neuro-tomography (FONT) platform that can simultaneously monitor calcium activity in all five dimensions, i.e., space, time and wavelength (spectrum), with a scalable spatiotemporal resolution performance. As shown in x arrays . Their i18. The collected data were further processed off-line with a self-developed accurate 3D model-based reconstruction algorithm19 for better quantitative assessment of dynamic absorption contrast and calcium concentration changes. This reconstruction procedure is based on numerically solving an inversion problem corresponding to the minimization of the least square difference between the measured signals and those predicted by a discretized version of the forward optoacoustic model. Model-based reconstruction further enables accounting for the effect of the finite aperture of individual detection elements20, which leads to contrast enhancement and better accuracy of the reconstructed images. A uniform non-attenuating acoustic medium was assumed for the image reconstruction since acoustic attenuation in agar is insignificant while its acoustic impedance is further very similar to that of water and soft tissues. The value of the speed of sound used for the reconstruction was determined during calibration experiments with microspheres and further fine-tuned for best image appearance of the zebrafish specimens.Real-time volumetric optoacoustic image rendering and pre-view during the experiment was realized with a GPU-accelerated implementation of standard filtered back-projection reconstruction12.Systematic comparison of optoacoustic and fluorescence contrasts was enabled by integrating the optoacoustic tomographic system with a rapid high-resolution sCMOS camera , synchronized with the pulsed-laser source. The illumination was altered for this purpose and directed at the sample laterally to avoid blinding the camera. As a result, large optically opaque samples (like the adult brain) were not always uniformly illuminated. The fluorescence emission was picked up by the camera using a longpass filter (cutoff wavelength 500 nm). In order to avoid damage to the camera, simultaneous optoacoustic and fluorescent imaging was performed with a single-excitation wavelength of 488 nm. The interrogation laser allows for fast wavelength tuning on a per-pulse basis, thus the system also effectively enables fast acquisition of five-dimensional tomographic data sets, namely, time-resolved volumetric data at multiple wavelengths. The multispectral imaging frame rates are then accordingly reduced by the number of acquired wavelengths. Yet, our imaging rates in all experiments were significantly higher than the characteristic response times of the calcium indicators21. The fertilized eggs from a HuC:GCaMP5G, originally generated by the laboratory of Michael B. Orger and obtained as a kind gift from the laboratory of Hernán López-Schier, were raised in embryo medium at 28 °C under standard light/dark cycle. All procedures involving animals and their care were conducted in conformity with the institutional guidelines and with approval from the Government of Upper Bavaria.Zebrafish maintenance and breeding were conducted under standard conditions3) chamber, placed at the focal area of the optoacoustic matrix array detector and filled with fish water. The animals were given 30–60 s to acclimate before starting the stimulation. During the experiments with immobilized fish, six-day-old HuC:GCaMP5G larvae were placed in fish water containing 0.5% of muscle relaxant  briefly before they were embedded in 0.5% low-melting-temperature agarose and positioned into the FOV of the optoacoustic detector. To elicit calcium transients, 50 mM Pentylenetetrazole (PTZ)22 was applied via a syringe pump , directly into the surrounding of the larvae containing ~200 μL of fish water.For the experiments with freely swimming larvae, the latter was released into a small  were killed. Their brains were isolated following a standard procedureThe resolution and FOV of the systems were characterized by imaging absorbing polyethylene microspheres . The particles were embedded in an agar matrix mimicking optical scattering in tissues. The resolution was estimated from the full width at half maximum (FWHM) of the reconstructed absorption profiles for the microspheres located approximately in the center of the detection array . In ordeTo explore the fundamental spectral signature of our experimental system, 5-day-old HuC:GCaMP5G zebrafish larvae were imaged using two distinct wavelengths: 488 nm peak absorption wavelength of GCaMP5G and 530 nm where GCaMP5G absorption is negligible . While tn=5). For this purpose, the larvae were exposed to a neuroactivating agent (PTZ) known to induce fast ictal-like spikes in the nervous system that alter their swimming behavior22, most likely by interfering with GABAergic signaling25. Exposure to PTZ caused robust calcium waves propagating from the posterior (site of drug injection) to the anterior spinal cord region (R2=0.98) and in the medial (R2=0.97) regions, providing confirmation that GCaMP5G calcium sensing can be read out via optoacoustics . Here we were able to perform high-speed volumetric imaging of larvae that were allowed to swim freely in an ~0.5-cm2-area chamber; following exposure to the neurostimulant, rapid movements followed by long-resting periods were observed. The optoacoustically recorded calcium signal dynamics appear to have characteristic timescales between 0.1 and 0.3 s during this behavior and revealed an increase of up to ΔOA/OA0=1.8 in the GCaMP5 signals in the spinal cord just before the animal moves  having approximate dimensions of 2 × 3 × 4 mm, which were exposed to the neurostimulant. Intense light scattering in those large brains makes them inaccessible by state-of-the-art two-photon or light-sheet/field microscopy methods, which are also currently incapable of capturing such large volumes in real time. Here FONT was found to provide time-resolved 3D reconstructions across these highly scattering brains (3 volumes). According to the optoacoustic signal traces in 0=8.5, whereas voxels close to the surface actually showed a slight decrease in activity (blue VOI). However, as compared with true 3D information provided by the tomographic optoacoustic reconstructions, whose spatial resolution is not affected by the intense light scattering, the planar fluorescence lacks optical sectioning and thus may result in wrong conclusions based on smeared subsurface information averaged over large volumes28. Only when activity appears mostly limited to superficial regions of the brain where light scattering is less significant, a relatively high correlation between the optoacoustic and epifluorescence readings was observed  slices of the time-resolved 3D optoacoustic whole-brain data (n=6 optoacoustic image slices originating from two isolated brain preparations) had onset time constants ranging from 120 to 260 ms with resolvable image features in the range of 90–135 μm . Moreover, this type of performance would necessitate implementation of high-frequency ultrasound arrays with extreme density of elements that might not have sufficient detection sensitivity. Instead, matrix array probes with different spatial resolution and FOV could be used to image different brain sizes. For example, whereas FONT2 and FONT3 are suitable for imaging the zebrafish nervous system, the lower-resolution FONT1 system with FOV >1 cm3 can readily be used for real-time visualization of an entire brain of a live intact mouse using near-infrared wavelengths.Another issue that has to be carefully considered when evaluating the performance of new imaging modalities, such as our method, is scalability. For example, a cellular-scale spatial resolution in all three dimensions would be currently unrealistic to anticipate when imaging an entire mouse brain. This is because the number of voxels to be reconstructed in real time would be tremendously high in this case (~1016. As a result, the currently existing calcium indicators are inadequate for deep-tissue optoacoustic imaging of the mouse brain because of the strong background absorption by blood and insufficient light penetration in the visible range. In addition, the vast majority of calcium indicators are optimized for fluorescence imaging, mainly exhibiting changes in their fluorescent quantum yield in response to calcium influx36 rather than their absorption cross-section, also making those indicators less practical for generating measurable optoacoustic signal variations. High-resolution imaging of the whole mouse brain is further challenged by acoustic distortions and attenuation of high-frequency ultrasound waves by the skull37. Nevertheless, our recent demonstration of in vivo imaging of cells labeled with near-infrared fluorescent proteins in deep mouse brain using multispectral optoacoustic tomography38 clearly suggests that development of analogous calcium-sensitive indicators in the near-infrared is expected to facilitate translation of the FONT technology into mammalian brains.When considering imaging of neural activity in the mouse brain, one crucial aspect is the strongly limited penetration of light into highly vascularized mammalian tissues in the visible range. Light at the GCaMP excitation wavelength is strongly absorbed by blood, generating a strong optoacoustic signal background13 and red-shifted probes like RGECO39, or future sensors optimized for in vivo deep-tissue optoacoustic detection providing high extinction coefficient changes in the near-infrared window as well as lower quantum yield. Furthermore, the ability of FONT to simultaneously track movements and neural activation in three dimensions from living unrestrained organisms could form a basis for behavioral studies not currently possible with other optical neuroimaging techniques owing to their limited fields of view. Whereas the current spatial resolution still does not allow to distinguish individual cells, future generations of the described tomographic approach could potentially utilize higher-frequency transducer technology and/or super-resolution strategies for fast functional observations at the cellular scale. However, FONT's high temporal resolution is important even without the ability to resolve individual cells, as neural population calcium dynamics imaged with low-resolution systems still clearly convey very rapid timescale information41 (see also 43. This may provide highly complementary information to the functional calcium imaging and thus enhance the amount of spectrally- and time-resolved volumetric information available for the five-dimensional optoacoustic studies looking at coupling between the vascular changes and nervous system in more complex animal models. By virtue of combining the contrast abilities of both microscopic and macroscopic functional neuroimaging methods with its unprecedented spatiotemporal resolution performance, FONT fills an important performance gap in the current neuroimaging technology and opens new prospects for large-scale observations of neural networks.In summary, we demonstrated a novel optoacoustic imaging platform for direct imaging of spatiotemporal neural activity across entire light-scattering brains while maintaining similar values of spatial resolution at highly scalable depths. Our study is also, to the best of our knowledge, the first to examine the optoacoustic signature of modern GECIs, showing that the strong changes in GCaMP5G fluorescence are directly related to their optoacoustic signature. As FONT uses a widely tunable nanosecond optical parametric oscillator laser technology, it can be conveniently tuned to work with a large array of other functional probes, including, for instance, the newer generation of GCaMP6 probessee also . In addiDR, SS and GGW proposed and designed the project. DR and XLDB designed the optoacoustic imaging system. AL and GGW provided the animal models. XLDB, GS, AL and MK carried out the experiments. GS and XLDB implemented image reconstruction and processing algorithms and analyzed the data. DR, SS, GGW and VN supervised the study. All authors discussed the results and contributed to writing the manuscript.


Diet composition impacts metabolic and cardiovascular health with high caloric diets contributing to obesity related disorders. Dietary interventions such as caloric restriction exert beneficial effects in the cardiovascular system, but alteration of which specific nutrient is responsible is less clear. This study investigates the effects of a low protein diet (LPD) on morphology, tissue composition and function of the neonatal and adult mouse heart. Mice were subjected to LPD (8.8% protein) or standard protein diet  throughout intrauterine and postnatal life. At birth LPD female but not male offspring exhibit reduced body weight whereas heart weight was unchanged in both sexes. Cardiomyocyte cross sectional area was increased in newborn LPD females compared to SPD, whereas proliferation, cellular tissue composition and vascularization were unaffected. Adult female mice on LPD exhibit reduced body weight but normal heart weight compared to SPD controls. Echocardiography revealed normal left ventricular contractility in LPD animals. Histology showed reduced interstitial fibrosis, lower cardiomyocyte volume and elevated numbers of cardiomyocyte and non-myocyte nuclei per tissue area in adult LPD versus SPD myocardium. Furthermore, capillary density was increased in LPD hearts. In conclusion, pre- and postnatal dietary protein restriction in mice causes a potentially beneficial myocardial remodeling. One of the most obvious examples is obesity induced by high caloric diets , which causes metabolic syndrome and is associated with most of the diseases listed above2. Furthermore, a high fat diet (HFD) has been shown to execute negative effects in animal models by increasing body weight and fat deposition, increasing blood glucose and lipid levels, causing insulin resistance and elevating blood pressure4. In the rodent heart, HFD causes variable outcomes likely caused by differences in diet composition, onset and duration as well as species, gender or genetic background3. Nevertheless, HFD appears to promote cardiac hypertrophy resulting in impaired contractility under baseline conditions6 and increase myocardial susceptibility to ischemia8.Diet composition has a major impact on metabolic and cardiovascular health in humans and influences the pathogenesis of various chronic diseases including diabetes mellitus, hypertension and coronary heart disease10 and extend lifespan in various model organisms11. In rodents, for example, CR prevents hypertension and cardiac hypertrophy in spontaneously hypertensive rats12, improves heart function after ischemia/reperfusion injury or myocardial infarction14 and ameliorates ageing associated cardiac dysfunction16. Furthermore, intermittent fasting has been shown to exert cardioprotective effects in the rat heart18. Interestingly, a Mediterranean diet is associated with beneficial influences on human cardiovascular health and lifespan19. Although a Mediterranean diet might contain less calories compared to most western diets, it has become increasingly clear that its composition  likely mediates the positive impact19.Caloric restriction (CR) has been shown to improve metabolic and cardiovascular health in humans21. In contrast, dietary protein restriction has been associated with longevity in various model organisms as well as humans by reducing overall mortality and cancer incidence24. It even appears that specific amino acids, such as branched-chain amino acids or methionine, might be primarily responsible for the positive outcome of dietary protein restriction23.In order to identify specific diet components that mediate the beneficial effects of CR, carbohydrate and protein content has been modified. Interestingly, low carbohydrate/high protein diets appear to increase the risk of cardiovascular disease in humansAlthough beneficial influences of a low protein diet (LPD) on overall metabolic health and longevity have been proposed in animal models and humans, the specific effects on the heart have not been investigated. The aim of this study was to expose mice to dietary protein restriction both throughout intrauterine development as well as postnatal life and investigate the consequences for the heart at birth and in early adulthood. Our data revealed normal left ventricular function and various potentially beneficial alterations in cell composition and vascularization of the LV myocardium in adult mice constantly on LPD.We subjected female mice to LPD (8.8% protein compared to 22% in the standard protein diet (SPD)) for ≥2 weeks prior to mating and throughout pregnancy. Mice could potentially compensate the effect of the altered diet composition by increasing food consumption, such that protein uptake would not be different compared to the SPD group. We measured food intake in pregnant and non-pregnant adult female mice but did not find a difference between groups . Importantly, litter size was comparable between both groups  to visualize cell membranes and measure cardiomyocyte cross sectional area (CSA) in the LV myocardium. Whereas no difference in cardiomyocyte CSA was observed between SPD and LPD males, CSA was significantly increased in LPD compared to SPD females at P1 Fig. . The latCellular tissue composition was determined in the left ventricular myocardium of newborn SPD and LPD hearts. Non-myocytes and cardiomyocytes were differentiated based on WGA staining of cell membranes in combination with DAPI staining of nuclei  gene26. Whereas the hemizygous inactivation of Hccs in the heart of male mice results in embryonic lethality, heterozygous Hccs knockout females (cHccs+/−) are born with reduced heart size due to a reduced number of cardiomyocytes27. Heart size normalizes during postnatal development until early adulthood mainly by compensatory hypertrophy of existing cardiomyocytes27. To test whether this process is dependent on dietary protein uptake, we included cHccs+/− female mice in this study and subjected them to pre- and postnatal dietary protein restriction.Given the sex specific alterations in body weight and cardiomyocyte size observed in LPD females but not males at birth, we subjected female mice born from LPD pregnancies to postnatal LPD until early adulthood (i.e. 11 weeks of age). Another rational to focus on females was the aim to investigate the effect of dietary protein restriction on postnatal compensatory growth of the heart after neonatal cardiac hypoplasia. Therefore, we used mice carrying a heart conditional knockout of the X-linked Holocytochrome c synthase  pathway is regulated by amino acid availability to induce translation and protein biosynthesis required for cell proliferation and cardiomyocyte hypertrophy33, a phenotype similar to the one seen in heart muscle in this study. The underlying proangiogenic mechanism was proposed to depend on an amino acid starvation response (AASR) involving phosphorylation of eIF2α by GCN2, increased translation of ATF4 and transcriptional induction of VEGF. In addition, activation of AMPK (AMP-regulated protein kinase) results in metabolic adaptations to support endothelial cell proliferation and migration33. We did not find evidence that a similar mechanism is active in adult hearts after long-term LPD treatment, as we did not detect differences in eIF2α phosphorylation  mediate some of the beneficial effects of dietary restriction on stress resistance and longevity36. The impact of diets on metabolic health is mostly determined at the systemic level  whereas changes at the organ or tissue level are less frequently investigated. Our present study suggests that dietary protein restriction positively affects myocardial tissue composition, as mice constantly on LPD had smaller cardiomyocytes, reduced interstitial fibrosis and increased capillary density in the left ventricle compared to SPD controls. Whether the observed changes are influenced by beneficial effects of LPD on body weight, blood pressure or circulating metabolites is unclear at this stage. Also, future studies are warranted to longitudinally follow up hearts constantly on LPD upon ageing and investigate their response to challenges like ischemia or pressure overload. Such analyses will address the possibility that certain myocardial changes upon long-term protein restriction observed in our study might increase stress tolerance of the adult heart thereby promoting cardiovascular health.It is well-established that diet impacts on human health and disease. The most striking examples are high caloric diets contributing to the pathogenesis of diseases associated with metabolic syndrome, such as type 2 diabetes, hypertension and coronary heart disease38. In contrast, caloric restriction accelerates revascularization after hindlimb ischemia in part by increasing capillary density39 and exerts proangiogenic functions in cerebral endothelial cells40. Importantly, caloric restriction ameliorates myocardial fibrosis in postischemic heart failure and upon ageing as well as age associated cardiomyocyte hypertrophy16. Similarly, intermittent fasting has positive effects on tissue remodeling after myocardial infarction in rats by attenuating cardiomyocyte hypertrophy, reducing fibrosis and increasing capillary density18. The impact of dietary protein restriction on myocardial tissue composition is less clear. Maternal LPD during pregnancy is often used in animal studies to induce intrauterine growth restriction and investigate the consequences for disease susceptibility in adulthood25. Typically, mothers and pups are switched to standard chow shortly after birth , such that LPD conditions are effective during pre- and perinatal life. Such diet regime results in maladaptive changes in the adult offspring heart, a pathological gene expression program and increased collagen deposition42. Dietary protein restriction initiated after birth and maintained throughout the lactation period or LPD started after weaning also negatively affect cardiomyocyte contractility and morphometry as well as myocardial fibrosis44. Our data show that constant pre- and postnatal LPD reduces cardiomyocyte size and myocardial fibrosis but increases capillary density in the adult heart compared to mice on SPD. Interestingly, LPD restricted to the prenatal period also increases capillary density in the adult heart41. A recent study has identified a proangiogenic function of dietary amino acid restriction when initiated in young adult mice resulting in increased capillary density in skeletal muscle33. In contrast, prenatal LPD has been proposed to reduce capillary density in adult skeletal muscle45. Therefore, although dietary protein restriction could exert certain positive effects on myocardial tissue composition the timing of initiation might be crucial to determine beneficial versus maladaptive changes in the adult heart.Dietary interventions have previously been reported to alter tissue composition in animal models. For example, a high fat diet decreases capillary density in heart and skeletal muscle and increases cardiomyocyte diameter46. Calculations based on heart weight and cardiomyocyte volume show a trend towards increased cardiomyocyte numbers in LPD compared to SPD hearts, although this difference misses statistical significance. Nevertheless, given that this approach is based on various simplistic assumptions, future studies are warranted to precisely determine cardiomyocyte numbers in the adult LPD heart using more sensitive methodologies, such as stereology or FACS sorting46. The latter would furthermore allow to more precisely quantify non-myocyte cell populations. Interestingly, cardiomyocyte cross sectional area is increased in LPD female hearts at birth but heart weight is not different compared to SPD controls. This might indicate compensatory hypertrophy to account for a reduced cardiomyocyte number. Prenatal LPD in rats has been reported to cause a cardiomyocyte deficit at birth47 which is normalized until weaning48. In agreement, others have reported reduced proliferation in the neonatal rat heart upon LPD followed by a boost around day 749. Our data raise the interesting hypothesis that combined pre- and postnatal dietary protein restriction in female mice reduces cardiomyocyte number at birth but through compensatory proliferation in the postnatal period eventually increases cardiomyocyte number in the adult heart, although this will have to be thoroughly tested in future studies.Adult female mice after pre- and postnatal LPD exhibit reduced cardiomyocyte size but normal heart weight compared to mice constantly on SPD. Such counterintuitive findings could be explained by an increase in cardiomyocyte and/or non-myocyte numbers or increased extracellular matrix (ECM) deposition. The latter can be excluded given that LPD hearts exhibit less myocardial fibrosis compared to SPD controls. In contrast, the number of cardiomyocyte nuclei per tissue area was significantly increased in adult LPD myocardium, although this could represent differences in nuclearity rather than cell number50. This is proposed to be caused by alterations of cellular metabolism, gene and protein expression and epigenetics induced in the fetal organism upon unfavorable growth conditions and persisting after birth50. According to the thrifty phenotype hypothesis, fetal programming primes the unborn organisms to live in a postnatal environment in which nutrients and resources are likely to be short. In today’s western world, however, a mismatch exists between poor intrauterine growth conditions and a postnatal environment of plenty51. It is proposed that after IUGR accelerated postnatal growth during infancy, overfeeding and an unhealthy lifestyle are the main drivers boosting maladaptive consequences of fetal programming52. That way, intrauterine adaptations initially intended to be beneficial for the growth restricted baby turn into a risk factor for various diseases in adulthood. Based on this logic, maintaining starvation conditions after birth should prevent fetal programming associated disease after IUGR. In support of such idea, studies in sheep have shown that a mismatch in pre- versus postnatal nutrient conditions induces alterations in blood pressure regulation, renal function and LV morphology that were not observed if nutrient conditions were the same before and after birth53. As described above, IUGR can be induced by maternal LPD during pregnancy in mice or rats25. Switching pups to standard chow after birth results in maladaptive changes in the adult heart, which also includes contractile dysfunction42. Interestingly, if LPD conditions are maintained for 2 weeks after birth no impairment of heart function was observed in adulthood54. Consistently, our data show that constant pre- and postnatal LPD conditions do not adversely affect the offspring´s heart in adulthood under baseline conditions, even though detrimental changes upon cardiac stress or ageing cannot be excluded. This supports the hypothesis that if pre- and postnatal nutrient availability and growth trajectories match, negative effects of IUGR on the adult heart can potentially be prevented or at least delayed. A limitation of our study in regard of such interpretation is that we did not include mice subjected to prenatal LPD but switched to SPD after birth to confirm maladaptive changes in the heart. Interestingly, we applied pre- and postnatal LPD conditions to cHccs+/− mice, which are born with smaller hearts due to a reduced cardiomyocyte number and require postnatal compensatory hypertrophy to normalize heart size until adulthood27. Heart size and function were normal in adult cHccs+/− mice on LPD compared to SPD, indicating that the increased demand for postnatal cardiac growth is not affected by dietary protein restriction and that LPD does not induce cardiac pathology in this mouse model.Our data might have implications for fetal or developmental programming, a phenomenon by which the intrauterine environment impacts postnatal health and disease. Intrauterine growth restriction (IUGR) resulting in low birth weight and small for gestational age babies increases susceptibility towards various chronic diseases in adulthood, including diabetes mellitus, hypertension and coronary artery disease25. This reflects the variety of outcomes reported when using the LPD model, which might depend on species (rats versus mice), diet regime , diet composition, time of analyses  and others25. Nevertheless, sex specific changes in response to IUGR have been reported previously and fetal programming is known to affect sexes differently50. We furthermore observed increased cardiomyocyte cross sectional area in LPD versus SPD females but not males at birth, which could potentially indicate a sex specific effect of LPD on cardiomyocyte growth and proliferation. Interestingly, maternal hypoxia (but not nutrient restriction) during the second half of gestation in guinea pigs reduces the number of cardiomyocytes in the heart of adult female but not male offspring55.After prenatal LPD we observed a reduction in neonatal body weight in female but not male offspring compared to SPD but no changes in heart weight in either sex. This is in contrast to various other IUGR studies that showed reduced body and heart weight in both sexes at birth56, it has become clear that factors other than protein content could have confounding effects on the resulting data57. The reduction in protein has to be adjusted for by increasing other ingredients  to ensure isocaloric conditions. The diets used in this study for example show differences in sugar content  containing 8.8% crude protein  or a standard protein diet (SPD) containing 22% crude protein . A detailed diet composition is provided in Supplementary Table 58. In order to investigate the effect of dietary protein restriction on both normal as well as compensatory postnatal cardiac growth and function, a specific mouse strain which generates a heart conditional Hccs (Holocytochrome c synthase) knock-out (KO) was used for all experiments. Mice were kept on a mixed 129 Sv/C57BL6 genetic background. The characterization of pre- and postnatal development in heart conditional Hccs KO mice has been described previously27. Briefly, mice carrying a “floxed” (fl) Hccs allele were bred to mice expressing Cre recombinase under the control of the Nkx2.5 promoter. Experiments in adulthood that included heart conditional Hccs KO mice were performed on heterozygous Hccs KO females  and their respective female littermate controls . The latter were furthermore used for all other experiments not including cHccs+/− females, implying that all mice were carrying the “floxed” Hccs allele. Similarly, the genotype of neonates used in the study was Hccsfl/wt/Nkx2.5wt/wt for all females and Hccsfl/Y/Nkx2.5wt/wt for all males.Basic considerations for mice used in this study and sample sizes have been described beforeHccs+/+ females n = 9 from 5 different litters, SPD cHccs+/− females n = 9 from 6 different litters, LPD Hccs+/+ females n = 9 from 5 different litters, LPD cHccs+/− females n = 13 from 7 different litters.The total number of mice included in the different experiments is as follows: neonates on postnatal day 1: SPD males n = 9 from 5 different litters, SPD females n = 10 from 6 different litters, LPD males n = 7 from 6 different litters, LPD females n = 8 from 6 different litters; adults: SPD 59. Thus, data was averaged for mice from the same litter if applicable, such that n represents the number of litters included. In case this results in small sample sizes (n ≤ 3), data represent individual mice. Whether n indicates litters or mice is stated in each figure legend.For animal studies of developmental programming it is recommended to use only one representative male and/or female per litter in order to compensate for differences in intrauterine or postnatal conditions specific for a particular pregnancy or dam. If more than one mouse per litter is used, this should be accounted for in the statistical analysesAll animal procedures were performed following institutional guidelines and had previously been approved by the responsible authorities  Berlin, approval number G 0027/10).To measure food intake of adult mice, 5 months old non-pregnant or pregnant females were kept in individual cages. A defined amount of food was weighed and provided to the mice. For non-pregnant mice the remaining food was weighed at the same time each day over the following 10 days and body weight was determined half way through the observation period (i.e. on day 5). For pregnant mice the defined amount of food was provided at 13.5 dpc (days post coitum) and the remaining food was weighed daily until 19.5 dpc. Body weight was determined at the end of pregnancy at 19.5 dpc. Absolute food intake was calculated from the daily decline in food weight and averaged over the observation period (given in gram food per day per mouse) and food intake was furthermore normalized to body weight.58. Adult mice (11 weeks old) were anesthetized by inhalation of a 2.5% isoflurane/oxygen mixture using the Vevo compact dual anesthesia system . Body temperature was kept constant at 37 °C using a heat lamp and a rectal temperature probe. Echocardiography was recorded using the Vevo 2100 high frequency ultrasound system  with a MicroScanTM transducer MS400 set to 30 MHz. Operators were blinded for mouse genotypes and treatment groups during echocardiographic recordings as well as data analyses.Echocardiography was performed as described58. Hearts were excised, rinsed in cold PBS and fixed in 4% paraformaldehyde/PBS (Sigma Aldrich) for 24–48 hours. The tissue was subsequently dehydrated through an increasing ethanol series, cleared in toluol and embedded in paraffin. 5 µm paraffin sections were stained with hematoxylin & eosin (Carl Roth) to assess overall cardiac morphology or with Sirius Red  to visualize myocardial fibrosis.Histology and immunofluorescence staining were performed as describedFor immunofluorescence staining paraffin sections were deparaffinized, rehydrated and heat mediated antigen retrieval was performed in sodium citrate buffer  for 20 minutes. After blocking in antibody solution containing 5% normal goat serum (Jackson ImmunoResearch) for 1 hour, sections were incubated over night with primary antibodies at 4 °C. Secondary antibody detection was performed at room temperature for 1 hour using Alexa Fluor 488 or 555 conjugated secondary antibodies . Nuclei were stained with DAPI or TO-PRO-3 (Life Technologies) and sections were mounted in ProLong Gold antifade reagent (Life Technologies).58. Light microscopy images of Sirius Red stained heart sections from 11 week old adult mice were taken using the Biozero BZ-8100 microscope . By applying 5x optical magnification, 6 random fields of the left ventricular (LV) myocardium, including free wall and interventricular septum (IVS), were imaged, thereby covering the entire LV tissue. Two non-adjacent cross-sections per heart were used. The percentage of interstitial fibrotic tissue was quantified using the color-threshold plugin of the ImageJ software (https://imagej.nih.gov/ij/), which measures the red stained area in relation to the total LV myocardial area. Values of all 12 images were averaged to give the mean fibrotic tissue content for each heart. Perivascular fibrosis was excluded and deleted from the images prior to analysis.Experiments for quantifying fibrosis have been described previouslyParaffin sections of postnatal day 1 and adult hearts were stained with an antibody against alpha smooth muscle actin  following standard procedures. For neonates 3 non-adjacent sections per heart and for adults 1 section per heart were included. Neonatal hearts were imaged at 40x and adult hearts at 20x magnification using a Zeiss Axio Scope A1 microscope. SMA positive coronary arteries within the entire myocardium of the LV free wall and interventricular septum were counted irrespective of their size and morphological appearance. The number of coronary arteries was related to the overall tissue area. For size measurements in the neonatal heart, only cross sectioned arteries with a circular profile were outlined along the outer margin of the SMA positive cell layer and the area occupied as well as the outer circumference were measured (using Zeiss ZEN blue software). Approximately 15 arteries per heart were included.Paraffin sections of neonatal and adult hearts were stained with Alexa Fluor 488 or 555 conjugated wheat germ agglutinin  and DAPI to visualize cell membranes and nuclei, respectively. Images of tissue areas with cross sectioned cardiomyocytes within the LV free wall and IVS were captured at 40x magnification using a Zeiss Axio Scope A1 microscope. In neonates 7–10 images and in adults 6–8 images per heart were analyzed. The total number of DAPI stained nuclei per image was automatically counted using the “find maxima” tool of the ImageJ software and manually corrected if necessary. The total tissue area was measured using ZEN blue software (Zeiss). Nuclei were assigned to the cardiomyocyte or non-myocyte cell population based on WGA staining: WGA staining directly adjacent to the nucleus with no visible cytoplasm was considered to indicate non-myocytes, whereas nuclei localized within large cells with a discernable cytoplasmic fringe were scored as cardiomyocytes . Secondary detection was performed by Tyramide signal amplification  according to the manufacturer’s instructions using a horseradish peroxidase (HRP)-linked secondary antibody . Nuclei and cell membranes were co-stained as described above.Fibroblast and myofibroblasts in the adult LV myocardium were detected using immunostaining for vimentin  and smooth muscle actin , respectively. The number of vimentin and SMA positive cells was manually counted on four confocal laser scanning microscopy (Leica SP5) images per heart and related to the total number of DAPI stained nuclei.To visualize endothelial cells in the myocardium, paraffin sections of postnatal day 1 and adult hearts were stained with Alexa Fluor 568 conjugated Isolectin B4 . Sections were co-stained with Alexa Fluor 488 conjugated wheat germ agglutinin  and DAPI to visualize cell membranes and nuclei, respectively. Images of tissue areas with cross-sectioned cardiomyocytes within the LV free wall and IVS were captured at 40x magnification using a Zeiss Axio Scope A1 microscope. In neonates 7–10 images and in adults 6–8 images per heart were analyzed using ZEN blue software (Zeiss) and ImageJ. The total number of nuclei as well as the number of cardiomyocyte and non-myocyte nuclei was determined for each image as described above. In adult hearts, the total number of cardiomyocyte profiles (with and without visible nuclei) was furthermore counted. Capillary profiles were manually counted and normalized to the underlying tissue area to indicate capillary density. In neonatal hearts the ratio of capillaries per cardiomyocyte nuclei and in adult hearts the ratio of capillaries per cardiomyocyte profiles and non-myocyte nuclei were calculated. Values were determined for each image and averaged per heart.58. Briefly, CSA was measured in the LV myocardium based on WGA staining whereas cardiomyocyte length was measured by visualizing intercalated discs based on N-cadherin immunostaining using rabbit polyclonal antibody sc-7939 (1:200) from Santa Cruz , length, volume and number per heart were determined as previously describedTo assess proliferation rates in neonatal hearts, immunofluorescence staining for phosphorylated histone H3 (p-HH3) was performed . Sections were co-stained with WGA and DAPI following standard procedures as described above. One longitudinal section per heart was imaged at 40x optical magnification using a Zeiss Axio Scope A1 fluorescence microscope resulting in approximately 10 random fields taken within the LV myocardium . Cells that exhibited co-localization of p-HH3 and DAPI were considered as cycling. The total number of DAPI stained nuclei as well as p-HH3 positive nuclei were manually quantified using the cell counter plugin of the ImageJ software. For each image the percentage of p-HH3 positive nuclei was calculated. Data from all images were averaged to give the mean proliferation rate for each heart.58. For isolation of cardiac protein extracts snap frozen tissue samples derived from apical LV myocardium were homogenized in RIPA buffer supplemented with protease  and phosphatase inhibitors  and incubated at 4 °C for 2 hours with gentle agitation. For samples on the same gel equal protein amounts were loaded (40 μg), separated using denaturing polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto nitrocellulose  or PVDF (Millipore) membranes. Membranes were blocked for 1 h in 5% non-fat dry milk (Carl Roth) in TBS-T and incubated with the following primary antibodies at 4 °C over night: phospho-S6 Ser235/236 , phospho-S6 Ser240/244 , total S6 , phospho-4E-BP1 Thr37/46 , phospho-4E-BP1 Ser65 , total 4E-BP1 , phospho-AMPKα Thr172 , total AMPKα , phospho-Akt Thr308 , phospho-Akt Ser473 , total Akt , phospho-p42/44 Thr202/Tyr204 , total p42/44 , phospho-p38 Thr180/Tyr182 , total p38 , phospho-eIF2α Ser51 , total eIF2α  ; collagen I , vimentin , osteopontin , fibronectin , VEGFA  ; smooth muscle actin , α-actinin  (both from Sigma Aldrich); troponin T . Antibodies against GAPDH , α-Tubulin  and Vinculin  (both from Sigma Aldrich) were used for loading control. Secondary detection was performed using horseradish peroxidase (HRP)-linked secondary antibodies . Enhanced chemiluminescence (ECL) reaction was performed and detected with the imaging system Odyssey Fc (LI-COR Biosciences) or ChemiDoc XRS + (BIO-RAD). Intensity of detected protein bands was quantified by densitometry using ImageJ software.Western blot analyses were performed as described previously58. Snap frozen tissue samples derived from apical LV myocardium were homogenized in TRIzol reagent (Invitrogen) and total RNA was isolated according to the manufacturer´s instructions. RNA was subsequently purified using RNeasy spin columns (Qiagen), including digestion of genomic DNA on the column . Isolated cardiac RNA was reversely transcribed using M-MuLV reverse transcriptase (New England BioLabs) and random hexamer primers. Quantitative real time PCR was performed using the Power SYBR® Green PCR Master Mix (Applied Biosystems) on the ViiA™ 7 real-time PCR system (Applied Biosystems). Primers were obtained from BioTeZ  and sequences are provided in Supplementary Table RNA isolation and qRT-PCR experiments have been described previously58. All data were analyzed with SPSS (IBM) or Excel 2010 (Microsoft) software and are presented as mean ± standard error of the mean (SEM). Data sets were tested for normal distribution by Kolmogorov-Smirnov test and homogeneity of variances between groups was assessed by Levene’s test. If these criteria were met, differences between two groups were evaluated with unpaired, two-sided student’s t-test and those among multiple groups with one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test. Differences between multiple groups with unequal variances were evaluated with non-parametric Kruskal-Wallis one-way analysis of variance followed by Mann-Whitney post-hoc test. Neonatal data involving males and females were analyzed by two-way ANOVA with the factors diet, sex and their interaction followed by Bonferroni post-hoc test for pairwise comparison. A probability (P) value less than 0.05 was considered to indicate statistical significance .Basic considerations for statistical analyses have been described previouslySupplementary Information