The HDL-specific phospholipid efflux (HDL-SPE) assay is a novel cell-free measure of HDL function that is inversely associated with coronary artery disease. However, the effect of exercise training on HDL-SPE is unknown. The purpose of this study was to examine the effect of exercise training on HDL-SPE in a large, diverse cohort free of overt disease. Clinical and functional measures of HDL were taken before and after 20 weeks of endurance exercise training in 508 participants from the HERITAGE Family Study. Associations of HDL-SPE with HDL-related traits were examined using Pearson's correlations at baseline and following exercise training (significance: P < 7.4 × 10−4). The effect of exercise training on HDL-SPE was examined using paired t-tests (significance: P < 0.05). Mean (SD) HDL-SPE was 1.40 (0.19) and higher in females compared with males and in White participants compared with Black participants. Baseline HDL-SPE was strongly associated with HDL-C (r = 0.45) and apoA-I (r = 0.43, both P < 6.9 × 10−24) but not with measures of cholesterol efflux. Mean HDL-SPE increased (0.023, P = 0.002) following exercise training, but these increases only occurred in those with the lowest baseline HDL-SPE levels. Change in HDL-SPE was associated with changes in HDL-C (r = 0.27), medium HDL concentration (r = 0.24), and apoA-I and HDL size (r = 0.17, all P < 1.3 ×10−4). HDL-SPE increased following regular exercise, and changes in HDL-SPE were related to changes in HDL size and subclass concentrations. Our findings demonstrate that individuals at higher risk for coronary artery disease may experience the largest benefits from exercise training as related to this novel biomarker of HDL function.

Exercise is an integral therapy for many cardiometabolic diseases, including obesity, type 2 diabetes, and hypertension. Despite its broad health benefits, the circulating factors that mediate exercise adaptations in humans remain incompletely defined, particularly across different exercise intensities. Here, we conducted a multi-cohort human exercise intervention incorporating sprint-interval exercise (SIE) and moderate-intensity exercise (MIE) to analyze intensity-dependent regulation of interorgan crosstalk. We found that exercise intensity distinctly influenced the plasma proteome and metabolome in untrained and trained participants. By integrating multi-organ gene and protein expression datasets with in vitro and in vivo tissue sampling, we mapped regulated proteins to their predicted tissues of origin and destination. Muscle fibers and adipocytes were particularly sensitive to exercise intensity and observed to undergo broad secretory and transcriptomic changes. Moreover, we leveraged a large-scale plasma-phenome database to identify intensity-dependent proteins associated with cardiometabolic health and disease, highlighting how exercise intensity differentially shapes interorgan communication and organismal health.

Regular physical activity represents one of the greatest mechanisms for maintaining human health, yet the underlying molecular transducers of these benefits remain incompletely understood. Multi-omic assays now provide new opportunities to study the coordinated molecular responses of body tissues to different exercise modalities. The Molecular Transducers of Physical Activity Consortium (MoTrPAC) was established to address this need by creating a molecular map of the response to physical activity. Described here is the first human cohort of MoTrPAC: sedentary adults enrolled prior to study suspension during the COVID-19 pandemic (N=175) randomized to either endurance or resistance exercise, or non-exercise control. From these participants, we detail their global acute molecular response in skeletal muscle, adipose tissue, and blood, integrated at multiple levels: tissue, exercise modality, timepoint, and omic category. These analyses characterize key molecular pathways, identify central regulators, and implicate novel candidate exerkines in mediating multi-organ exercise effects.

Exercise benefits numerous organ systems and tissues, however limited knowledge exists about its underlying molecular pathways. Identifying the exercise-induced biochemical changes that occur in the circulation may provide further insights into how exercise confers systemic health changes. Here, we perform large-scale plasma proteomic, metabolomic, and whole blood transcriptional profiling in sedentary human participants undergoing acute endurance exercise (EE), resistance exercise (RE), or a non-exercise control (CON) in up to 7 timepoints over a 24 hour period. We observe 7066 transcript, 189 protein, and 448 metabolite changes in response to EE or RE compared to CON. Our analyses reveal numerous shared biochemical responses between EE and RE modes, but also differences in immune cell responses, lipid metabolism, and pathways reflective of tissue repair and angiogenesis. Taken together, our findings highlight novel temporal and exercise mode-specific blood-based molecular responses to acute exercise, and provide a new resource for the scientific community.

While the cardiometabolic benefits of exercise volume and intensity are well established, the clinical significance of exercise timing remains poorly understood, largely due to the limitations of short-term accelerometry. We leveraged minute-level heart rate data from 14,489 participants from the All of Us Research Program to define habitual exercise timing over a one-year period. Compared to daytime exercise, habitual morning exercise was associated with lower odds of coronary artery disease (OR 0.69; CI 0.55-0.87), hypertension (OR 0.82; CI 0.72-0.94), type 2 diabetes (OR 0.70; CI 0.58-0.85), hyperlipidemia (OR 0.79; CI 0.69-0.90), and obesity (OR 0.65; CI 0.55-0.77). These associations were independent of total physical activity volume and remained consistent across hour-of-day analyses, with the lowest risk nadir occurring between 07:00-08:00 for coronary artery disease. These findings suggest that exercise timing may represent a distinct, underappreciated dimension of exercise behavior linked to cardiometabolic health.

BACKGROUND:Cardiorespiratory fitness is an integrative measure of cardiometabolic health and predictor of survival, yet little is known about its molecular underpinnings. Small molecule metabolites and lipids are increasingly recognized as exercise-stimulated signaling molecules and candidate molecular transducers of cardiorespiratory fitness. METHODS:We performed nontargeted liquid chromatography mass spectrometry-based plasma metabolomics in 654 participants (mean age, 35 years; 55% women) from the HERITAGE Family Study (Health, Risk Factors, Exercise Training, and Genetics) who had cardiorespiratory fitness (maximal oxygen uptake [VO2max]) measured by cardiopulmonary exercise testing and underwent 20 weeks of supervised endurance training. Metabolite-VO2max relationships were assessed using linear regression and tested for replication in FHS (Framingham Heart Study) participants who also underwent cardiopulmonary exercise testing. Metabolite relationships with incident all-cause mortality ascertained in JHS (Jackson Heart Study) and MESA (Multi-Ethnic Study of Atherosclerosis) were tested using Cox regression. Experimental studies of cellular respiration and mitochondrial function were performed in C2C12 myotubes. RESULTS:An unknown mass spectrometry peak (mass-to-charge, 385.3056; retention time, 3.69 minutes) had the strongest, positive relationship with VO2max (mL×kg-1min-1) after adjustment for age, sex, race, and lean body mass (β=1.29; false discovery rate q=5.3×10-6); was identified as N-palmitoyl glutamine (N-pal-gln) using tandem mass spectrometry and bioinformatics; and was confirmed with an authentic chemical standard. The biological role of N-pal-gln has not been described previously. The relationship of N-pal-gln with VO2max was validated in 408 participants from the FHS (β=1.2; P=3.8×10-5), and its levels increased after exercise training (log fold change=0.22; q=5.3×10-12). N-pal-gln levels were inversely associated with all-cause mortality in JHS and MESA (hazard ratio, 0.91 and 0.65 [P=0.029 and P=0.028], respectively). Previous studies have shown that structurally related biochemicals modulate energy homeostasis; thus, we performed mitochondrial experiments. N-pal-gln administration led to a dose-dependent increase in mitochondrial:nuclear DNA ratio compared with control treated cells (15% and 20% increases at 6.5 nM and 26 nM N-pal-gln, respectively [P=0.04 and P=0.02]) and improved bioenergetics (N-pal-gln at 26 nM increased the phosphate:oxygen ratio across ADP concentrations from 0 to 100 μM; ANOVA P=0.0027). CONCLUSIONS:We identified a novel, lipidated amino acid, N-pal-gln, that is positively associated with VO2max, increases after regular aerobic exercise, and is inversely associated with incident mortality. N-pal-gln stimulates mitochondrial biogenesis and efficiency, demonstrating its potential role as an exercise-stimulated transducer of cardiorespiratory fitness.

Obesity and metabolic dysfunction are associated with pulmonary vascular remodeling, yet molecular mechanisms remain poorly understood. We sought to study trans-right ventricular (RV) metabolite gradients to elucidate potential molecular pathways operant among individuals with obesity and pulmonary hypertension. In this study, 38 individuals with obesity (mean age 58 years, 68% women, average BMI 36.6 kg/m2) underwent invasive right heart catheterization. Multi-site blood sampling from the superior vena cava and pulmonary artery was performed to assess trans-RV gradients, with targeted metabolite profiling using liquid chromatography-mass spectrometry. We found 56 metabolites with significant trans-RV gradients (FDR q < 0.05), including intermediates of fatty acid oxidation, the tricarboxylic acid cycle, and nucleotide metabolism. Further, trans-RV gradients in lipid and purine metabolism were associated with BMI and related cardiometabolic traits, such as waist circumference, insulin resistance, and serum lipids. Finally, differential levels of bile acids, intermediates of lipid peroxidation, and nucleotide metabolism across the RV were associated with pulmonary hypertension. In conclusion, trans-RV metabolite gradients among individuals with obesity reveal alterations in metabolites representative of molecular pathways such as fatty acid oxidation, and others correlated with cardiometabolic traits and/or pulmonary hypertension, including orotic acid, bile acids, and acylcarnitines.

BACKGROUND:Identifying the cause of dyspnea (i.e., cardiac vs. non-cardiac) can be challenging in the absence of significant resting cardiac abnormalities. Exercise cardiovascular magnetic resonance (Ex-CMR) enables quantification of cardiac volumetric indices under physiological stress. Using Ex-CMR, we sought to develop a non-invasive imaging marker, referred to as the myocardial dynamic index (MDI), and to demonstrate its potential for evaluating cardiac dyspnea. METHODS:MDI is a metric derived from Ex-CMR work-volume loop model that integrates rest and stress left ventricular (LV) end-diastolic and end-systolic volumes with workload measured during supine exercise, while accounting for body size and LV mass. To evaluate MDI as a marker of cardiac dyspnea, we retrospectively analyzed data from a prospective multicenter study measuring MDI in patients with cardiac or non-cardiac dyspnea. All had invasive exercise testing before Ex-CMR. Cardiac dyspnea was defined by established invasive and non-invasive criteria, including HFpEF (early to advanced) and HFmrEF. Non-cardiac dyspnea patients had normal invasive hemodynamics and cardiac function. Univariable and multivariable logistic regression identified clinical and imaging predictors of cardiac dyspnea. A base model incorporating clinical and rest CMR variables was compared to a model that included the base model plus MDI. Diagnostic performance was assessed using receiver operating characteristic analysis and compared using the DeLong test. MDI scan/re-scan reproducibility over one year, inter- and intra-observer reproducibility, and correlation with VO₂ max were evaluated. RESULTS:Among 93 patients (66 with cardiac dyspnea, 27 with non-cardiac dyspnea), MDI was lower in patients with cardiac dyspnea (25.9±9.5 vs. 45.1±10.7 mL·W/g/m², p<0.0001). The base model included age, body mass index, NYHA class, and left atrial strain. In multivariable analysis, MDI emerged as the only independent predictor of cardiac dyspnea when added to the base model. Inclusion of MDI improved the AUC from 0.86 to 0.93 (p=0.012), while MDI alone yielded an AUC of 0.91. A strong correlation was observed between MDI and the VO₂ max index (r=0.84, p<0.0001). Reproducibility was excellent. CONCLUSION:Ex-CMR MDI is independently associated with cardiac dyspnea and strongly correlates with the VO₂ max index. It aids in differentiating cardiac from non-cardiac dyspnea and provides incremental diagnostic value beyond conventional clinical and resting imaging parameters.

Exercise is recognized as first-line therapy for many cardiometabolic diseases, including obesity, type 2 diabetes, and hypertension. Despite the abundant health-promoting effects of exercise, in-depth characterization of circulatory factors that mediate these benefits in humans remains incomplete. Moreover, how different modes and intensities of exercise uniquely regulate these processes is unclear. Here, we address these questions by conducting a multi-cohort human exercise intervention, incorporating sprint-interval exercise (SIE) and moderate-intensity exercise (MIE) to analyze intensity-dependent regulation of interorgan crosstalk. We find that exercise intensity distinctly influences the plasma proteome and metabolome in both untrained and trained participants. SIE led to immediate and robust changes to the plasma proteome, whereas MIE resulted in delayed secretory kinetics. By leveraging large, multi-organ gene and protein expression datasets, in combination with in vitro and in vivo tissue sampling, we map the differentially regulated proteins to their predicted tissue of origin and destination. We find that adipocytes are particularly sensitive to exercise intensity, undergoing broad transcriptomic remodeling following in vitro incubation with SIE as compared to MIE plasma. These findings underscore the integrated whole-body response following acute exercise and highlight exercise intensity as a key factor influencing interorgan communication.

BACKGROUND:We report IMPROVE-DiCE (Improve Diabetic Cardiac Energetics), a 2-part open-label, phase 2a trial evaluating the safety and effectiveness of ninerafaxstat, a novel therapeutic designed to enhance cardiac energetics. Between May and September 2021, part 1 enrolled patients with type 2 diabetes and obesity without heart failure with preserved ejection fraction (HFpEF). Between January 2023 and June 2024, part 2 enrolled patients with type 2 diabetes, obesity, and HFpEF. METHODS:Forty-two participants received 200 mg ninerafaxstat twice daily (part 1, n=21, 43% women, 72±0.5 years of age, 4-8 weeks; part 2, n=21, 29% women, 71±6 years of age, 12 weeks). Myocardial energetics (phosphocreatine-to-ATP ratio [PCr/ATP], primary outcome) and function (rest and dobutamine stress) were assessed before and after treatment using magnetic resonance imaging, 31P- and 1H magnetic resonance spectroscopy. In part 1, hyperpolarized [1-13C]pyruvate magnetic resonance spectroscopy to assess in vivo pyruvate dehydrogenase flux (n=9) and plasma metabolomics and proteomics were also performed. RESULTS:In part 1, in patients with diabetes and obesity but without HFpEF, the heart was characterized by impaired pyruvate dehydrogenase flux, reduced PCr/ATP, triglyceride deposition, and diastolic impairment. Treatment with ninerafaxstat was associated with improved PCr/ATP (+0.39±0.49 [95% CI, 0.16-0.62]; Cohen's d, 0.79; P=0.002) and lower myocardial triglyceride (by 34%, P=0.03). In part 2, in patients with diabetes, obesity, and symptomatic HFpEF, the heart was characterized by reduced PCr/ATP, diastolic impairment, and failure of systolic augmentation to exercise. Consistently, treatment with ninerafaxstat was associated with improvement in PCr/ATP (+0.15±0.25 [95% CI, 0.03-0.26]; Cohen's d, 0.60; P=0.02), improved systolic augmentation to exercise (+1.4 L/min, P=0.04), improved exercise capacity (6-minute walk distance +16 m, P=0.02), and improved New York Heart Association class symptom burden. CONCLUSIONS:These mechanistic phase 2a study results show that ninerafaxstat is safely tolerated and improves myocardial energetics in participants with obesity and diabetes without or with clinically manifest HFpEF. REGISTRATION:URL: https://www.clinicaltrials.gov; Unique identifier: NCT04826159.

Despite the wide effects of cardiorespiratory fitness (CRF) on metabolic, cardiovascular, pulmonary and neurological health, challenges in the feasibility and reproducibility of CRF measurements have impeded its use for clinical decision-making. Here we link proteomic profiles to CRF in 14,145 individuals across four international cohorts with diverse CRF ascertainment methods to establish, validate and characterize a proteomic CRF score. In a cohort of around 22,000 individuals in the UK Biobank, a proteomic CRF score was associated with a reduced risk of all-cause mortality (unadjusted hazard ratio 0.50 (95% confidence interval 0.48-0.52) per 1 s.d. increase). The proteomic CRF score was also associated with multisystem disease risk and provided risk reclassification and discrimination beyond clinical risk factors, as well as modulating high polygenic risk of certain diseases. Finally, we observed dynamicity of the proteomic CRF score in individuals who undertook a 20-week exercise training program and an association of the score with the degree of the effect of training on CRF, suggesting potential use of the score for personalization of exercise recommendations. These results indicate that population-based proteomics provides biologically relevant molecular readouts of CRF that are additive to genetic risk, potentially modifiable and clinically translatable.

Introduction: Exercise is critical to cardiovascular health. However, the underlying molecular mechanisms are not well described. The Molecular Transducers of Physical Activity Consortium (MoTrPAC) seeks to create a detailed molecular map of the response to exercise. Described here is the first human cohort of MoTrPAC, enrolled prior to the COVID-19 shutdown (N=175). Methods: Healthy, sedentary adults were randomized to an 8-exercise circuit of resistance exercise (RE, N=73), a 40 minute submaximal endurance exercise bout (EE, N=65), or to non-exercising control (N=37). Blood, muscle, and adipose tissue were sampled at 4-7 time points relative to exercise, depending on tissue/modality. Samples were deep phenotyped across multiple omic domains including chromatin accessibility, transcriptomics, proteomics, phosphoproteomics, and metabolomics. Results: The cohort was 72% female, with a mean±sd age of 41±15 years and BMI of 27.1±4.0 kg/m2. Exercise affected over 34,000 molecular features in ≥1 tissue/time point including a high proportion of transcriptomic and phosphoproteomic features (Figure A). Molecular signatures were compared between EE and RE: enrichment analysis of muscle phosphoproteomics showed a greater activation of MAP kinases in RE compared to EE at all time points. To identify plausible exerkines (secreted molecules signaling an acute exercise bout), differentially abundant features in any sampled tissue cells were compared to temporally-matched cognate protein levels in plasma, yielding 110 features. A known exerkine, CX3CL1 (fractalkine) was identified, in addition to novel candidates, such as cellular communication network factor 1 (CCN1), a secreted extracellular matrix protein linked to plasma triglyceride levels, which showed increased abundance early post exercise (Figure B). Network analysis across tissues and omes identified novel transcription factor “hubs” as candidate master regulators of exercise response. Conclusions: These first MoTrPAC data represent an unparalleled multi-tissue, multi-omic, multi-time point, multi-modality map of acute exercise, enhancing our understanding of the molecular transducers that may link exercise and cardiovascular health.

Background: The lipoprotein insulin resistance (LP-IR) score has been shown to assess insulin resistance, predict future type 2 diabetes, and improve with regular exercise. The lipidomic profile is known to differ with insulin resistance and type 2 diabetes, but few studies have examined lipidome associations with LP-IR, particularly in response to an exercise intervention. Methods: Plasma lipids were measured using the C8-positive LC-MS method in 671 participants from the HERITAGE Family Study (56% Female, 35% Black, 35.2 yrs) before and after a 20-week exercise intervention. LP-IR, a weighted index of six lipoprotein parameters, was measured before and after training through nuclear magnetic resonance spectroscopy (Labcorp, NC). Linear mixed models were used to test the associations of 193 known plasma lipids with LP-IR before and after exercise training. All models were adjusted for age, sex, and race, while post-training models also adjusted for baseline lipid and baseline LP-IR. Results: A total of 162 lipids (84%) were associated (FDR<0.05) with LP-IR at baseline ( Figure 1 ). The top positive associations were found for TG species, while cholesterol ester species showed the top inverse associations. Following training, the change in 143 (74%) lipids were associated (FDR<0.05) with change in LP-IR. There were 129 lipid species associated with both baseline and changes in LP-IR, while 33 were only associated with baseline measures and 14 only associated with changes in LP-IR following exercise ( Figure 1 ). Conclusions: We found that most lipids were associated with LP-IR regardless of exercise training status. Importantly, we identified a subset of lipids that were only associated with changes in LP-IR, which may represent exercise responsive biomarkers of exercise induced changes in LP-IR. However, further research is needed to identify the biological mechanisms connecting the changes in these lipid species and metabolic changes following exercise training.

Physical activity, including structured exercise, is associated with favorable health-related chronic disease outcomes. Although there is evidence of various molecular pathways that affect these responses, a comprehensive molecular map of these molecular responses to exercise has not been developed. The Molecular Transducers of Physical Activity Consortium (MoTrPAC) is a multicenter study designed to isolate the effects of structured exercise training on the molecular mechanisms underlying the health benefits of exercise and physical activity. MoTrPAC contains both a preclinical and human component. The details of the human studies component of MoTrPAC that include the design and methods are presented here. The human studies contain both an adult and pediatric component. In the adult component, sedentary participants are randomized to 12 wk of Control, Endurance Exercise Training, or Resistance Exercise Training with outcomes measures completed before and following the 12 wk. The adult component also includes recruitment of highly active endurance-trained or resistance-trained participants who only complete measures once. A similar design is used for the pediatric component; however, only endurance exercise is examined. Phenotyping measures include weight, body composition, vital signs, cardiorespiratory fitness, muscular strength, physical activity and diet, and other questionnaires. Participants also complete an acute rest period (adults only) or exercise session (adults, pediatrics) with collection of biospecimens (blood only for pediatrics) to allow for examination of the molecular responses. The design and methods of MoTrPAC may inform other studies. Moreover, MoTrPAC will provide a repository of data that can be used broadly across the scientific community. NEW & NOTEWORTHY The Molecular Transducers of Physical Activity Consortium (MoTrPAC) will be the first large trial to isolate the effects of structured exercise training on the molecular mechanisms underlying the health benefits of exercise and physical activity. By generating a compendium of the molecular responses to exercise, MoTrPAC will lay the foundation for a new era of biomedical research on Precision Exercise Medicine. Presented here is the design, protocols, and procedures for the MoTrPAC human studies.

BACKGROUNDMost GWAS of plasma proteomics have focused on White individuals of European ancestry, limiting biological insight from other ancestry-enriched protein quantitative loci (pQTL).METHODSWe conducted a discovery GWAS of approximately 3,000 plasma proteins measured by the antibody-based Olink platform in 1,054 Black adults from the Jackson Heart Study (JHS) and validated our findings in the Multi-Ethnic Study of Atherosclerosis (MESA). The genetic architecture of identified pQTLs was further explored through fine mapping and admixture association analysis. Finally, using our pQTL findings, we performed a phenome-wide association study (PheWAS) across 2 large multiethnic electronic health record (EHR) systems in All of Us and BioMe.RESULTSWe identified 1,002 pQTLs for 925 protein assays. Fine mapping and admixture analyses suggested allelic heterogeneity of the plasma proteome across diverse populations. We identified associations for variants enriched in African ancestry, many in diseases that lack precise biomarkers, including cis-pQTLs for cathepsin L (CTSL) and Siglec-9, which were linked with sarcoidosis and non-Hodgkin's lymphoma, respectively. We found concordant associations across clinical diagnoses and laboratory measurements, elucidating disease pathways, including a cis-pQTL associated with circulating CD58, WBC count, and multiple sclerosis.CONCLUSIONSOur findings emphasize the value of leveraging diverse populations to enhance biological insights from proteomics GWAS, and we have made this resource readily available as an interactive web portal.FUNDINGNIH K08 HL161445-01A1; 5T32HL160522-03; HHSN268201600034I; HL133870.

Background and aims: Previous studies have derived and validated an HDL apolipoproteomic score (pCAD) that predicts coronary artery disease (CAD) risk. However, the associations between pCAD and markers of cardiometabolic health in healthy adults are not known, nor are the effects of regular exercise on pCAD. Methods: A total of 641 physically inactive adults free of cardiovascular disease from the HERITAGE Family Study completed 20 weeks of exercise training. The pCAD index (range 0-100) was calculated using measurements of apolipoproteins A-I, C-I, C-II, C-III, and C-IV from ApoA-I-tagged serum (higher index = higher CAD risk). The associations between pCAD index and cardiometabolic traits at baseline and their training responses were assessed with Spearman correlation and general linear models. A Bonferroni correction of p < 8.9 x 10(-04) was used to determine statistical significance. Results: The mean +/- SD baseline pCAD index was 29 +/- 32, with 106 (16.5 %) participants classified as high CAD risk. At baseline, pCAD index was positively associated with blood pressure, systemic inflammation, and body composition. HDL size, VO2max, and HDL-C were negatively associated with pCAD index at baseline. Of those classified as high CAD risk at baseline, 52 (49 %) were reclassified as normal risk after training. Following training, pCAD index changes were inversely correlated (p < 1.4 x 10(-04)) with changes in HDL-C, HDL size, and LDL size. Conclusions: A higher pCAD index was associated with a worse cardiometabolic profile at baseline but improved with regular exercise. The results from this study highlight the potential role of HDL apolipoproteins as therapeutic targets for lifestyle interventions, particularly in high-risk individuals.