Agrobacterium tumefaciens is a specialized plant pathogen that causes crown gall disease and is commonly used for Agrobacterium-mediated transformation. As a pathogen, Agrobacterium triggers plant immunity, which affects transformation. However, the signaling components and pathways in plant immunity to Agrobacterium remain elusive. We demonstrate that two Arabidopsis mitogen-activated protein kinase kinases (MAPKKs) MKK4/MKK5 and their downstream mitogen-activated protein kinases (MAPKs) MPK3/MPK6 play major roles in both Agrobacterium-triggered immunity and Agrobacterium-mediated transformation. Agrobacteria induce MPK3/MPK6 activity and the expression of plant defense response genes at a very early stage. This process is dependent on the MKK4/MKK5 function. The loss of the function of MKK4 and MKK5 or their downstream MPK3 and MPK6 abolishes plant immunity to agrobacteria and increases transformation frequency, whereas the activation of MKK4 and MKK5 enhances plant immunity and represses transformation. Global transcriptome analysis indicates that agrobacteria induce various plant defense pathways, including reactive oxygen species (ROS) production, ethylene (ET), and salicylic acid- (SA-) mediated defense responses, and that MKK4/MKK5 is essential for the induction of these pathways. The activation of MKK4 and MKK5 promotes ROS production and cell death during agrobacteria infection. Based on these results, we propose that the MKK4/5-MPK3/6 cascade is an essential signaling pathway regulating Agrobacterium-mediated transformation through the modulation of Agrobacterium-triggered plant immunity. Agrobacterium tumefaciens is a plant pathogen that causes crown gall disease in a wide range of species , which is perceived by the plant receptor EFR    cascades play essential roles in the processes of plant growth and development as well as in responses to environmental stimuli such as cold, heat, drought, and especially pathogen attack , the level of 1-amino-cyclopropane-1-carboxylic acid (ACC), an ET precursor, is elevated in the presence of both virulent and disarmed Agrobacterium strains  and ethylene (ET) function as essential signaling molecules in plant defense to Arabidopsis MKK4 and MKK5, as well as their downstream kinases MPK3 and MPK6, play major roles in Agrobacterium-triggered immunity and transformation. They are involved in plant immunity to Agrobacterium through the regulation of plant defense pathways, including defense-responsive gene expression, reactive oxygen species (ROS) production, and the synthesis of ET and SA. Together, these data indicate that the MKK4/5-MPK3/6 cascade is an essential signaling pathway contributing to plant immunity to Agrobacterium and regulating Agrobacterium-mediated transformation through the modulation of Agrobacterium-triggered immunity in Arabidopsis. Based on these findings, we propose that the MKK4/5-MPK3/6 module can be modified in the future to promote the application of Agrobacterium-mediated transformation technology in other species.Here, we report that Arabidopsis thaliana Col-0 ecotype was used in this study. Arabidopsis plants were grown in a growth chamber at 22°C, with 10 h-day/14 h-night photoperiod and 100 μmol m−2 s−1 light intensity. Mutants mpk3-1 , mpk6-2 , and acs2/6/4/5/9 (CS16644) were obtained from the Arabidopsis Biological Resource Center (ABRC). MPK6SR (mpk3 mpk6 PMPK6:MPK6YG), MKK4-DD, NahG, and mkk4/mkk5 double mutants were kindly provided by Dr. Suqun Zhang . To generate MKK5-DD and MEK2-DD transgenic lines, Arabidopsis MKK5 and tobacco NtMEK2 complementary DNAs (cDNAs) were cloned as described in previous studies  liquid medium for 10 days. The resulting 10-day-old seedlings were mock treated (1/2 MS liquid medium only) or treated with 2 × 108 cfu/ml GV3101 agrobacteria cells suspended in 1/2 MS liquid medium without shaking. Samples were harvested at four time points  with agrobacteria). Three replicates were performed for each treatment. The seedlings were harvested at each time point and immediately frozen in liquid nitrogen; within each replicates, all seedlings were pooled together for RNA extraction and quantitative real-time PCR (qRT-PCR) analysis.For gene expression analysis, Arabidopsis seedlings using the Trizol reagent . First-strand cDNA was synthesized from the extracted messenger RNA (mRNA) using oligo dT primer and reverse transcriptase . qRT-PCR was performed using a Bio-Rad CFX96 Real-Time System and iQ™ SYBR® Green Supermix  with 40 cycles. Templates were normalized with Arabidopsis UBQ10. Three biological and technical replicates were performed. Data were analyzed using the BioRad CFX Manager 2.0 Software. The comparative cycle threshold method (ΔΔCt) was used to obtain the relative fold change. Primer information is provided in Total RNA was extracted from infected Arabidopsis seedlings of Col-0 and mkk4/5 were mock treated or treated with agrobacteria for 24 h. The seedlings were collected individually and frozen immediately in liquid nitrogen for RNA-seq. Three biological replicates were performed. Total RNA was used to make RNA-seq libraries with the Illumina TruSeq RNA Sample Preparation Kit v2  following the recommendations of the manufacturer. These libraries were sequenced by Beijing Genomics Institute .For RNA-sequence (RNA-seq) analysis, 10-day-old A. thaliana TAIR10 genome by hisat2 (version 2.1.0) with default parameters. However, the above mapped SAM files were sorted and indexed by SAMtools (version 1.5). The expression data were calculated using featureCounts (v1.6.1) in default setting , seedlings were harvested for histochemical β-glucuronidase (GUS) staining and activity testing.To evaluate transformation frequency in whole A. tumefaciens tumorigenic strain A208 was used for stable transformation assays. Roots from 14-day-old Arabidopsis plants grown in sterile square Petri dishes containing sterile Gamborg's B5 medium were cut into ~5-mm segments. Root segments were assayed as described by Tenea et al.  solution at 37°C overnight and then destained with 70% ethanol for observation. β-galactosidase activity was assessed with a fluorescent substrate (4-methylumbelliferyl-β-D-glucuronide (MUG assay)) to quantitatively determine GUS activity. These assays were performed according to the method of Jefferson et al. . All of 3VO4, 10 mM NaF, 10% glycerol, and 1 × protease inhibitor cocktail ]. The concentration of each protein extract was determined using the Bio-Rad protein assay kit  with bovine serum albumin as the standard.Seedlings cultured for 10 days in liquid 1/2 MS medium were used for protein extraction. These 10-day-old seedlings were mock treated (1/2 MS liquid medium only) or treated with agrobacteria cells suspended in 1/2 MS liquid medium. Samples were harvested at four time points . Total protein was extracted from the whole seedlings in an extraction buffer [50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 10 mM EDTA, 1 mM NaFor the immunoblot analysis, anti-FLAG  and anti-pTEpY  antibodies were used to detect MKK4-DD, MKK5-DD, and NtMEK2-DD protein expression and MPK3/MPK6 activation, respectively.Arabidopsis with Agrobacterium GV3101 carrying pBISN1 (GV3101-pBISN1) Arabidopsis was performed as described previously  were injected into 4-week-old plant leaves. Then, the leaves were used for H2O2 detection and cell death analysis.The leaf infiltration of 2O2 was detected by the 3,3′-diaminobezidine (DAB) uptake method, which is an endogenous peroxidase-dependent in situ histochemical staining procedure using DAB  or mock infected (1/2 MS liquid medium only). Samples were harvested at 24 h after inoculation and frozen in liquid nitrogen. Approximately 100 mg of each sample was suspended in 90% (V/V) methanol at a ratio of 1:1000. Samples were vortexed, sonicated for 15 min, and centrifuged at 15,000 × g for 20 min two times. The supernatant was transferred to a tube, and the SA solution was filtered and separated on a C18 analytical column  using high performance liquid chromatography (HPLC) and detected using fluorescence . The mobile phase used for HPLC was 90% methanol and 0.1% formic acid with a flow rate of 0.8 ml/min.Ten-day-old t-test.All data were analyzed using Student's Agrobacterium, currently we used Arabidopsis model to profile MPK3/MPK6 activation and defense-responsive gene expression during the Agrobacterium infection process. In this system, 10-day-old Arabidopsis seedlings of wild-type (WT) (Col-0) cultured in liquid medium were infected with the disarmed hyper-virulent Agrobacterium strain GV3101. Samples were harvested at various postinfection time points. Then, MPK3/MPK6 activity and defense-responsive gene expression were assessed. The results showed that, compared with mock treatment, agrobacteria infection triggered a strong and rapid activation of MPK3/MPK6 , but as infection time progressed, this induction faded somewhat  and the induction of defense-responsive gene expression are the early steps in the immune response after plants sense an invading pathogen  and mkk4/5 with the agrobacteria strain GV3101, samples were collected at various time points. MPK3/MPK6 activity and defense-responsive gene expression were measured. The results indicated that MPK3/MPK6 activity was induced and reached a high level at 15 min after the inoculation with agrobacteria in WT Col-0. However, this activity was abolished in mkk4/mkk5 double mutants  and stable transformation (tumorigenesis induced by agrobacteria), to evaluate how the impaired immunity of this mutant affects the transformation. Firstly, we evaluated the transient transformation frequency in the whole seedling using the AGROBEST infection method . Then, GUS activity was assessed to monitor the transient expression efficiency at 3 dpi. Compared with WT plants, stronger and more concentrated spots of GUS staining were detected in mkk4/5 double-mutant seedlings at 3 dpi , MKK4-DD, and MKK5-DD transgenic plants were infected with GV3101-pBISN1. The GUS staining results showed that in DEX-treated samples, MKK4-DD and MKK5-DD plants exhibited a much lower transformation frequency at 3 dpi  seedlings than in NtMEK2-DD transgenic seedlings after DEX treatment (NtMEK2-DD transgenic seedlings compared with the WT (Col-0) after DEX induction  , with more tumors in the roots. NA-PP1 itself had no effect on tumorigenesis in WT (Col-0). These results indicate that MPK3 and MPK6 act downstream of MKK4 and MKK5 to regulate Agrobacterium-mediated transformation.We confirmed this result through the tumorigenesis analysis. As shown in Agrobacterium-mediated transformation process, the host plants exhibit dynamic gene expression patterns in response to Agrobacterium. Changes in host gene expression patterns throughout the transformation process have effects on transformation frequency. To clarify the mechanisms of MKK4/MKK5 in regulating the Agrobacterium-mediated transformation process, global transcriptome analysis was performed to compare WT and mkk4/5 double-mutant plants. After data analysis, genes were considered significantly differentially expressed if they had a false discovery rate of less than 0.05 and a change in expression between the two treatments of at least two-fold. Overall, 7,702 unique genes in WT (blue circle) and 7,810 (yellow circle) in mkk4/5 were differentially expressed in the agrobacteria treatment relative to the mock treatment  analysis to categorize the 3,905 agrobacteria-induced genes in WT to investigate wide-scale changes in gene expression related to specific cellular and biological processes. pression . Thus, mAgrobacterium-mediated ROS production and cell death, leaves from a 4-week-old seedling of WT (Col-0), MKK4-DD, and MKK5-DD were inoculated with agrobacteria GV3101 through leaf infiltration, and then the H2O2 production and cell death were assessed. As shown in 2O2 production, were visible in the leaves of MKK4-DD, MKK5-DD transgenic plants pretreated with DEX as an inducer. As controls, the leaves of Col-0 plants were pretreated with DEX and processed alongside inoculated leaves, and no H2O2 generation was observed in these control leaves. The leaves from Col-0, MKK4-DD, and MKK5-DD transgenic plants clearly exhibited no H2O2 generation in the absence of DEX  and cell death at a late infection stage (5 dpi), and that the activation of MKK4/MKK5 promotes agrobacteria-induced ROS production and cell death. Thus, ROS production and cell death are involved in plant immunity and regulated by MKK4/MKK5. This pathway may be one factor influencing Agrobacterium. To explore whether SA is involved in MKK4/MKK5-regulated Agrobacterium-mediated transformation, we first examined the expression of SA synthesis genes AtPBS3, AtICS1, AtEDS5 , but this induction effect was impaired in mkk4/5 double mutants. AtICS1 and AtEDS5 gene expressions were induced by agrobacteria inoculation in the WT, but the expression of AtICS1 was higher in the mkk4/5 mutant than in WT and AtEDS5 expression showed no difference between mkk4/5 double mutants and the WT (Col-0) mutant  mutant . We thenculation . Agrobac5 mutant , indicatmkk4/5 double mutants. The results showed that applying SA to WT (Col-0) and mkk4/5 double-mutant seedlings caused the transformation frequency of both the WT (Col-0) and mkk4/5 to decrease relative to plants without SA treatment, with ~1.6- and 1.8-fold decreases, respectively, in GUS activity , with a GUS activity increase of 2.9-fold compared with the WT (Col-0)  . Stable in roots . From thAgrobacterium has been found to regulate ET metabolism during the transformation process. Levels of the ET precursor ACC (1-amino-cyclopropane-1-carboxylate) were elevated during infection by agrobacteria  during agrobacteria infection. The results show that with agrobacteria inoculation, the ACC biosynthesis genes ACS2, ACS4, and ACS6 were strongly induced compared with the mock treatment. These genes expression levels peaked at 6 hpi and decreased with further time after inoculation  mutant or mkk4/5 double mutants at 3 hpi  (acs2/6/4/5/9. This mutant exhibited very low ET production compared with the WT (Col-0)   , indicat (Col-0) . Tumorig than WT . These rAgrobacterium tumefaciens has been widely used in plant transformation for decades, and the biological process of Agrobacterium-mediated transformation has been extensively studied  and the mpk6 single mutant exhibited a slight increase in the transformation frequency  BioProject database under accession number GSE179628.KD, TL, and LC designed the research. TL, LC, and JJ performed most of the research all authors contributed to material preparation and data analysis. KD wrote the paper. All authors read and approved the article.This research was supported by grants from the National Natural Science Foundation of China (31901957).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Crohn’s disease (CD) is a chronic disorder characterized by full thickness patchy inflammation of the gastrointestinal tract. The pathogenesis is multifactorial and involves defective innate immune responses, microbiome alterations, and dysregulated activation of the acquired component of mucosal immunity. One of the molecular mediators that is involved at different levels in the initiation and progression of intestinal inflammation characteristic of CD is tumor necrosis factor (TNF). The present manuscript provides a comprehensive review focused on the potential role of TNF in the different phases of CD pathogenesis, particularly in light of its potential clinical implications. Currently available drugs blocking TNF are evaluated and discussed, specifically for open issues that still remain utilizing such therapy. TNF exerts a paramount role in the established phase of intestinal inflammation that characterizes CD patients, and anti-TNF biologics have definitely changed patient management, offering effective and safe options of treatment. Nonetheless, many patients still do not respond to anti-TNF therapy or experience unwanted side-effects. This could partially be due to the role that TNF plays in intestinal homeostasis that is particularly important during the early phase of the inflammatory process. In fact, emerging evidence supporting the dichotomous role of TNF and the identification of molecular markers will guide a more tailored and refined therapy for CD patients in the near future. Crohn’s disease (CD) is a complex, chronic disorder of the intestine with still unknown etiology. It is one of the etiopathogenic forms of inflammatory bowel disease (IBD), a group of disorders wherein dysregulated mucosal immune responses causes a persistent inflammation of the gut with a clinical relapsing-recurrent pattern of disease . Unlike Among the so-called conventional therapies, corticosteroids are still the mainstay of treatment for acute inflammatory flares, and budesonide , charactIn general, the goal of a successful therapeutic approach is induction and maintenance of disease remission; however, the specific definition of “disease remission” consistently has changed throughout the years. The target for successful therapy has changed from simple ‘symptom control’ to the current identification of solid therapeutic endpoints such as clinical and biochemical normalization, mucosal and transmural healing patient reported outcomes (PROs), and quality of life. These are better indicators of complete short- and long-term disease control and are also related to a decreased rate of complications  ,12,13. DBiologic (or biotechnologic) drugs are large molecules  produced from biologic sources . They are characterized by a complex and specific production process, resulting in the generation of compounds with a certain range of structural variability and a defined biological effect . For incIn regard to targeted therapy, the knowledge of disease pathogenesis has expanded throughout the years thanks to intense research and progress in laboratory technology, even though substantial pitfalls persist . GeneticDisease pathogenesis of CD has been associated early on with prevalent Th1 immune responses, so that dysregulation of acquired immunity has been indicated as a prevalent mechanism for disease occurrence. Indeed, several experimental observations support a role for Th1 cells in disease pathogenesis, and CD patients show a high prevalence of Th1 cells in the gut lamina propria that produce large amounts of IFNγ ,21,22,23The increase in knowledge of CD pathogenesis has led to a clearer understanding of the complexity of the disease. Genetic predisposition, dysregulation of innate and adaptive immunity, microbiota alterations, and possible environmental factors all interplay, resulting in a number of clinical pictures that falls under the umbrella of Crohn’s disease. The simplified schematic view of CD in the past has been replaced by a modern vision in which multiple molecular pathways, redundancy, plasticity, and dynamicity concur to a different degree in the initiation, development, and maintenance of the condition. In line with this new outlook, different cell types and their mediators may have diverse (and even opposing) roles in the various disease stages and in different topographic compartments. Tumor necrosis factor (TNF) is a perfect example of a molecular mediator with multiple, and still not completely discovered, effects at the intestinal mucosa with potentially relevant roles in maintenance of homeostasis and inflammatory disease occurrence. Aim of the present review was to analyze the role of TNF in the onset and the maintenance of the inflammatory process characteristic of CD, and to describe the clinical application of the block of TNF, depicting the currently available drugs and the potential future implications.TNF is a homotrimer of 157 amino acid subunits, primarily produced in soluble or transmembrane form by activated macrophages, although other immune cells, such as dendritic cells, T cells, B cells, and mast cells—but also epithelial and endothelial cells and fibroblasts—can be induced to express TNF . TNF exeTNF was identified in 1975 and purified and cloned in 1985 ,52. InitAside from its proinflammatory function, consistent data exist regarding the role of TNF in mucosal homeostasis. TNF represents a fundamental cytokine of innate immunity that has an important role in maintaining the homeostatic balance between luminal contents  and the mucosal immune system, actively participating in intestinal barrier function. In an experimental model of intestinal inflammation, such as dextran sulfate sodium (DSS)-induced colitis, an incremental increase in colitis severity was observed in mice after monoclonal anti-TNF administration ,57. AddiTaken together, those findings suggest a definite distinct role of TNF in the early vs. late phase of the inflammatory process leading to Crohn’s disease . In the There are currently three anti-TNF drugs approved by the US Food and Drug Administration (FDA) for the treatment of CD patients: infliximab, adalimumab, and certolizumab pegol. Certolizumab pegol is not approved by European regulatory agency (European Medicine Agency—EMA) . Infliximab is a chimeric IgG1 monoclonal antibody against TNF, and it was the first anti-TNF drug approved by FDA for the treatment of CD , first demonstrating efficacy in 1997 . It can Adalimumab is a human IgG1 anti-TNF antibody that was approved for clinical use in CD patients by the FDA in 2002 and by the EMA in 2003. It has the ability to fix complement and lyse cell expressing TNF, and it is administered by subcutaneous injection every other week. Among many studies demonstrating efficacy and safety of that drug in CD patients, the CLASSIC I and II ,76 trialCertolizumab pegol is a PEGylated Fab’ fragment of a humanized monoclonal anti-TNF antibody with a high binding affinity for soluble and transmembrane TNF and less immunogenicity side effects due to the lack of the Fc portion. It does not induce apoptosis nor fix complement. It is administered subcutaneously and, having a longer half-life than adalimumab due to the PEG addition, has a maintenance dosing every month. The registrative trials PRECISE 1 , PRECISEData of the main clinical trials of the three anti-TNF agents currently used in CD patients are summarized in Other anti-TNF drugs did not show efficacy in RCTs in CD patients including: etanercept , a solubDespite the widespread use of anti-TNF agents since the last two decades, some open issues are still up for debate. According to clinical trials and real-life studies, about 20% of patients do not respond to anti-TNF ab initio (primary failure), and about 20–30% of patients are likely to lose response every year (secondary failure). Therefore, a consistent portion of patients would need a second-line therapy or a surgical approach. In order to increase efficacy of therapy and to ideally affect the natural course of the disease, “early treatment” has been proposed—by 2 years from the initial diagnosis—in selected CD patients with features of severe disease . In the SUTD (Step Up vs. Top Down) trial, an early immunosuppression plus infliximab administration was associated with a higher clinical remission rate at 2 years in 133 CD patients compared to patients with conventional management . In the The observation that anti-drug antibodies are associated with infusion reactions and loss of response—and that therapeutic efficacy is coupled with adequate blood drug level—has pushed toward the concept that the measurement of such parameters  would optimize anti-TNF drug utilization . TherapeDespite anti-TNF drugs are generally considered safe, adverse events are still possible and some pre-existing clinical condition may contraindicate therapy or require special attention. In fact, drug- , class- , and disease-specific  adverse events may occur, sometimes leading to discontinuation of therapy and/or hospitalization . TherefoTNF is one of the most extensively investigated molecular mediators in CD patients. The clinical application of such intense research is rendered evident with the development of the anti-TNF class of biologic drugs, which presently represent the main reference for therapy of moderate–severe disease not responding to conventional therapy. As the comprehension of the pathogenesis of the disease increases, novel potential mechanisms of action with a possible role in the onset and maintenance of the chronic mucosal inflammation are investigated, eventually leading to the proposal of novel drugs for the management of CD patients. Anti-TNF block do not represent the exclusive mechanism of action of biologic drugs in CD, but therapeutic approach targeting the leucocyte homing and the IL12-23 pathway are already available in the market, and more drugs with new mechanisms of action are ready to be proposed or are being investigated in clinical trials . In ordeBesides classical pro-inflammatory role in the onset and maintenance of chronic intestinal inflammation, more recent studies have highlighted a possible protective role of TNFα at mucosal level. This finding from one side may confirm the high complexity of the homeostatic mechanism of the intestinal mucosa, in which multiple pathways may reciprocally interact and contribute in turn either to the protection of the mucosa or to its alteration by unregulated inflammation. On the other hand, the perception of the multiple and even opposite effect that single molecular mediators may provoke opens the door to the implementation and the refining of the positioning of drugs interfering with that specific molecular pattern. Thus, both for clinical/economic reasons and for the growing comprehension of the multiple molecular function at mucosal level, discussing “potential for intervention” for TNF in CD is not anachronistic. In fact, as the basic and translational research progress, a new impulse and novel possible implications may arise even for a molecular mechanism that has already been investigated for a long time, such as TNF. To further confirm that, the National Institute of Health (NIH) database  presently has a total of 220 trials involving anti-TNF in CD patients registered, among which 173 are completed  and 47 are still recruiting patients .As we just entered a new decade, we can imagine a future scenario in which more novel drugs will become available for the treatment of CD patients and more biological drugs’ patents will expire, further expanding therapeutic medical options and therapy availability. Key points for the future developments of positioning and implementation of TNF-based therapeutic strategies would involve clinical and basic research. For clinical research, the design and implementation of fair, well-designed, head-to-head clinical trials will help clinicians to orientate among the increased therapeutic options available. To date, only the Varsity study, in UC patients, directly compared two different biologic drugs in the same set of patients , but morIn addition, a higher definition of the multiple molecular interactions in CD may lead to the design and development of pre-clinical and clinical studies including multiple biologics association. At present, few case reports are available for association therapy of biologic drugs in CD ,117,118,Despite consistent research and some advance in the comprehension of IBD, many aspects still remain insufficiently known—in particular, the disease pathogenesis. The more our knowledge seems to shed light in this obscure cave, the more novel questions arise, making more puzzling, and still fascinating, our path toward the truth. CD appears more and more as a complex condition where multiple different factors, reciprocally interacting, contribute to the variable clinical phenotype of disease that we face in our clinical daily practice. Still, some aspect of this complex puzzle has been clarified, leading to important discoveries in the therapeutic field. This is the case of biologic drugs that, interfering with specific molecular pathways of the inflammatory cascade, has dramatically expanded our therapeutic impact on the disease and profoundly changed the management approach to CD patients. Among those, the anti-TNF agents have been the first drugs discovered and remain the standard of therapy for moderate–severe disease patients who did not respond to the conventional therapy. The consistent efficacy of those drugs straightens the important role of the TNF for the disease maintenance in CD. Nonetheless, in line with the complexity of the disease, the same TNF has multiple complex molecular effects and potentially different roles in the initiation and maintenance of the mucosal chronic inflammation. The further progress of the research in IBD would hopefully help to better unravel the multiple complex role of TNF and of other molecular mediators. The desirable final result is to implement and optimize the current available drugs utilization as well as the future development of novel drugs and strategies.


Heart failure (HF) with preserved left ventricular ejection fraction (HFpEF) is becoming the predominant form of HF. However, medical therapy that improves cardiovascular outcome in HF patients with almost normal and normal systolic left ventricular function, but diastolic dysfunction is missing. The cause of this unmet need is incomplete understanding of HFpEF pathophysiology, the heterogeneity of the patient population, and poor matching of therapeutic mechanisms and primary pathophysiological processes. Recently, animal models improved understanding of the pathophysiological role of highly prevalent and often concomitantly presenting comorbidity in HFpEF patients. Evidence from these animal models provide first insight into cellular pathophysiology not considered so far in HFpEF disease, promising that improved understanding may provide new therapeutical targets. This review merges observation from animal models and human HFpEF disease with the intention to converge cardiomyocytes pathophysiological aspects and clinical knowledge. Heart failure (HF) afflicts an estimated 64 million people world-wide  and the The last decades have seen the successful evolution of medical treatment, which improved survival and decreased morbidity in HFeEF. But until today, understanding of HFpEF remains poor and treatment options that improve prognosis remain limited to HF patients with a LVEF of 50–60%, or even lower, as attested repeatedly in large randomized trials such as the TOPCAT study testing spironolactone, the PARAGON-HF trial comparing LCZ696 with enalapril, and the EMPEROR-Preserved study with empagliflozine in the test group ,11. The HFpEF usually does not trace back to a primary injury of the cardiomyocyte as is often the case with HFrEF . InsteadVery recently, major pathobiochemical aspects of HFpEF were comprehensively summarized . The goaHeart failure is a clinical syndrome that consists of cardinal symptoms  accompanied by typical clinical signs . Its origin lies with the structural and functional pathology of the heart, which results in increased cardiac filling pressure and inadequate cardiac output at rest and/or during exercise . This de2 undergoing coronary artery bypass grafting [Independent of this diagnostic dilemma, enormous progress has been made in the comprehension of HFpEF disease in recent years with translational studies that investigated the origin of diastolic dysfunction. A remarkable study in the human investigated left ventricular epicardial anterior wall biopsy specimens obtained from patients with LVEF ≥ 50%, normal wall motion, and end-diastolic volume index < 75 mL/mgrafting . Patientgrafting .passive), while PKC mediated phosphorylation at the PEVK domain increases stiffness [passive and thus stiffness [In fact, the giant cytoskeletal protein titin, which is the largest known protein , acts astiffness ,34. Thertiffness ,34. PKG tiffness ,35,36,37tiffness .PKG also functions as a brake on myocardial hypertrophy and lower PKG activity was furthermore shown to correlate with larger cardiomyocyte diameter , which iHomeostasis of cardiac contractile function requires refined regulation of sarcomeric protein phosphorylation such as of the troponin complex of the thin filaments, which are substrates for multiple kinases . In HFrEEarlier animal models investigated aspects of HFpEF disease, in particular LV hypertrophy and diastolic dysfunction either in the TAC mouse model  or in thIn humans, aging is associated with an increased prevalence of LV hypertrophy and diastolic dysfunction, even in the absence of arterial hypertension ,49. Old Systemic hypertension is the single most common comorbidity in HFpEF with a prevalence ranging from 60 to 96% in epidemiological studies, HF registries and large randomized controlled trials ,55. The 2) behave differently when compared to HFpEF patients with elevated systolic blood pressure and left ventricular hypertrophy. Their hemodynamic burden is less important, the systolic force-Ca2+ dependence is reduced by 50%, indicating depressed contractility, and passive stiffening is less important [However, neither the rodent aging models nor the hypertension models contain aspects of the metabolic comorbidity in HFpEF, in particular obesity and diabetes. In fact, large outcome trials and registries reveal that being overweight or obese is a major risk factor of HFpEF . There amportant ,64. The mportant  , animals show increase of inflammatory cytokines levels, whereas there is decrease at older age (28 weeks). Furthermore, LV hypertrophy develops only at older age along with enlarged cardiomyocytes, an increase of cardiac fibrosis and capillary rarefication. In addition, this mouse shows abnormal ventriculoarterial coupling due to decreased arterial compliance and increased LV stiffness , which rcp), the lean female Zucker diabetic fatty rat (+/fa) and the lean male spontaneously hypertensive HF rat (+/facp) derived from the obese SHR carrying the corpulent facp gene. These animals develop a cardiac phenotype compatible with HFpEF [Another useful model for understanding the HFpEF phenotype is the ZSF1 rat, which was developed by crossing rat strains with two different leptin mutations , with the SGLT2 protein playing a less prominent role. Empagliflozin’s pharmacological action mainly affected cardiomyocyte oxidative stress modulation, and greatly influenced cardiomyocyte stiffness, myocardial extracellular matrix remodeling, heart concentric hypertrophy, and systemic inflammation. Validation of these in silico data was performed in vivo in patients with HFpEF by measuring the declining plasma concentrations of NOS2, the NLPR3 inflammasome, and TGF-β1 during 12 months of empagliflozin treatment. Using AI modelling, the main effect of empagliflozin in HFpEF is exerted via NHE1 and is focused on cardiomyocyte oxidative stress modulation supporting the potential use of empagliflozin in HFpEF [With the large success of the SGLT2 inhibitor empagliflozin improving prognosis for patients suffering from HF, various pathophysiological concepts have been proposed mostly based on animal data . In the in HFpEF , in acco2+ entry via L-type calcium channels, which in turn stimulate release of Ca2+ ions from the sarcoplsmaic reticulum with subsequent myofilament activation and contraction with sarcomere shortening [2+-mediated myofilament interaction, the amount of cytosolic Ca2+ must decline which requires inactivation of L-type calcium channels and sequestration of cytosolic Ca2+ by the sarcoplasmic reticulum Ca−ATPase (SERCA2a) back into the sarcoplasmic reticulum. Relaxation is a key player of HFpEF pathophysiology, therefore, it is not surprising that modifications in proteins that mediate Ca2+ homeostasis have been reported. ZSF1-obese rats show higher cytosolic Ca2+ level resulting from a disbalanced interaction between phospholamban (PLB) and sarcoplasmic endoplasmic reticulum calcium pump (SERCA2a). This disbalance reduces the reuptake of Ca2+ ions via the SERCA2a back into the sarcoplasmic reticulum. In ZSF1-obese rats, the SERCA2a protein content was unchanged between groups when compared to the lean controls, while PLB expression was higher in the ZSF1 obese rat. Furthermore, hypophosphorylated PLB has also an inhibitory effect on SERCA2a activity, suggesting that disbalance of the PLB/SERCA2a expression ratio together with hypophosphorylation increase cytosolic Ca2+ levels in diastolic dysfunction. Since isolated hearts from rats with metabolic syndrome and diabetes have shown incapacity to upregulate ATP synthesis and decreased free energy of ATP hydrolysis (ΔG∼ATP) at higher workloads, exercise will worsen diastolic relaxation in addition [2+ ions via the central pore of the cardial L-type calcium channel [Notwithstanding of the role of hyperacetylization, the calcium metabolism may still play a role as suggested from a recent study investigating mitochondrial and cytosolic calcium handling in intact cardiomyocytes from ZSF1-obese animals ,84. Mitoortening . To termaddition ,87. Addiaddition . Coherenaddition , while r channel . Taken t channel  may expl2+ is not only associated with altered excitation-contraction coupling, but also with increased mitochondrial Ca2+. The latter is a regulator of NADH (complex 1) activity, which may explain the observation that mitochondrial respiration and oxidative phosphorylation are reduced in HFpEF [However, increased cytosolic Cain HFpEF ,92,93,94in HFpEF . In summin HFpEF  with thein HFpEF .Cardiac fibrosis is defined as a state in which excess deposition of collagen in the extracellular matrix occurs. In the resting state of a healthy heart, fibroblasts constantly modify the extracellular environment, while an overall disproportionately increased extracellular matrix volume is a hallmark of HFpEF . The impHowever, fibrosis is mild to moderate in the majority of HFpEF patients, while only 26% suffer from severe fibrosis , questioData from the longitudinal health-ABC study following an aged urban population provide further support for a role of sustained systemic inflammation and vascular endothelitis in development of HFpEF . Increas+ cells and leucocytes positive for CD11a+, CD45+, and CD68nells was shown in right ventricular biopsy samples of HFpEF patients [An increasingly popular idea about HFpEF is that systemic inflammation not only disrupts endothelial signaling, but also creates a stressful environment for the cardiomyocytes. In fact, an increased level of CD3patients . Furtherpatients .Last not least, systemic inflammation with the increased levels of pro-inflammatory mediators such as TNFα affects endothelial function of the coronary microvasculature with increased expression of endothelial adhesion molecules such as vascular cell adhesion molecule (VCAM) and E-selectin, as shown in myocardial biopsy samples from HFpEF patients ,107,129.In parallel, endothelial cells are also affected by the systemic inflammation resulting in endothelial production of reactive oxygen species (ROS) due to activation of nicotinamide adenine dinucleotide phosphate oxidase . This maThe expanding prevalence of HFpEF throughout the world and the lack of truly effective therapeutic options clearly ask for a focus on this syndrome. Current evidence suggests that HFpEF syndrome is at least partially a systemic syndrome, and there is clearly a need to broaden the focus of research and to create animal models incorporating the complex interactions between different comorbidities and organ systems. Although small rodents are undeniably the premier model system for discovering new mechanisms and targets, the major push is in-depth characterization of HFpEF patients using cutting-edge imaging based on latest cardiovascular magnetic resonance (CMR) ,141 conj


Ambio is a leading journal in environmental science and policy, sustainable development, and human-environment interactions. The paper at hand aims to run a bibliometric analysis to inspect the main publications features of Ambio in Science Citation Index Expanded SCI-EXPANDED. For this scope, a bibliometric survey has been carried out to investigate the journal’s historic characteristics in the Web of Science (WoS) categories of environmental sciences and environmental engineering for Ambio from 1980 to 2019. These are the categories for which the journal has been indexed throughout the indexed time frame. The paper proposes technical and methodological innovations, including improvements in the methods and original characteristics analyzed. Documents published in Ambio were searched out from SCI-EXPANDED. Six publication indicators were applied to evaluate the publication performance of countries, institutes, and authors. Three citation indicators were used to compare publications. As a parameter, the journal impact factor contributor was applied to compare the most frequently cited publications. The journal impact factor contributing publications were also discussed. Results show that Sweden ranked top in six publication indicators and that the top three productive institutes were located in Sweden. A low percentage of productive authors emerged as a journal impact factor contributor. Similarly, a low relationship between the IF contributing publications and the highly cited publications was also found. Less than half of the top 100 highly cited publications in Ambio did not lie within the high impact in most the recent year of 2019. Three members of the advisory board in Ambio were the main productive authors. T.V. Callaghan contributed to most of the publications while papers published by J. Rockstrom as first and corresponding author contributed the most to the journal impact factor. An article authored by Steffen et al. (n et al.  scored t Ambio is a primary source for environmental scientists and policy analysts, as well as for scholars interested in sustainable development, ecosystem functioning and human-environment interactions. The journal is published by The Royal Swedish Academy of Sciences and was founded in 1971.Ambio has been indexed in the Science Citation Index Expanded (SCI-EXPANDED) in 1977 and classified in the Web of Science (WoS) categories of environmental engineering and environmental sciences, performing an impact factor of 4.778 in 2019. Currently, Ambio is indexed in four WoS categories: ecology; environmental chemistry; geography, planning and development; medicine.Sustainable development, sustainability studies and, more broadly, ecological and resource policy are ascending multidimensional disciplines that deserve devoted research, classification and data . All document information from SCI-EXPANDED and each year’s citation times for every publication sorting from the Web of Science Core Collection was checked and downloaded into Microsoft Excel 2016. Additional coding was manually performed.The documents used in this study were derived from the SCI-EXPANDED of the WoS Core Collection, Clarivate Analytics. The searching keyword phrase “The SCI-EXPANDED database was originally designed for researchers to find documents, but it cannot be directly used for bibliometric research Ho . TherefoThe performed research has been undertaken through a brand-new set of feature analysis that has been completed by the traditional key publication features explorations. This includes taking into account: (i) selected characteristics percentages and ranks together; (ii) independent studies; (iii) international collaborations; (iv) most performative first authors and corresponding authors; (v) characteristics of the most productive countries; (vi) characteristics of the most productive institutions; (viii) most recurrent words within the publication’s title.The collaboration type, country and institution were determined by the authors’ affiliation. In the SCI-EXPANDED database, the corresponding author is designated as the “reprint author”. This study uses the term “corresponding author” instead. In a single author publication where the authorship is unspecified, the single author was considered both as first author and corresponding author. Similarly, in a singly institutional publication, the institution is classified as the first-author institution and the corresponding-author institution. In multi-corresponding-author articles, only the last corresponding author, institute, and country were considered see Ho .The affiliations that originated in England, Scotland, Northern Ireland and Wales were reclassified as UK (United Kingdom). For publications that occurred before 1997, Hong Kong affiliates were included in China. The affiliations in Trinid & Tabago were included in Trinidad Tobago. The affiliates of Fed Rep Ger  and Ger Dem Rep (German Democratic Republic) were also included in Germany. France includes the affiliation of French Guiana. The affiliations in Greenland were included in Denmark. The affiliation of Czechoslovakia was checked and reclassified as the Czech Republic. The affiliation in the USSR  were checked and reclassified as in Russia and Estonia—depending on the institution’s nationality. The members of W Ind Assoc St (West Indies Associated States) were reclassified as St Lucia (Saint Lucia) affiliates. Similarly, Geol Survey Czechoslovakia  was checked and reclassified as Czech Geol Survey . Acad Sci USSR (the Academy of Sciences of the Soviet Union) was checked and reclassified as Russian Acad Sci (the Russian Academy of Sciences). The same applied to the Acad Med Sci USSR —it was checked and reclassified as Russian Acad Med Sci . Publications by Stockholm Univ and Univ Stockholm were also reclassified as pertaining to Stockholm Univ (Stockholm University in Sweden).Ambio published 4154 documents in 10 Web of Science document types.From 1980 to 2019, APP) of 3.3. It is worth noting that the documents in the Web of Science Core Collection can be divided into two document types. Among those, 137 documents are classified as conference papers and articles. Therefore, the total percentage is higher than 100%.Table APP of 7.0 followed by reviews with 5.8. The average number of authors per publication in the journal was 3.2, and the maximum number of authors was 53.Document type of discussions with one document had the highest CPP2019 of the proceedings paper was much higher than other document types, at 65. Reviews had similar CPP2019 than articles. A review entitled “Coupled human and natural systems” ”  is defined as in the Journal Citation Reports (JCR) year—i.e., the average number of citations of a journal article published in the past two years. The denominator includes document types of articles or reviews. Some categories of documents that are not normally cited, for example, letters, editorial materials, and other document types are not included in the denominator of the impact factor. Due to the definition of the journal impact factor, only the following document types were considered for further analysis: articles, reviews, and proceedings papers.According to the Web of Science’s definition,IF) is defined as the sum of all the citations coming from articles published by the journal in the previous two years in the selected JCR year, divided by the total number of academic articles published by the journal . Periodicals of the first two years have observed the following: .According to the 2019 Journal Citation Reports (JCR), JCR used 178 Web of Science categories in SCI-EXPANDED to index 9381 journals. The journal impact factor  that means the citation number of Cyear-1 + Cyear-2. The denominator is made of three document types such as reviews, articles, and proceedings papers. A research product pertaining to any other document type is excluded from the denominator.IF) contributing papers were listed in Table IF contributing papers were ranked top 20 in terms of CN (Cyear-2 + Cyear-1), respectively. However, only 25% and 50% of them were ranked as top 20 in terms of TC2019 and C2019, respectively. Only five of the top 20 most contributing papers for the journal impact factor (IF) contributing papers including four articles by Mergler et al.  is a paper-level metric. It does not apply to individual papers or subgroups of papers that appeared in the publication. Additionally, it does not apply to authors of papers, research groups, institutions, or universities.The top 20 journal impact factor  fluctuated and slightly increased in the long run, reaching a minimum of 0.929 in 1999 to a maximum of 4.778 in 2019. The ranking of Ambio in the Web of Science subject categories of environmental sciences and environmental engineering were not improved, especially in the last five years. The journal had a higher ranking in the category of environmental engineering with respect to environmental sciences since 2012. In 2014, notable changes occurred in Ambio’s policies, including the enclosure of six major new features  with the peak year in the 7th year  in the 4th year  in the 5th year   and their citations per publication (CPP2019 = TC2019/TP) have been proposed to discover the development of a journal , country independent papers (IP), internationally collaborative papers (CP), first-author papers (FP), corresponding-author papers (RP), and single-author papers (SP) have been applied as criteria to compare the publication’s performance of countries and institutes for a research topic  were single country papers from 87 countries, while 1019 (31%) were internationally collaborative papers from 117 countries. Six publication indicators: number total of papers , an IP of 639 papers (29% of 2242 country independent papers), a CP of 441 papers , an FP of 879 papers (27% of 3261 first-author papers), an RP of 872 papers (27% of 3227 corresponding-author papers), and an SP of 186 papers (22% of 838 single-author papers).Table CPP2019 of 25 and 1814 papers (56%) from inter-institutional collaborations with a CPP2019 of 34. The percentage of the inter-institutional collaboration rate of the Ambio (56%) was found higher than that of J. Membr. Sci. (38%) (Fu and Ho Pol. J. Environ. Stud. (31%)   came from independent institutions with a TP of 308 papers (9.4% of 3261 papers), an IP of 68 papers , a CP of 240 papers , an FP of 159 papers (4.9% of 3261 first-author papers), and an RP of 157 papers (4.9% of 3227 corresponding-author papers) while the Swedish University of Agricultural Sciences in Sweden took the leading position with an SP of 27 papers (3.2% of 838 single-author papers).The characteristics of the top 10 productive institutions with the six publication indicators were listed in Table TP), first-author papers (FP), corresponding-author papers (RP), and single-author papers (SP) were applied for the analysis of authors’ characteristics for papers in a research field.With the exception of seven anonymous papers , 3398 papers were published by 8431 authors including 2798 authors who published first-author papers, 2733 authors who published corresponding-author papers, and 834 authors who published single-author papers. The first author and the corresponding author are the most significant author positions Gaeta . Three bAmbio. T.V. Callaghan , who is a member of the advisory board in Ambio, affiliated to the Royal Swedish Academy of Sciences in Sweden, was the largest contributor with 40 papers including seven reviews and 33 articles. Callaghan also ranked top in first-author papers with 20 items and corresponding-author papers with 19 publications. F. Barnaby  ranked the top in single-author papers with 12. M. Johansson  with 27 papers ranked second in total papers and T.R. Christensen  with 22 papers ranked third, both of Johansson and Christensen were from Lund University in Sweden. C. Folke  with 20 papers from the Stockholm University in Sweden and T.D. Prowse  with 18 papers from the University of Victoria in Canada were also sitting in the advisory board of Ambio.Table CPP2019 = 424; CN = 100) from Stockholm University in Sweden was the most productive associate editor in Ambio, and published seven articles. Comparing the top 24 most productive authors, an advisory board member—C. Folke—with one review and 19 articles, had the highest CPP2019 of 227, followed by T. Elmqvist  and F. Wulff  from Stockholm University, respectively. T. Elmqvist from Stockholm University was the journal impact factor best contributor with the highest CN (Cyear-1 + Cyear-2) of 290, followed by C. Folke from the same university with a CN of 278, and T.V. Callaghan from Royal Swedish Academy of Sciences with a CN of 247. The most productive authors, C. Folke and T. Elmqvist also published the top largest number of papers falling within the 100 journal impact factor contributing papers with five contributions, respectively. A.D. Fox, C. Folke, J.N. Galloway, T.V. Callaghan, and W. Steffen published two first-author papers and corresponding-author papers in the top 100 journal impact factor contributing papers.E. Andersson  of 3405 papers were highly cited papers including one classic article with a TC2019 of 1126. The 202 highly cited papers were published by 836 highly cited authors from 412 institutions in 45 countries. The USA had 86 highly cited papers (43% of 200 highly cited papers with affiliation information in SCI-EXPANDED) followed by Sweden , Canada , the UK , and Germany . Stockholm University in Sweden published 32 of 202 highly cited papers  followed by the Swedish University of Agricultural Sciences, Royal Swedish Academy of Sciences, and Lund University in Sweden with 10 papers. C. Folke published the most cited papers in the Ambio with 12 papers including the most four first-author and corresponding-author papers, respectively.In CN (Cyear-2 + Cyear-1), respectively while five and 10 of them were ranked top 20 in terms of CN and C2019, respectively. The five of the top 20 most frequently cited papers including four articles by Lindberg et al. , followed by “ecosystem services” , “Baltic sea” , “eutrophication” , “sustainability” , “biodiversity” , “governance” , “phosphorus” , “conservation” , and “arctic” . The results of the analysis considered the author’s keywords and words in the paper title. Papers having the topics of “Baltic sea” and “climate change” were the most popular in Ambio, with respective scores of 231 and 345.Several scholars proposed the distribution of words in article titles, abstracts, author keywords, and Ambio is a top journal in environmental sciences and policy and sustainability studies. Several investigations have conducted bibliometric analyses on journals’ publications performances over the years. These facts motivated this paper’s rationale in scrutinizing the main publication features of Ambio for the time span 1980–2019. This time frame is the available indexing period for the journal and refer to the Web of Science categories of environmental sciences and environmental engineering—the two original categories for which the journal has been historically indexed.Research in environmental policy and sustainable development is on the rise. This trend calls for accurate mapping of existing publications in these domains (Gatto et al. Ambio from 1980 to 2019 was analyzed. The peak year of citations per publication was found to be the 3rd full year since its publication. The papers published in 2007 and 2002 had a higher impact on the journal. Sweden dominated the performance indicators, ranking first in six publication indicators. Stockholm University and the Swedish University of Agricultural Sciences in Sweden were the best performers. T.V. Callaghan did not only publish the largest number of papers in Ambio but was also the best performer for the categories of first-author paper and corresponding-author papers. The less productive author, J. Rockstrom was, however, the largest journal impact factor contributor.A total of 3405 papers including 3263 articles and 142 reviews in IF contributing papers were found in the top 100 highly cited papers. Only 45% of the top 100 most impactful papers in 2019 were listed within the top 100 highly cited papers. Three members of the advisory board in the Ambio—T.V. Callaghan, C. Folke, and T.D. Prowse—were ranked as productive authors. One article published by Rockstrom et al. in 2017 was the top journal impact factor contributing article. An article by Steffen et al. in 2007 was not only the most frequently cited article but also the most impactful publication in 2019. The results show that the journal impact factor contributing papers were not the highly cited papers or the highly impactful publications in Ambio in the most recent available year—2019.One-third of the top 100 The results in the study provided some initial evidence that highly cited papers in a journal might not necessarily greatly contribute to the journal impact factor. This observation leads to a striking conclusion: it is not always appropriate to use the journal impact factor as an indicator to evaluate the publication performance of institutes and authors. This conclusion may lead to important research, policy, and practical implications in the field of bibliometrics, scientometrics, information science, and publications studies that may be carried on in other papers.This work also attempted to provide some technical and analytical innovations with respect to the existing scholarship. Some of the novelties proposed in this study are the procedural improvements performed with the scope to render more solid and reliable outputs and avoid overlappings and errors. Additionally, the paper benefited from an enlarged set of new characteristics—that has been investigated along with traditional publication features. These distinct elements allowed for increased precision in the analyses and for broadening the investigation spectrum, returning more accountable results.Bibliometric analyses can offer a bird’s eye perspective on specific timely subjects. This includes specific journals’ characteristics and publications trends, as well as quantitative indications on the analyzed literature. However, bibliometrics is unable to yield recommendations on germane publications features, including papers’ contents, indications and quality. This is why bibliometrics suggests for integration of qualitative studies on the same bodies of research and mixed methods. In this regard, the paper at hand makes no exception. Future papers may address this issue, disentangling selected characteristics of the examined works. Furthermore, other studies may target different environmental policy and science and sustainability studies journals, as well as further disciplines. Alternative bibliometric techniques, publications databases, papers characteristics, software, and time frames could also be considered. Differentiating the data and the analyses would contribute to better assess the trends and determinants of the sorting sectoral publications.


Post-Traumatic Stress Disorder (PTSD) is a debilitating mental health disorder that occurs after exposure to a traumatic event. Patients with comorbid chronic pain experience affective distress, worse quality of life, and poorer responses to treatments for pain or PTSD than those with either condition alone. FDA-approved PTSD treatments are often ineffective analgesics, requiring additional drugs to treat co-morbid symptoms. Therefore, development of new treatment strategies necessitate a better understanding of the pathophysiology of PTSD and comorbid pain. The single prolonged stress (SPS) model of PTSD induces the development of persistent mechanical allodynia and thermal hyperalgesia. Increased Nociceptin/Orphanin FQ (N/OFQ) levels in serum and CSF accompany these exaggerated nociceptive responses, as well as increased serum levels of the pro-inflammatory cytokine tumor necrosis factor (TNF-α). Therefore, the primary goal was to determine the role of TNF-α in the development of SPS-induced allodynia/hyperalgesia and elevated serum and CNS N/OFQ using two approaches: TNF-α synthesis inhibition, and blockade with anti-TNF-α antibody that acts primarily in the periphery. Administration of TNF-α synthesis blocker, thalidomide (THL), immediately after SPS prevented increased TNF-α and development of allodynia and hyperalgesia. The THL effect lasted at least 21 days, well after thalidomide treatment ended (day 5). THL also prevented SPS-induced increases in serum N/OFQ and reversed regional N/OFQ mRNA expression changes in the CNS. Serum TNF-α increases detected at 4 and 24 h post SPS were not accompanied by blood brain barrier disruption. A single injection of anti-TNF-α antibody to male and female rats during the SPS procedure prevented the development of allodynia, hyperalgesia, and elevated serum N/OFQ, and reduced SPS-induced anxiety-like behaviors in males. Anti-TNFα treatment also blocked development of SPS-induced allodynia in females, and blocked increased hypothalamic N/OFQ in males and females. This suggests that a peripheral TNF-α surge is necessary for the initiation of allodynia associated with SPS, as well as the altered central and peripheral N/OFQ that maintains nociceptive sensitivity. Therefore, early alleviation of TNF-α provides new therapeutic options for investigation as future PTSD and co-morbid pain treatments. Chronic or persistent pain is one of the most commonly co-occurring physical problems for patients with PTSD, and an even higher incidence of this comorbidity was observed in the veteran population . FurtherThe single prolonged stress (SPS) model of PTSD has been employed as a preclinical model of PTSD for over 20 years , 9. PrevAnti-TNF-α therapy in rheumatoid arthritis patients decreases brain-derived TNF-α and alleviates pain . ConsequAdult Sprague–Dawley rats weighing 220–250 g at the initiation of SPS were obtained from Charles River Labs . Animals were housed in the animal facility under a 12-h light: 12-h dark cycle (lights on at 06:00 h) with free access to food and water. After arrival, rats were acclimated to the animal facility for 7–10 days prior to initiation of experiments. Experimental protocols were approved by the Institutional Animal Care and Use Committee of the University of Oklahoma Health Sciences Center and the US Army Medical Research and Materiel Command Animal Care and Use Review Office. Research was compliant with the Animal Welfare Act Regulations and other Federal Statutes relating to animals and experiments involving animals, and adhered to the principles set forth in the Guide for Care and Use of Laboratory Animals, National Research Council, 1996. All experiments conformed to the guidelines of the International Association for the Study of Pain. Every effort was made to minimize animal discomfort and reduce the number of animals used.Animals were randomized into groups. The SPS procedure was followed as described , 27 withn = 6/group) or 21 (n = 3/group), 9 total rats per group were assessed for nociceptive sensitivity on days 0, 3, 7 and 9 . Four groups of three rats/group also were tested on days 13 and 21 as a pilot experiment to determine if latent allodynia or hyperalgesia appeared over the subsequent 12 days.The THL dose effectively reduced TNF-α synthesis . THL  were randomly divided into 4 groups: control + IgG, control + anti-TNF-α, SPS + IgG and SPS + anti-TNF-α (N = 5~6/group/sex). Anti-TNF-α antibody (30 μg)  or the sRats were assessed changes in nociceptive responses to tactile and thermal stimuli in all groups after placement in clear plastic boxes with a glass floor for thermal tests and a wire mesh floor for tactile assessments. Animals acclimated to the boxes for 15–30 min prior to assessment. A plantar analgesia meter  was utilized to measure paw withdrawal latency (PWL) to an infrared light beam  directed toward the right hind paw with the lamp set at 25% active intensity. Cut-off time was set at 30 s to prevent tissue damage . Blood was withdrawn from the heart with an 18-gauge needle (between 15:00 and 17:00 h), placed in Eppendorf tubes and maintained at room temperature for 30 min. Blood samples were then centrifuged at 5,000 × g at 4°C for 5 min, the serum was collected and stored at −80°C. CSF was withdrawn by inserting a 26-gauge needle into the cisterna magna and was immediately stored at −80°C. Brains and spinal cords were extracted and stored at −80°C. Brains were thawed on ice and sliced with a vibratome to dissect hippocampus, hypothalamus, amygdala, prefrontal cortex and periaqueductal gray (PAG) regions according to the Paxinos and Watson rat brain atlas  for N/OFN/OFQ content in sera, CSF and selected brain regions was determined by radioimmunoassay kit  as previously described . ResultsBlood samples were obtained by tail bleed (200–300 μL) under isoflurane anesthesia or from cardiac puncture. Serum and CSF was collected and prepared as described above. Levels of TNF-α in serum and CSF (50 μL) were quantified using the Rat TNF-α ELISA kit  as directed by the manufacturer.TRI reagent  was immediately added to dissected tissues for mRNA extraction. cDNA was synthesized using Super-Script III Reverse Transcriptase . Real-time PCR was performed using SYBR Green Master Mix  and 125 nM forward and reverse primers  in QuantStudio StepOne qPCR (Applied Biosystems). The average of GAPDH and 28S CT values served as an internal standard to which expression of other genes were normalized. Data were analyzed using the comparative Ct method as described .Blood brain barrier permeability was determined by measuring the ratio of CSF albumin to serum albumin. Albumin levels in CSF (100 μL of a 1:500-fold diluted sample) and serum  were collected from control or SPS-treated male rats euthanized at 1, 4, and 24 h  after being placed in isolation in their cages following recovery from ether anesthesia. Samples were analyzed by ELISA  based on manufacturer's instructions. The standard curve ranged from 0 to 200 ng/ml. Levels of CSF albumin in four rats  were outside the range of detection. With an insufficient volume of CSF remaining to re-assay, the albumin CSF:serum ratio for those rats could not be determined.post-hoc analysis, as indicated. Non-parametric data were analyzed using the Kruskal-Wallis test. Results were considered significantly different if p < 0.05. Analysis was performed with Prism v. 9.2 for Windows .Results of D'Agostino & Pearson omnibus normality tests determined if subsequent data analysis should utilize parametric or non-parametric approaches. Outliers were identified by ROUT. Data were analyzed by one- or two-way ANOVA with Tukey's F = 44.64, P < 0.0001], as well as significant SPS  and treatment  effects. Treatment of SPS rats with THL prevented (#p < 0.0001) the TNF-α increase observed in SPS-treated rats (*p < 0.0001) 3 days post-SPS, but had no effect on TNF-α levels in in non-stressed rats (THL) compared to vehicle-treated controls (Veh), N = 9/group.We previously reported on preliminary studies that prior to appearance of anxiety-like behaviors at day 9, allodynia was evident as early as 3 days post-SPS, and was accompanied by elevated serum TNF-α levels in SPS-treated rats . Since tN = 9/group) with Tukey's Multiple Comparison post-hoc test. PWT and PWL were significantly reduced in the SPS groups compared to Veh- and THL-treated groups (*p < 0.0001). Acute treatment of SPS with THL almost completely prevented the appearance of tactile allodynia (#p < 0.0001) compared to SPS on day 3, by day 7 no differences between SPS+THL, Veh- and THL-treated groups were noted  = 306.4, P < 0.0001] and thermal  sensitivity. Significant effects of THL treatment on tactile  and thermal sensitivity  and of time: tactile  and thermal sensitivity , were noted. To confirm that nociceptive sensitivity in THL-treated SPS rats did not gradually return to SPS levels over time, 3 rats per group also were assessed for nociceptive sensitivity on days 13 and 21  = 274.9, p < 0.0001] and thermal  sensitivity.Nociceptive sensitivity to tactile (A) and thermal (B) stimuli was assessed prior to SPS (0) and on days 3, 7, and 9  or THL treatment alone (ΔΔΔΔp < 0.0001), and THL treatment blocked SPS-induced increases in serum N/OFQ (####p < 0.0001), as determined by two-way ANOVA  = 19.23, P = 0.0003]. Significant SPS  and treatment effects [F = 20.92 P = 0.0002] also were observed.SPS increased serum N/OFQ levels compared to Veh- (ay ANOVA . There wt-test), they were grouped together to obtain sufficient sample size for analysis. A significant effect of stress was observed . SPS increased CSF N/OFQ levels compared to Veh- (***p < 0.001) and THL-treated groups (ΔΔp < 0.01). Transient THL treatment lowered the SPS-induced increase in CSF N/OFQ levels, but SPS+THL still differed significantly from Veh (*p < 0.05). THL treatment did not alter baseline N/OFQ levels when compared to vehicle-treated controls from CSF or serum.Hemoglobin adulteration of CSF samples from 4 rats in the Veh-treated control group and 1 rat from each of the other 3 day 9 groups precluded assay. Since there were no differences between d9 and d21 vehicle-treated group CSF N/OFQ samples  = 4.442, p = 0.0486] as determined by two-way ANOVA (N = 5–6/group)  , but no *p < 0.05 by F = 4.705, p = 0.0165] and prefrontal cortex  compared to VEH. Decreased TNF-α mRNA is consistent with compensatory down-regulation following gene activation. Serum and CSF samples from day 9 rats also were assayed for TNF-α levels by ELISA, using a 2-way ANOVA. There was a significant difference between groups for CSF F = 8.960, p = 0.0086, but no post-hoc differences were noted. No significant differences in TNF-α between treatment groups for serum were found (data not shown).The hippocampus (HC), amygdala (AMY), and prefrontal cortex (PFC) process responses to traumatic stress and pain, and express both the NOP receptor and N/OFQ. N/OFQ mRNA in PAG and HC, and NOP mRNA in AMY and PAG were elevated at day 21 post-SPS , but earF = 3.969, p = 0.0259], with an increase in PNOC noted in the THL+SPS group compared to Veh alone (#p < 0.05).Though no differences were noted for N/OFQ in HIP , a signi*p = 0.0351), but no post-hoc differences were found. No changes in TNF-α, PNOC or NOP receptor mRNA were found in AMY (data not shown). These changes are consistent with differential regional modulation of NOP receptor peptide and receptor by TNF-α.No changes in NOP receptor mRNA were noted in HIP . Kruskalpost-hoc analyses did not confirm that SPS groups differed from Veh-treated controls in any parameter shown. THL treatment was a significant factor in all parameters assessed  or SPS-treated rats (#p < 0.05), making it difficult to interpret the impact of THL treatment on anxiety-like symptoms produced by SPS.Since acute THL treatment prevented SPS-induced increases in serum TNF-α, nociceptive sensitivity and modulated NOP receptor and peptide mRNA, its effect on the development of anxiety-like behaviors was assessed in Veh-, THL-, SPS-, and SPS+THL-treated rats using the EPM test . SPS wasassessed  and postCSF:serum ratio) (Q = 1%), and was excluded. Because three of the four groups failed the Shapiro & Wilk normality test, analysis was performed using the Kruskal-Wallis test. No significant differences between groups were found, consistent with an intact BBB following SPS.The blood-brain barrier (BBB) acts a selective physical barrier between the CNS and the periphery whereby it regulates the transport of molecules between both compartments . While am ratio) . Elevatem ratio) –43. Traum ratio) . Therefom ratio) , and albF = 379.3, P < 0.0001] using one-way ANOVA with Tukey's multiple comparison test. Serum TNF-α levels increased 4 and 24 h after SPS compared to the control group . The 24 h post-SPS group also differed significantly from the 4 h post-SPS group (####p < 0.0001), confirming that levels began to decline after 4 hr. Both 4 and 24 h time points (*p < 0.05) as determined by unpaired t-test.While levels of TNF-α in serum at 3 days post-SPS were higher than in untreated rats, they were still lower than would be expected immediately following exposure to a traumatic stress. Serum TNF-α often increases shortly after an injury or stressful event, and it was likely that the day 3 time point represented the trailing end of an earlier surge in TNF-α levels. Thus, serum TNF-α from rats in Ctrl, 1, 4, and 24 h post-SPS groups was determined by ELISA . As antie points  were hige points . TNF-α le points . TNF-α mTo support the hypothesis that circulating TNF-α released 1–4 h post-SPS resulted in SPS-induced allodynia, hyperalgesia and anxiety-like behaviors, male and female SD rats were divided into one of four groups: Sham Control + normal rat serum (Ctr/IgG), Sham Ctr + anti-TNF-α antibody (Ctr/α-TNF-α), SPS+IgG and SPS + α-TNF-α. Three hundred μl (30 μg) IgG or anti-TNF-α was injected into the tail vein (i.v.) during the last few min of the 2 h period of restraint in step one of the SPS protocol . Four hoF = 7.640, p = 0.0002] and a significant interaction between treatment and time for thermal hyperalgesia in males . Significant effects of time and treatment also were noted for thermal hyperalgesia in males. For females, data analysis revealed significant interactions between anti-TNF-α antibody treatment and time for tactile allodynia  and thermal hyperalgesia . Increased sensitivity in an SPS+anti-TNF-α group was noted only to thermal stimuli in female rats at day 7, but it was back to baseline by day 9  post-SPS, and assessment of anxiety-like behaviors took place on the morning of day 9, rats were euthanized on day 9 following EPM assessment. As anticipated, SPS produced tactile allodynia and thermal hyperalgesia in males  = 3.90, p = 0.0392], PAG  and hypothalamus  post-SPS as determined by one-way ANOVA (#p < 0.05), and HYPO in males (#p < 0.05) (p = 0.2203).There were no differences in N/OFQ levels between Ctr+IgG and Sham+α-TNF-α, so data from those groups were pooled (designated as Ctr/α-TNF-α) for male and female serum . SPS incay ANOVA . Since T < 0.05) . CSF samt-test, so those groups were not pooled for either brain region in F = 8.507, p = 0.0085] and of α-TNFα treatment . N/OFQ levels in PAG from SPS rats differed significantly from Ctr + α-TNFα (ΔΔp = 0.01). For female hypothalamic N/OFQ, there was a significant effect of SPS  and of α-TNFα . SPS differed from control (**p < 0.01), Ctr + α-TNFα (ΔΔΔp < 0.001), and from SPS + α-TNFα (##p < 0.01).However, PAG and HYP from female rats exhibited the same SPS- and α-TNFα treatment induced changes in N/OFQ as males . Female F = 8.139, p = 0.0068] and of group . No post-hoc differences between males and females were noted within any treatment group. Similar results were obtained when analyzing N/OFQ levels in the PAG, with effects of sex  and group  but no differences between males and females within each group. In the hypothalamus, no differences were noted with sex as a variable, but there was a significant group effect .Comparisons between male and female N/OFQ for serum, PAG and HYP were performed by 2 way ANOVA. For serum N/OFQ, no interaction between sex and treatment group was found, but there were significant effects of sex [##p < 0.01). Similar effects were found with ppN/OFQ mRNA in female CTX . In the male HYP, SPS + α-TNFα treatment significantly increased ppN/OFQ mRNA compared to SPS (#p < 0.05). Despite increased N/OFQ peptide in male PAG with SPS  and amygdala in male and female rats treated with or without anti-TNF-α antibody and SPS were quantified by qPCR to determine if circulating TNF-α contributes to changes noted with SPS. SPS-induced increases in ppN/OFQ mRNA in the female PAG  that parwith SPS , ppN/OFQwith SPS . No othe*p < 0.05) and in the HYP of male rats (*p < 0.05), but this increase was not blocked by anti-TNF antibody treatment in either case . It also shows that that blockade of TNF-α synthesis or action prevented the development of SPS-induced allodynia and alleviated the development of hyperalgesia in male and female rats, as well as prevented upregulation of N/OFQ in serum of male rats, and in the HYP of male and female rats.The pathophysiology of co-morbid PTSD and chronic pain is unclear. Patients with PTSD often exhibit excessive inflammatory activities of the immune system including increased circulating pro-inflammatory cytokines, however few studies have tested the relationship between inflammatory cytokines, PTSD and pain experimentally in preclinical models. Previous work in our lab and others has shown that the SPS preclinical model for PTSD induces the development of persistent allodynia, thermal hyperalgesia, visceral sensitivity and increased anxiety-like behaviors , 17, 18.TNF-α may arise from peripheral and central sources, producing pro- and anti-inflammatory effects, including transcriptional activation. In the periphery, TNF-α may arise from several sources following severe stress such as spleen, lymph nodes, macrophages, NK cells, CD4+ lymphocytes, neutrophils, mast cells, and eosinophils , 45–52. Besides neurons , 53, N/OInflammatory mediators released by glial cells  induce profound up-regulation of N/OFQ mRNA and peptide , 53, 57.Neuroinflammation in the CNS refers to the microglial response, and to a lesser extent that of astrocytes and oligodendrocytes . AlthougThe ratio of albumin in CSF compared to serum serves as an index of the integrity of the blood-CSF (BBB) barrier. Increases in this ratio denote increased CSF permeability and thus, decreased BBB integrity. No changes in BBB permeability were detected over the 24 h period following SPS. This is consistent with a role of peripheral TNF-α in the initiation of allodynia and hyperalgesia. Though TNF-α may be transported across the BBB , increasThalidomide crosses the BBB  to produThe efficacy of a single administration of a circulating antibody against TNF-α to prevent initiation of allodynia and almost completely alleviate hyperalgesia following SPS in the presence of an intact BBB suggests that the initial surge in serum TNF-α is an integral factor in initiating the pathophysiological cascade of events of SPS. This is in concordance with other experimental models implicating TNF-α with the emergence of pain symptoms and in particular the neuroinflammatory and nociceptive properties that play a role in central sensitization , 80. SerAntibodies in general cross the BBB very poorly unless the barrier is leaky to very large proteins, such as with chronic inflammation or brain tumor, or the antibody can target a receptor-mediated transport protein to do so . Our datOne limitation of the CSF:Serum albumin ratio method is that it does not directly measure the location and physical extent of BBB disruption, and small disruptions of the barrier may be masked. Additional methods may be useful to fully ascertain the sites and impact of stress/trauma on BBB integrity in this model in the future –88.Of particular interest to this study is (1) previous evidence that TNF-α increased prepronociceptin mRNA and N/OFQ peptide , and thaWe previously reported that SPS increased levels of N/OFQ in the CSF and brain on days 9–28, and blockade or loss of NOP receptor prevented this increase in male rats. In the current study, serum N/OFQ levels also significantly increased by day 9 of SPS, but only in male rats. THL blocked increased N/OFQ in serum and CSF, correlating with alleviation of TNF-α-induced allodynia and hyperalgesia. Though the results with anti-TNF-α antibody treatment appear more nuanced, they reflect differences between males and females with SPS. We previously reported that female SPS rats did not show increased levels of N/OFQ in serum and CSF as the males did, and their anxiety-like symptoms were not evident at day 9 post-SPS , though In summary, this study suggests that TNF-α is integral in the pathophysiology of PTSD leading to the development of SPS-induced allodynia and hyperalgesia, and modulates changes in N/OFQ peptide and transcript. Acute treatment with a short-acting, small molecule TNF-α blocking drug biological may offer a novel therapeutic method to prevent or reduce symptoms of PTSD and co-morbid pain.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s. Some of the data was previously presented in poster form.The animal study was reviewed and approved by Institutional Animal Care and Use Committee of the University of Oklahoma Health Sciences Center and the US Army Medical Research and Materiel Command Animal Care and Use Review Office.PD and YZ performed animal experiments and biochemical and molecular studies. PD, YZ, and KS analyzed the data. KS, PD, RG, and MI designed the experiments. PD and KS wrote the manuscript. All authors discussed and commented on the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Heart failure with preserved ejection fraction (HFpEF) does not exist as a singular clinical or pathological entity but as a syndrome encompassing a wide range of clinical and biological phenotypes. There is an urgent need to progress from the unsuccessful ‘one-size-fits-all’ approach to more precise disease classification, in order to develop targeted therapies, personalise risk stratification and guide future research. In this regard, this review discusses the current and emerging roles of cardiovascular imaging for the diagnosis of HFpEF, for distilling HFpEF into distinct disease entities according to underlying pathobiology and for risk stratification. Heart failure with preserved ejection fraction (HFpEF) does not exist as a singular entity but as a syndrome that encompasses a broad cohort of patients with a range of clinical and biological phenotypes , 2.diagnosis: to determine whether patients’ symptoms and signs are due to heart failure (HF) and to identify specific causes of HF in the context of a normal or near normal left ventricular (LV) ejection fraction (EF); (2) biological classification: to characterise the underlying disease mechanisms; and (3) risk stratification: to guide prognosis.The utility of cardiovascular imaging in HFpEF serves three primary functions: (1) The growing prevalence of HFpEF and its poor outcome dictate an urgent need for more precise disease classification in order to develop therapies that target specific pathophysiological mechanisms and to personalise risk stratification. The failure of multiple large phase III trials to identify an effective therapy serves to demonstrate that the “one-size fits all” approach to HFpEF is inadequate.This review discusses the current and emerging roles of cardiac imaging for the diagnosis of HFpEF, for distilling HFpEF into distinct disease entities and for risk stratification.Patients with suspected HFpEF represent a considerable diagnostic challenge, often having multiple co-morbid reasons for breathlessness, peripheral oedema and other clinical features consistent with HF. Whilst natriuretic peptides have increased diagnostic confidence for HF, they lack sensitivity and specificity in important subgroups such as obesity. Furthermore, their elevation may represent an advanced pathophysiological stage that may be less modifiable, where decompensation has already occurred , 4.e′ velocity , average E/e′ ratio > 14, LA maximum volume index > 34 mL/m2 and peak tricuspid regurgitant velocity > 2.8 m/s ) and combined cardiovascular hospitalisation and/or death  in multivariable analysis . Th. Th29]. HFpEF involves multiple pathophysiological mechanisms, which drive the heterogeneous phenotypes that are evident clinically . By char . El. El  showed t . As suchβ , and ECV was the only independent predictor of β on multivariable analysis. To illustrate the potential utility of ECV for identifying phenotypic subtypes of HFpEF, when patients in the study by Rommel et al. were dichotomized according to median ECV, both groups showed a pathological upward shift of the end-diastolic pressure–volume relationship during exercise; however, the dominant pathophysiology was an increase in myocardial passive stiffness in patients with elevated ECM volume, whereas the dominant mechanisms were arterial stiffness and impaired active relaxation in patients with a below-median ECM volume.Rommel et al.  demonstrImportantly, myocardial fibrosis, measured using ECV, strongly associates with adverse outcome in patients with HFpEF, including death and hospitalisation for HF , and as 31P-MRS) can non-invasively quantify the phosphocreatine (PCr) to ATP ratio, an index of energetic status that reflects the creatine kinase energy shuttle  and prolonged time to peak diastolic filling (a measure of active relaxation) during exercise. Proton magnetic resonance spectroscopy (1H-MRS) provides non-invasive measurement of myocardial triglyceride content and the identification of myocardial steatosis. Mahmod et al. . Ph. Ph31P-Md et al. , who meaE) trial  includesn = 9) compared with hypertensive and normotensive controls  per 1% decrease on multivariable analysis , 61 , 63. A n . CMR gen . Further . PWR are . Weber e  showed s  showed snt HFpEF , 70, 71.No change in aortic distensibility was found after 12 months of enalapril or 9 months of spironolactone, compared with placebo, in two randomised controlled trials of HFpEF , 72. SacCardiac imaging, in conjunction with clinical indices, has the potential to more precisely risk stratify patients. Shah et al.  identifiresting echocardiographic indices and natriuretic peptide levels, exercise echocardiographic indices are useful for distinguishing patients that truly have dynamic LAP elevation; however, the technique is far from perfect and current measurements (e.g. E/eʹ) may generate a high rate of false-positive results. Assessment of diastolic dysfunction during exercise requires further refinement.The diagnosis of HFpEF is complex and inconsistent imaging measurements lead to greater ambiguity. Indices of diastolic function are very much in keeping with the ethos of ‘measuring what we can measure, rather than what we want to measure’. Indeed, no echocardiographic index is specific to any particular component of diastolic function, and all are confounded by factors unrelated to diastolic function . The ‘goAs a result of the limitations of current measurements of diastolic function, HF symptoms may be wrongly classed (or ‘dismissed’) as ‘non-cardiac’ , 28, 58.So, what for the future? Whilst, as discussed, a number of imaging measurements hold promise for more meaningful assessment of diastolic function, there is no non-invasive index on the horizon that seems likely to overcome the current limitations. But perhaps that is the wrong approach in any case. Having identified a patient with symptoms ± signs of HF, raised natriuretic peptide levels and a preserved LVEF, perhaps the next step in the diagnostic pathway should be to search for the underlying disease mechanism, for it is this that an intervention would aim to target, and it is this that will determine prognosis. Does the patient have fibrotic-HFpEF, do they have arterial dysfunction-HFpEF, do they have atrial dysfunction-HFpEF? We currently do not routinely investigate for the cause of HF in the context of a preserved EF, and as such, it is entirely unsurprising that we do not have a treatment. Indeed, as an analogy, we currently stop at ‘anaemia’, rather than going on to determine the cause of the anaemia and hence the underlying diagnosis. HFpEF is not a diagnosis.Imaging can help. As described, contemporary imaging techniques can delve into the myocardium and identify those patients with myocardial fibrosis, with cellular hypertrophy, with impaired energetics, and assess the wider cardiovascular system to identify those patients with atrial dysfunction and with arterial dysfunction. Myocardial fibrotic-HFpEF is a good example. Myocardial fibrosis can be measured non-invasively with CMR and myocardial fibrosis is strongly associated with prognosis in HFpEF. If we routinely assessed for myocardial fibrosis, we could potentially target those patients displaying it with antifibrotic interventions, as is under investigation in the ongoing PIROUETTE trial (NCT02932566) .Reclassifying the complexity of HFpEF into more appropriate diagnoses that take into account underlying disease mechanisms and clinical factors is likely to require techniques such as machine learning, as used by Shah et al. and others. Such techniques allow “agnostic’ variable selection, in which optimal groups of variables are chosen through iterative statistical modelling, rather through human endeavour . Groupin