MiR-199a-3p Induces Mesenchymal to Epithelial Transition of Keratinocytes by Targeting RAP2B.

Cutaneous squamous cell carcinoma (CSCC) is an epidermal skin cancer that evolves from normal epidermis along several pre-malignant stages. Previously we found specific miRNAs alterations in each step along these stages. miR-199a-3p expression decreases at the transition to later stages. A crucial step for epithelial carcinoma cells to acquire invasive capacity is the disruption of cell-cell contacts and the gain of mesenchymal motile phenotype, a process known as epithelial-to-mesenchymal transition (EMT). This study aims to study the role of decreased expression of miR-199a-3p in keratinocytes' EMT towards carcinogenesis. First, we measured miR-199a-3p in different stages of epidermal carcinogenesis. Then, we applied Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) assay to search for possible biochemical targets of miR-199a-3p and verified that Ras-associated protein B2 (RAP2B) is a bona-fide target of miR-199a-3p. Next, we analyzed RAP2B expression, in CSCC biopsies. Last, we evaluated possible mechanisms leading to decreased miR-199a-3p expression. miR-199a-3p induces a mesenchymal to epithelial transition (MET) in CSSC cells. Many of the under-expressed genes in CSCC overexpressing miR-199a-3p, are possible targets of miR-199a-3p and play roles in EMT. RAP2B is a biochemical target of miR-199a-3p. Overexpression of miR-199a-3p in CSCC results in decreased phosphorylated focal adhesion kinase (FAK). In addition, inhibiting FAK phosphorylation inhibits EMT marker genes' expression. In addition, we proved that DNA methylation is part of the mechanism by which miR-199a-3p expression is inhibited. However, it is not by the methylation of miR-199a putative promoter. These findings suggest that miR-199a-3p inhibits the EMT process by targeting RAP2B. Inhibitors of RAP2B or FAK may be effective therapeutic agents for CSCC.

H3K27Ac modification and gene expression in psoriasis.

BACKGROUND: Numerous alterations in gene expression have been described in psoriatic lesions compared to uninvolved or healthy skin. However, the mechanisms which induce this altered expression remain unclear. Epigenetic modifications play a key role in regulating genes' expression. Only three studies compared the whole-genome DNA methylation of psoriasis versus healthy skin. The present is the first study of genome-wide comparison of histone modifications between psoriatic to healthy skins. OBJECTIVE: Our objective was to explore the pattern of H3K27Ac modifications in psoriatic lesions compared to uninvolved psoriatic and healthy skin, in order to identify new genes involved in the pathogenesis of psoriasis. METHOD: Using ChIP-seq with anti H3K27Ac we compared the acetylation of lysine 27 on histone 3 (H3K27Ac) modification between psoriatic to healthy skins, combined with mRNA array. RESULTS: We found a differential H3K27Ac pattern between psoriatic compared to uninvolved or healthy skins. We found that many of the overexpressed and H3K27Ac enriched genes in psoriasis, harbor a putative GRHL transcription factor-binding site. CONCLUSIONS: In the most overexpressed genes in psoriasis, there is an enrichment of H3K27Ac. However, the loss of H3K27 acetylation modification does not correlate with decreased gene expression. GRHL appears to play an important role in the pathogenesis of psoriasis and therefore, might be a new target for psoriasis therapeutics.

Comprehensive Analysis of Correlations in the Expression of miRNA Genes and Immune Checkpoint Genes in Bladder Cancer Cells.

Personalised medicine is the future and hope for many patients, including those with cancers. Early detection, as well as rapid, well-selected treatment, are key factors leading to a good prognosis. MicroRNA mediated gene regulation is a promising area of development for new diagnostic and therapeutic methods, crucial for better prospects for patients. Bladder cancer is a frequent neoplasm, with high lethality and lacking modern, advanced therapeutic modalities, such as immunotherapy. MicroRNAs are involved in bladder cancer pathogenesis, proliferation, control and response to treatment, which we summarise in this perspective in response to lack of recent review publications in this field. We further performed a correlation-based analysis of microRNA and gene expression data in bladder cancer (BLCA) TCGA dataset. We identified 27 microRNAs hits with opposite expression profiles to genes involved in immune response in bladder cancer, and 24 microRNAs hits with similar expression profiles. We discuss previous studies linking the functions of these microRNAs to bladder cancer and assess if they are good candidates for personalised medicine therapeutics and diagnostics. The discussed functions include regulation of gene expression, interplay with transcription factors, response to treatment, apoptosis, cell proliferation and angiogenesis, initiation and development of cancer, genome instability and tumour-associated inflammatory reaction.

IL6R is a target of miR-197 in human keratinocytes.

Psoriasis is a chronic inflammatory disorder with cutaneous and systemic manifestations and substantial negative effects on patients' quality of life. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that play a role in the pathogenesis of psoriasis. Previously studies, from others and by us, highlighted specific miRNAs that are dysregulated in psoriatic lesions. MicroRNA-197-3p (miR-197) expression is downregulated in psoriatic lesions compared to normal or uninvolved skin in patients with psoriasis. We have previously reported that miR-197 could modulate IL-22 and IL-17 signalling in psoriasis. Herein, we identify additional biochemical targets of miR-197 in psoriasis. We applied a transcriptome-wide biochemical approach, Protein argonaute-2 photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (Ago2 PAR-CLIP), to search for new targets of miR-197 in live keratinocytes, and validated its results using reporter assay and analysing by Western blot protein levels in cells overexpressing miR-197. Ago2 PAR-CLIP identified biochemical targets of miR-197, including the alpha subunit of the IL-6 receptor (IL6R). This work provides evidence that IL6R in bona-fide biochemical target of miR-197. IL6R is known to be up-regulated in psoriasis and even was considered as a possible therapeutic target. From the present data and our previous studies, it appears that miR-197 is a major regulator of the interaction between immune system cells and keratinocytes.

Schistosomal extracellular vesicle-enclosed miRNAs modulate host T helper cell differentiation.

During the chronic stage of Schistosoma infection, the female lays fertile eggs, triggering a strong anti-parasitic type 2 helper T-cell (Th2) immune response. It is unclear how this Th2 response gradually declines even though the worms live for years and continue to produce eggs. Here, we show that Schistosoma mansoni downregulates Th2 differentiation in an antigen-presenting cell-independent manner, by modulating the Th2-specific transcriptional program. Adult schistosomes secrete miRNA-harboring extracellular vesicles that are internalized by Th cells in vitro. Schistosomal miRNAs are found also in T helper cells isolated from Peyer's patches and mesenteric lymph nodes of infected mice. In T helper cells, the schistosomal miR-10 targets MAP3K7 and consequently downmodulates NF-κB activity, a critical transcription factor for Th2 differentiation and function. Our results explain, at least partially, how schistosomes tune down the Th2 response, and provide further insight into the reciprocal geographic distribution between high prevalence of parasitic infections and immune disorders such as allergy. Furthermore, this worm-host crosstalk mechanism can be harnessed to develop diagnostic and therapeutic approaches for human schistosomiasis and Th2-associated diseases.

Checkpoint Genes at the Cancer Side of the Immunological Synapse in Bladder Cancer.

Immune checkpoint inhibitors have revolutionized cancer therapy, but not all cancers respond to the currently available drugs, and even within cancers considered responsive to such modality, response rates range between 15 and 40%, depending on the cancer type, the line of treatment, and yet unknown clinical/molecular factors. Coordinated expression of checkpoint proteins was shown to occur on T cells, probably allowing fine-tuning of the signal transmitted to the cell. We performed a bioinformatic analysis of the expression of putative checkpoint mRNAs at the cancer side of the immunological synapse from the bladder cancer tumorgenome atlas (TCGA) database. Fifteen mRNAs, corresponding to both coinhibitory and costimulatory checkpoints, were shown to be expressed above a designated threshold. Of these, seven mRNAs were found to be coexpressed: CD277, PD-1L, CD48, CD86, galectin-9, TNFRSF14 (HVEM), and CD40. The expression of 2 of these mRNAs-BTN3A1 (CD277) and TNFRSF14 (HVEM)-was positively correlated with overall survival in the TCGA database. All these seven mRNA share putative binding sites of a few transcription factors (TFs). Of these, the expression of the TF BACH-2 was positively correlated with the expression of checkpoint mRNAs from the network. This suggests a joint transcriptional regulation on the expression of checkpoint mRNAs at the bladder tumor side of the immunological synapse.

Giardia lamblia miRNAs as a new diagnostic tool for human giardiasis.

BACKGROUND: Giardia lamblia is a very common cause of gastrointestinal symptoms worldwide. There are several methods for the diagnosis of Giardia infection, however none are ideal. We aim to find a new, microRNA-based method that will improve the currently available diagnostic methods for giardiasis. METHODS: Deep-sequence profiling of Giardia small-RNA revealed that miR5 and miR6 are highly expressed in Giardia. These miRNAs were tested by qRT-PCR in duodenal biopsies of patients with giardiasis who were positive by microscopic pathological evaluation. The gastric biopsies of the same patients served as negative control tissues. Additionally, these miRNAs were evaluated in stool samples of patients with proven giardiasis. RESULTS: All histologically proven duodenal biopsies of patients with Giardia infection were positive for Giardia miR5, with a mean threshold cycle (Ct) of 23.7, as well as for Giardia DNA qPCR (16S-like gene, mean Ct 26.3). Gastric biopsies which were tested as a control all were negative. Stool evaluation of miR6 in patients with giardiasis showed 90% specificity but only 66% sensitivity, and a lower accuracy rate was obtained with miR5. CONCLUSION: Giardia miR5 testing in duodenal biopsies may be a new method for the diagnosis of giardiasis. It seems to be more sensitive when compared with testing for Giardia DNA by qPCR in duodenal biopsies. It will be important to investigate the contribution of routine Giardia miRNA testing in duodenal biopsies from patients with persistent abdominal symptoms.

Relation of Neutrophil to Lymphocyte Ratio to Risk of Incident Atrial Fibrillation.

Clinical and experimental data support a critical role for inflammation in cardiovascular disease. The purpose of the current study was to examine the relation between an inflammatory marker, neutrophil-to-lymphocyte ratio (NLR), and incident atrial fibrillation (AF) in asymptomatic adults. We investigated 21,118 self-referred men and women who were annually screened in a tertiary medical center. All subjects were free of AF at baseline and had their serum NLR calculated at the first annual visit. Subjects were divided into 2 groups based on their baseline NLR: Low (<2.83; n = 17,524) and high (≥2.83; n = 3,594; Upper Sextile). The primary endpoint was new onset AF during follow-up. Mean age of study population was 48 ± 10 years and 72% were men. A total of 563 (2.7%) incident events occurred during an average follow-up of 7.5 ± 5 years. Unadjusted Cox regression analysis demonstrated that each 1 unit increase in NLR was associated with a significant 14% increase in risk of occurrence of a first AF event (95% confidence interval 1.06 to 1.23, p < 0.001) and 20% increased risk of death. Kaplan-Meier's survival analysis showed that the cumulative probability of incident AF was significantly higher among subjects with high NLR compared with low NLR group (p = 0.006). Interaction analysis with adjustment to clinical parameters showed that NLR-related risk was age-dependent, such that in the younger age-group (< =50 years) high NLR group had two folds increased risk for AF event compared with low NLR group (95% confidence interval 1.08 to 3.51; p = 0.027) whereas among older subjects the rate of events was similar between both NLR groups (p = NS; p for interaction = 0.024). In conclusion, our findings suggest that high NLR is associated with increased risk of new onset AF. This finding is more pronounced among young adults.

Ultrasound targeting of Q-starch/miR-197 complexes for topical treatment of psoriasis.

Psoriasis is a common, worldwide autoinflammatory, incurable skin disease. miR-197 has therapeutic potential for psoriasis since it can down-regulate the expression of both IL-22RA1 and IL-17RA, subunits of the receptors of IL-22 and IL-17, respectively, which are key cytokines in the disease. Although miR-197 has the potential to treat the disease, several inherent physical barrier properties of the skin challenge miRNA's delivery to the target skin cells. In the present study, we evaluated a therapeutic approach that combines the use of ultrasound (US) as a means to enhance skin permeability with quaternized starch (Q-starch) as an miRNA delivery carrier. This resulted in decreased expression of the miR-197 target proteins and in a significant reduction in the psoriatic activity markers. Our results demonstrate the potential of combinations of US and Q-starch/miR-197 complexes for the topical skin treatment of psoriasis.

Region-Specific, Life-Threatening Diseases among International Travelers from Israel, 2004-2015.

We characterized posttravel hospitalizations of citizens returning to Israel by summarizing the returning traveler hospitalization dataset of the national referral Center for Travel Medicine and Tropical Diseases at Sheba Medical Center in Israel. Of 722 hospitalizations, 181 (25%) infections were life-threatening; most would have been preventable by chemoprophylaxis and pretravel vaccination.

Decreased A-to-I RNA editing as a source of keratinocytes' dsRNA in psoriasis.

Recognition of dsRNA molecules activates the MDA5-MAVS pathway and plays a critical role in stimulating type-I interferon responses in psoriasis. However, the source of the dsRNA accumulation in psoriatic keratinocytes remains largely unknown. A-to-I RNA editing is a common co- or post-transcriptional modification that diversifies adenosine in dsRNA, and leads to unwinding of dsRNA structures. Thus, impaired RNA editing activity can result in an increased load of endogenous dsRNAs. Here we provide a transcriptome-wide analysis of RNA editing across dozens of psoriasis patients, and we demonstrate a global editing reduction in psoriatic lesions. In addition to the global alteration, we also detect editing changes in functional recoding sites located in the IGFBP7, COPA, and FLNA genes. Accretion of dsRNA activates autoimmune responses, and therefore the results presented here, linking for the first time an autoimmune disease to reduction in global editing level, are relevant to a wide range of autoimmune diseases.

The contribution of feedback loops between miRNAs, cytokines and growth factors to the pathogenesis of psoriasis.

The present review describes in detail the existent data regarding feedback loops between miRNAs and cytokines or growth factors in the psoriatic inflammation. We have chosen to describe the roles of miR-31, miR-21, miR-146a, miR-155, miR-197 and miR-99a in this process. This choice derives from the fact that among around 250 miRNAs being altered in the psoriatic lesion, the comprehensive functional role was described only in those detailed above. In addition, considering the molecular targets and the pathways, which may possibly be regulated by those miRNAs, it seems that they may be chosen as preferred targets for the therapy of psoriasis.

Schistosomal MicroRNAs Isolated From Extracellular Vesicles in Sera of Infected Patients: A New Tool for Diagnosis and Follow-up of Human Schistosomiasis.

BACKGROUND: Schistosomiasis traditionally has been diagnosed by detecting eggs in stool or urine. However, the sensitivity of these examinations is limited, especially in travelers with a low worm burden. Serologic tests have a greater sensitivity, but their results remain positive regardless of treatment and thus cannot be used for follow-up of patients. We hypothesized that detection of worm microRNAs (miRNAs) in serum can overcome the drawbacks of the existing diagnostic methods. METHODS AND RESULTS: Twenty-six returning travelers with schistosomiasis (based on positive results of serologic tests or detection of ova) and 17 healthy controls were included in the study. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) amplification of miRNA extracted directly from 500 µL of serum had limited sensitivity and specificity. However, qRT-PCR analysis of RNA extracted from 200 µL of serum extracellular vesicles detected 4 schistosomal miRNAs; the sensitivity and specificity of the 2 highest expressed miRNAs (bantam and miR-2c-3p) were 86% and 84%, respectively. In 7 patients with posttreatment serum available for analysis, we observed outcomes ranging from a reduction in the schistosomal miRNA level to full recovery from disease. CONCLUSIONS: qRT-PCR of pathogen miRNAs isolated from extracellular vesicles in sera from infected individuals may provide a new tool for diagnosing schistosomiasis in patients with a low parasite burden. This assay could also be used for evaluating the outcome of therapy, as well as disease-control programs.

Gender disparities in the functional significance of anemia among apparently healthy adults.

BACKGROUND: Data on the functional impact of anemia on cardiorespiratory fitness (CRF) and survival in healthy individuals are limited. Our aim was to evaluate the association between anemia thresholds, low CRF, and survival in otherwise healthy adults. METHODS: Study population included 16 334 apparently healthy subjects attending annual periodic health screening examinations (71 200 annual visits), including exercise stress testing (EST). Anemia was defined by the World Health Organization (WHO) or Beutler and Waalens' (BW) criteria. Low CRF was defined as the lowest fitness quintile according to the Bruce protocol. RESULTS: The mean age was 46±10 years, and 70% were men. Mean Hb levels were 13±1 and 15±1 among women and men, respectively, with higher proportion of anemia among women. The majority of anemic subjects had mild anemia. When analyzing repeated annual visits, anemia was associated with a significant 39% and 64% increased risk of low CRF according the WHO and BW criteria only in women (n=18 672). Baseline anemia at first visit was associated with 2.6- and 1.9-fold increased risk of all-cause mortality using the WHO and BW criteria, exclusively in men (n=11 511). CONCLUSIONS: Overall, the functional and prognostic impact of anemia is gender dependent, based on the WHO and BW arbitrary criteria, suggesting differing mechanism and responses.

Gender-Related Cardiovascular Risk in Healthy Middle-Aged Adults.

Men tend to develop cardiovascular disease (CVD) earlier in life than women. Whether this difference is attributable only to gender is a matter of debate. The purpose of this study was to evaluate gender differences in cardiovascular risk in a large cohort of asymptomatic men and women and explore gender-related risk in prespecified risk factor subgroups. We investigated 14,966 asymptomatic men and women free of diabetes, hypertension, or ischemic heart disease who were annually screened. The primary end point of the present study was the occurrence of ischemic or cerebrovascular disease as composite end point. Multivariate Cox proportional hazards regression modeling was used to assess the gender difference regarding CVD and to examine the association between CVD risk factors and gender. Mean age of the study population was 47 ± 10 years and 30% were women. Kaplan-Meier survival analysis showed that at 6.2 ± 3.9 years' follow-up, the rate of CVD events was 6.1% among men compared with 1.8% among women (log-rank p <0.001). Consistently, multivariate analysis demonstrated that male gender was independently associated with a significant threefold increased risk for development of CVD events (hazard ratio 3.05, CI 2.25 to 4.14). The CVD risk associated with male gender was consistent in each risk subset analyzed, including older age, low high-density lipoprotein, impaired fasting glucose, and positive family history for ischemic heart disease (all p values for gender-by-risk factor interactions <0.05). Higher performance on treadmill test had a protective effect regarding CVD development in both men and women. In conclusions, healthy middle-aged men experienced increased risk for the development of CVD events compared with women independently of traditional CVD risk factors. However, better exercise capacity is associated with a protective effect.

Metabolic syndrome, obesity, and the risk of cancer development.

BACKGROUND: Metabolic syndrome and its components are severe global health issues that are increasing in frequency as the prevalence of obesity increases. Various studies have established a correlation between metabolic syndrome and diseases including, diabetes mellitus, non-alcoholic fatty liver disease, cirrhosis, and cardiovascular disease. In recent years, correlations have also been detected between obesity and metabolic syndrome and the prevalence of certain types of cancer. The current study examines whether obesity and metabolic syndrome components are risk factors for cancer among the adult population in Israel. METHODS: A cohort study analysis was performed of 24,987 initially healthy men and women who underwent yearly medical assessments at the Institute for Medical Screening in the Sheba Medical Center. Data from the Institute for Medical Screening database was correlated with that from the Israel Cancer Center in the Ministry of Health updated to December 2013. The correlation between metabolic syndrome, obesity, and the overall risk of cancer as well as the risks of specific types of cancer were examined. RESULTS: Of 20,444 subjects for whom complete data were available, 1535 were diagnosed with cancer during the mean follow-up time of 104.3months. In a multi-variant analysis, no significant correlation was found between metabolic syndrome or obesity and the incidence of cancer. When the data were stratified by gender and cancer type, however, a significant association between metabolic syndrome and breast cancer in women was observed (P=0.03, HR=1.67, 95% CI=1.05-2.67). CONCLUSION: Metabolic syndrome correlates with higher than expected breast cancer incidence in women.

Mutations in STN1 cause Coats plus syndrome and are associated with genomic and telomere defects.

The analysis of individuals with telomere defects may shed light on the delicate interplay of factors controlling genome stability, premature aging, and cancer. We herein describe two Coats plus patients with telomere and genomic defects; both harbor distinct, novel mutations in STN1, a member of the human CTC1-STN1-TEN1 (CST) complex, thus linking this gene for the first time to a human telomeropathy. We characterized the patients' phenotype, recapitulated it in a zebrafish model and rescued cellular and clinical aspects by the ectopic expression of wild-type STN1 or by thalidomide treatment. Interestingly, a significant lengthy control of the gastrointestinal bleeding in one of our patients was achieved by thalidomide treatment, exemplifying a successful bed-to-bench-and-back approach.

Body mass index and the risk of new-onset atrial fibrillation in middle-aged adults.

BACKGROUND: Increased body mass index (BMI) and obesity are associated with increased risk of new-onset atrial fibrillation (AF) among middle-aged adults. OBJECTIVES: The objective of the study is to investigate the association between BMI and the risk for new-onset AF among middle-aged adults. METHODS: We investigated 18,290 men and women who were annually screened in a tertiary medical center. Participants were divided at baseline into 3 groups: normal weight (BMI ≥18 and <25 kg/m(2), n = 7,692), overweight (BMI ≥25 and <30 kg/m(2), n = 8,032), and obese (BMI ≥30 kg/m(2), n = 2,566). The primary end point was new-onset AF during follow-up. RESULTS: Mean age of study population was 49 ± 11 years, and 73% were men. A total of 288 incident events (1.6%) occurred during 6 ± 4 years. Kaplan-Meier survival analysis showed that the cumulative probability of AF at 6 years was highest among obese participants, intermediate among overweight participants, and lowest among participants with normal weight (2.1%, 1.7%, and 0.8% respectively, P < .001). Multivariable Cox regression analysis showed that overweight and obesity were independently associated with increased AF risk (hazard ratio 1.54 [P = .004] and 2.41 [P < .001], respectively). Assessment of BMI change as a time-dependent covariate in the multivariable model showed that each 1 kg/m(2) reduction in BMI during follow-up was associated with a significant 7% reduction in the risk for the occurrence of a first AF event (hazard ratio 0.93, 95% CI 0.88-0.99, P = .019). Consistently, similar analysis showed that each 5-kg weight loss during follow-up was independently associated with a significant 12% reduced risk of new-onset AF (95% CI 0.81-0.98, P = .02). CONCLUSIONS: Our findings suggest that overweight and obesity are associated with increased AF risk, whereas weight reduction is independently associated with reduced risk of de novo AF.

Use of exercise capacity to improve SCORE risk prediction model in asymptomatic adults.

AIMS: The SCORE risk estimation system is used for cardiovascular risk stratification in apparently healthy adults and is based on known cardiovascular risk factors. The purpose of the current study was to evaluate whether exercise capacity can improve the accuracy of the SCORE overall survival risk estimation. METHODS AND RESULTS: We investigated 22 878 asymptomatic men and women who were annually screened in a tertiary medical centre. All subjects were free of known ischaemic heart disease, and had completed maximal exercise stress test according to the Bruce protocol. The SCORE risk estimation system was used to evaluate individual cardiovascular risk for all subjects. The primary endpoint was mortality, after exclusion of patients with metastatic cancer during follow-up. The incremental contribution of exercise capacity in predicting the risk of death was evaluated by net reclassification improvement (NRI) and area under the receiver operating curve (AUROC). Mean age of the study population was 47.4 ± 10.3, and 71.6% were men. There were 505 (2.21%) deaths during a mean follow-up of 9.2 ± 4.1 years. Kaplan-Meier survival analysis showed that both SCORE and low exercise capacity were associated with reduced survival. When added to the SCORE risk prediction, exercise capacity allowed more accurate risk stratification: NRI analysis showed an overall improvement of 56.8% in the accuracy of classification and the AUROC increased (0.782 vs. 0.766). CONCLUSION: Both SCORE and exercise capacity are strong independent predictors of all-cause mortality. The addition of exercise capacity to the SCORE risk model can improve the accuracy of the model.

Poor Heart Rate Recovery Is Associated With the Development of New-Onset Atrial Fibrillation in Middle-Aged Adults.

OBJECTIVE: To investigate the association between heart rate recovery (HRR) and new-onset atrial fibrillation (AF) in middle-aged adults. PATIENTS AND METHODS: Heart rate recovery was calculated using the exercise stress test in 15,729 apparently healthy self-referred men and women who attended periodic health screening examinations between January 2000, and December 2015. All participants completed the maximal exercise stress test according to the Bruce protocol and were followed clinically on a yearly basis for a median of 6.4±4 years. The primary end point was new-onset AF. Participants were grouped according to HRR at 5 minutes, dichotomized at the median value (<73 beats/min). RESULTS: Participants with low HRR were older, were more commonly men, had a higher rate of comorbidities, and were less fit. Kaplan-Meier survival analysis revealed that the cumulative probability of AF at 6 years was higher in participants with low HRR (2.1%) than in those with high HRR (0.6%) (log-rank, P<.001). Older age, male sex, obesity resting heart rate, and ischemic heart disease were all associated with increased AF risk in a univariate Cox regression model (P<.05 for all). Multivariate Cox regression analysis revealed that low HRR was independently associated with increased AF risk (hazard ratio, 1.92; 95% CI, 1.3-2.8; P<.001) after adjustment for multiple confounders. CONCLUSION: Lower HRR is independently associated with the development of new-onset AF during long-term follow-up in middle-aged adults.