We investigated specific eicosanoid and eicosanoid-related metabolites, bioactive lipids that regulate upstream initiation of systemic inflammation, and their associations with HFpEF in a hospital-based sample. Our findings are three-fold: First, we identified 70 distinct eicosanoid and eicosanoid-related metabolites that were associated with HFpEF status, including prostaglandins, epoxides, and oxylipins, with replicated findings for 18 eicosanoids with incident HF in the MESA cohort. Second, eicosanoid and related metabolite profiles characterized distinct exercise manifestations related to HFpEF. Finally, we show that distinct eicosanoids and eicosanoid-related metabolites may mediate the association of cardiometabolic risk factors including BMI, HTN, and DM with HFpEF. Unique aspects of this investigation included the application of a directed nontargeted LC-MS approach to quantify eicosanoid and related metabolites at scale, deployed in a hospital-based sample of individuals with comprehensive deep physiologic phenotyping to study exercise contributions to HFpEF and further corroboration of findings in a large community-based sample. Taken together, our findings highlight important upstream inflammatory pathways associated with HFpEF and HFpEF-related exercise traits that may offer novel insights into the pathogenesis of HFpEF and potential future therapeutic targets.
Mounting evidence supports the central role of systemic inflammation in the development of HFpEF and its varied manifestations. Specifically, systemic inflammation has been proposed as the common thread linking comorbidities to cardiovascular remodeling and progression to HFpEF. This comorbidity-inflammation paradigm asserts that activation of systemic inflammation, induced by comorbidities including HTN, obesity, and DM, contributes to myocardial inflammation, microvascular and endothelial dysfunction, and pulmonary vascular remodeling, all purported drivers of HFpEF pathogenesis.6 In prior studies, downstream markers of inflammation including CRP, IL-6, and TNF-a have been associated with incident HFpEF, disease severity, and outcomes.8-10 However, neither the causal role of inflammatory mediators nor their role as therapeutic targets have been established. The initiation of inflammation in humans is governed by small-molecule derivatives of arachidonic acid and other polysaturated fats (PUFAS), termed eicosanoids.13 Synthesized by a set of highly conserved enzymes (cyclooxygenases, lipoxygenases, and cytochrome P450 enzymes), eicosanoids and eicosanoid-related metabolites regulate both the activation and suppression of systemic inflammation. Previous studies of eicosanoids and related metabolites in HF demonstrated greater levels of pro-inflammatory metabolites including prostaglandins PGI2 and PGE2, but investigations of have been limited in scale.28,29 The advent of LC-MS has enabled global profiling of eicosanoids and eicosanoid-related metabolites including products of cytochrome P450 monoxygenases, cyclooxygenases, and lipoxygenases. Leveraging this novel LC-MS approach, we present the first comprehensive examination of eicosanoid and related metabolites and HFpEF.
Among 890 eicosanoids and eicosanoid-related metabolites assayed, we identified 70 unique eicosanoid and related metabolites associated with HFpEF status, including prostaglandins, oxylipins, eicosatetraenoic acids, docosanoids, resolvins, and other classical and non-classical eicosanoids. Prostaglandins 15R-PGF2a and 11ß-dhk-PGF2a were most strongly associated with HFpEF status, while epoxide 8(9)-EpETE demonstrated the greatest negative association with HFpEF. PGF2a, a COX-derived arachidonic acid metabolite, and its receptor, are primarily expressed in the female reproductive system and kidney. In women, PGF2a regulates the development of the corpus luteum via progesterone secretion and stimulation of angiogenic factors, physiologic effects of which have been exploited for pharmaceutical induction of labor and termination of pregnancy. The role of PGF2a in cardiovascular homeostasis is less well characterized, but a previous mouse model found that PGF2a elevated blood pressure and promoted atherogenesis, potentially via modulation of the renin-angiotensin-aldosterone system.30,31 In this context, the associations of 15R-PGF2a and 11ß-dhk-PGF2a with HFpEF are notable given the established role of HTN in HFpEF pathobiology and female susceptibility to HFpEF.32-34 By contrast, epoxy-eicosatetraneoic acids (EpETE) are cytochrome P450-derived products of the cardioprotective ɷ-PUFAs eicosapentaneoic acid (EPA) and docosahexaenoic acid (DHA) that induce vasodilation, stimulate angiogenesis, and suppress inflammation. The beneficial effects of ɷ-3 PUFAs have been demonstrated in both observational studies and clinical trials, with notable reductions in hospital admission and death among patients with chronic HF treated with ɷ-3 PUFAs vs placebo.35,36
Recognizing that HFpEF is a highly heterogeneous condition characterized by the hallmark feature of exercise intolerance, we further dissect the association of eicosanoid profiles with cardiac and extra-cardiac contributors to exercise intolerance. Identifying biologic pathways underlying exercise deficits may enable development of therapies for HFpEF more specifically targeted at subphenotypes.37 In this context, we specifically examined cardiac and extracardiac exercise responses and found that exercise traits carried distinct eicosanoid profiles that collectively contributed to the metabolite profile of overall HFpEF status. For example, 12,13 EpOME and 11ß-dhk PGF2a, two HFpEF-related eicosanoids, were uniquely associated with PCWP/CO, a marker of diastolic reserve. Similarly, 8(9)-EpETE was associated with % predicted peak VO2 and % predicted HR, but not with other queried exercise traits. Most data examining eicosanoids with exercise traits have focused on peak VO2, with early studies demonstrating significant associations of prostaglandin PGI2 and its hydration product 6-keto-PGF1a with VO2 max.38,39 We too found significant associations of prostaglandins and prostaglandin-like metabolites with peak VO2, but in contrast to the early studies that demonstrated positive associations with VO2 max, prostaglandin metabolites including 15-R PGF2a and 6ß-PGI1 were negatively associated with % predicted peak VO2 in our sample in keeping with the hypothesis that inflammation promotes development of exercise intolerance in HFpEF. However, prostaglandins, while long regarded as pro-inflammatory metabolites, are known to exert additional anti-inflammatory and vasodilatory effects that are context dependent and may explain the discordant findings.40 More recently, a lipidomic analysis found that moderate intensity exercise dramatically increased circulating levels of oxylipin 12,13-diHOME in both mouse and human subjects.41 Investigators further demonstrated that surgical removal of brown adipose tissue in mice reversed the exercise-induced increase in 12,13 diHOME levels and treatment with 12,13-diHOME increased skeletal muscle fatty acid uptake and oxidation. In our sample, we showed that 12,13-diHOME was strongly associated with both % predicted peak VO2 and % predicted HR, pointing to the important role of 12,13-diHOME and related pathways in the development of exercise intolerance in HFpEF.
While our investigation is the first large-scale evaluation of eicosanoids and eicosanoid-related metabolites in HFpEF, eicosanoid and related metabolites have been previously associated with clinical precursors of HFpEF including obesity, HTN, and diabetes.42-44 For example, in early experimental studies, investigators observed higher baseline levels of urinary 11-dehydro-thromboxane B2 (11dgTxB2), an eicosanoid metabolite and in vivo index of platelet activation, among patients with type II diabetes vs controls.45 Notably, metabolic control with insulin therapy and low-dose aspirin reduced levels of 11dgTxB2 by nearly 50% and 80%, respectively. Similarly, levels of F(2)-isoprostane, a class of eicosanoids involved in lipid peroxidation and oxidative stress, were increased in patients with diabetes and significantly reduced with improved metabolic control and vitamin E supplementation.42 LC-MS profiling has extended these initial findings and enabled identification of novel metabolites associated with HTN and DM. In a study of 8099 healthy participants from the FINRISK 2002 cohort and 2859 participants from the Framingham Heart Study, LC-MS profiling of 545 eicosanoid metabolites and related oxylipin mediators identified 187 unique metabolites with SBP.18 An eicosanoid risk score comprised of 6 metabolites independently associated with SBP, derived from the FINRISK 2002 discovery cohort, accurately discriminated odds of HTN with >2-fold increased odds of HTN among individuals with highest vs lowest quartile eicosanoid risk. A complementary evaluation of eicosanoids and related metabolites and DM in the FINRISK 2002 cohort found 76 eicosanoids and eicosanoid-related metabolites individually associated with incident type 2 diabetes.46 A three-eicosanoid risk score, comprised of 8-iso-prostaglandin A1 (8-iso-PGA1), 12-hydroxy-5,8,10-heptadecatrienoic acid (12-HHTrE), and a novel unidentified eicosanoid, was associated with a >50% increased hazards of incident diabetes.
Given the purported inflammatory link between cardiometabolic disease and HFpEF, we examined whether eicosanoids and eicosanoid-related metabolites potentially mediate the association between cardiometabolic risk factors (BMI, HTN, and DM) with HFpEF. In mediation analyses, we identified 6 eicosanoid and related metabolites as potential mediators, including prostaglandins, oxylipins, epoxides, docosanoids, and ɷ-3 PUFAs. Two metabolites specifically, oxylipin 12,13 diHOME and epoxide 8(9)-EpETE, were found to negatively mediate >10% of the association between BMI, DM, and HTN with HFpEF. Specifically, in mediation analyses, we showed that greater BMI and presence of DM and HTN were associated with greater odds of HFpEF via lower levels of 12,13 diHOME and 8(9)-EpETE. As previously discussed, increased 12,13 diHOME levels, induced by physical exercise or other stimuli like exposure to cold, has been associated with improved metabolic health. Its identification as a potential mediator of the relationship between cardiometabolic risk factors and HFpEF highlights the intimate relationship between inflammation, obesity, and metabolic changes related to physical exercise with HFpEF development.47 Moreover, strategies to increase levels of 12,13-diHOME hold great promise for the prevention and treatment of HFpEF. Similarly, the discovery of 8(9)-EpETE as a mediator of cardiometabolic comorbidities with HFpEF highlights ɷ-3 PUFAs as a potential target for therapeutic intervention in HFpEF.
Our study has several limitations. First, the variance of measured eicosanoid and eicosanoid-related metabolites is known to be significantly influenced by sample processing. Our samples were stored and processed using an established protocol, and all analyses were performed using standardized rather than absolute values of metabolites.23 Second, we studied patients referred for clinically indicated level 3 CPET. While this allowed us to define HFpEF using rigorous physiologic criteria with careful assessment of exercise physiology, we acknowledge that referral bias may limit generalizability to other HFpEF samples, including HFpEF registries or clinical trials. In that context, we further note individuals in MGH CPET who did not meet physiologic HFpEF criteria are not necessarily ‘healthy’ controls. Third, the MGH CPET and MESA incident HF cohorts represented different patient populations, and necessitated alignment of eicosanoid metabolites across different samples. Further, given limited number of HF events in the MESA sample we were not powered to specifically examine incident HFpEF. Finally, accurate identification and classification of eicosanoid and related metabolites using the LC/MS platform is inherently challenging, but MS signals have been consistently mapped to known and putative eicosanoids in previous investigations. We discovered a number of novel eicosanoid and related metabolites associated with HFpEF and acknowledge that exact molecular identity is not known for all of our findings at this time, and that future studies will be needed to identify exact identities.