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JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY VOL. 67, NO. 3, 2016 ª 2016 BY THE AMERICAN COLLEGE OF CARDIOLOGY FOUNDATION PUBLISHED BY ELSEVIER ISSN 0735-1097/$36.00 http://dx.doi.org/10.1016/j.jacc.2015.10.079 Prognostic Implications of Long-Chain Acylcarnitines in Heart Failure and Reversibility With Mechanical Circulatory Support Tariq Ahmad, MD, MPH,*y Jacob P. Kelly, MD,yz Robert W. McGarrah, MD,yx Anne S. Hellkamp, PHD,z Mona Fiuzat, PHARMD,z Jeffrey M. Testani, MD, MTR,* Teresa S. Wang, MD,k Amanda Verma, MD,y Marc D. Samsky, MD,y Mark P. Donahue, MD,y Olga R. Ilkayeva, PHD,x Dawn E. Bowles, PHD,{ Chetan B. Patel, MD,yz Carmelo A. Milano, MD,{ Joseph G. Rogers, MD,yz G. Michael Felker, MD, MHS,yz Christopher M. O’Connor, MD,z# Svati H. Shah, MD, MPH,yx William E. Kraus, MDyx ABSTRACT BACKGROUND Heart failure (HF) is characterized by perturbations in energy homeostasis and metabolism. The reversibility and prognostic value of circulating markers associated with these changes remain unclear. OBJECTIVES This study sought to describe the metabolomic profiles of patients along the spectrum of systolic HF, determine their association with adverse outcomes in a clinical trial of HF, and evaluate whether identified metabolites change with treatment for end-stage systolic HF. METHODS To assess association of metabolites with clinical outcomes, we evaluated a population of 453 chronic systolic HF patients who had been randomized to exercise training versus usual care. To assess change in metabolites with mechanical circulatory support, 41 patients with end-stage HF who underwent left ventricular assist device (LVAD) placement were studied. Targeted, quantitative profiling of 60 metabolites using tandem flow injection mass spectrometry was performed on frozen plasma samples obtained prior to randomization, as well as prior to and $90 days post-placement in the LVAD group. Principal components analysis was used for data reduction. RESULTS Five principal components analysis–derived factors were significantly associated with peak VO2 levels at baseline in fully adjusted models. Of these, factor 5 (composed of long-chain acylcarnitines) was associated with increased risk of all 3 pre-specified clinical trial outcomes: all-cause mortality/all-cause hospitalization, all causehospitalization, and cardiovascular death or cardiovascular hospitalization. Individual components of factor 5 were significantly higher in patients with end-stage HF prior to LVAD placement and decreased significantly post-implantation. CONCLUSIONS In chronic HF patients, circulating long-chain acylcarnitine metabolite levels were independently associated with adverse clinical outcomes and decreased after long-term mechanical circulatory support. These metabolites may serve as potential targets for new diagnostics or therapeutic interventions. (Exercise Training Program to Improve Clinical Outcomes in Individuals With Congestive Heart Failure; NCT00047437) (J Am Coll Cardiol 2016;67:291–9) © 2016 by the American College of Cardiology Foundation. From the *Section of Cardiovascular Medicine, Yale School of Medicine, New Haven, Connecticut; yDepartment of Internal Medicine, Division of Cardiology, Duke University Medical Center, Durham, North Carolina; zDuke Clinical Research Institute, Listen to this manuscript’s Duke University, Durham, North Carolina; xDuke Molecular Physiology Institute, Duke University, Durham, North Carolina; audio summary by kDepartment of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; {Division of Cardiac Surgery, Duke University JACC Editor-in-Chief Medical Center, Durham, North Carolina; and the #Inova Heart and Vascular Institute, Falls Church, Virginia. This study was Dr. Valentin Fuster. performed with a grant from the Daland Fellowship in Clinical Investigation. The HF-ACTION study was funded by grants from the National Heart, Lung, and Blood Institute. Dr. Ahmad has received consulting fees from Roche. Dr. Patel has served as a consultant for Thoratec Corp. and HeartWare Inc. Dr. Milano has served as a consultant for HeartWare, Inc. Dr. Felker has received grant support from Roche Diagnostics; and has served as a consultant for Singulex. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose. Evelyn Horn, MD, served as Guest Editor for this paper. Manuscript received June 8, 2015; revised manuscript received September 7, 2015, accepted October 22, 2015. 292 Ahmad et al. JACC VOL. 67, NO. 3, 2016 JANUARY 26, 2016:291–9 Metabolite Profiles of Systolic Heart Failure H ABBREVIATIONS AND ACRONYMS FA = fatty acid eart failure (HF) is a global health circulatory support for end-stage HF with long-term problem with an estimated preva- left ventricular assist device (LVAD) support. lence of 38 million patients world- wide, a number that is increasing with HF = heart failure METHODS the aging of the population. The most com- LVAD = left ventricular assist mon diagnosis in patients 65 years or To assess the prognostic significance of metabolites, older admitted to a hospital in high-income we analyzed a subgroup of the HF-ACTION (Heart nations (1), HF possesses a prognosis worse Failure: A Controlled Trial Investigating Outcomes of than that of most cancers. Pharmaceutical Exercise Training) trial of chronic systolic HF pa- treatments have primarily focused on neurohormonal tients. Details of the participants have been previ- device PCA = principal components analysis blockade with b-blockers, angiotensin-converting ously discussed (10). Specifically, 452 of 2,331 patients enzyme inhibitors, angiotensin-II receptor blockers, enrolled in the HF-ACTION study had agreed to and aldosterone antagonists. Because HF is a complex participate in a biomarker substudy that required syndrome, however, identification of novel molecular collection and banking of peripheral blood samples mechanisms might lead to new therapies. for purpose of research, and on whom metabolomics profiling was performed. Patients were 18 years or SEE PAGE 300 older, with left ventricular systolic dysfunction (left Patients with failing hearts are characterized by ventricular ejection fraction [LVEF] <35%) and structural, functional, inflammatory, and metabolic ambulatory HF; they were randomized to exercise derangements that develop and worsen during dis- training versus usual HF care. Outcomes of interest ease progression (2). Despite the multifactorial causes included changes in exercise capacity measured by for HF, it has been suggested that as hearts begin to distance walked during a 6-min walk test and peak fail, altered energetics play an increasingly important oxygen consumption (V O2) measured from a cardio- role in pathogenesis, that is, the heart becomes pulmonary exercise test; clinical endpoints included “an engine out of fuel” (3). The heart is among the all-cause mortality, cardiovascular mortality, cardio- most metabolically active organs in the body, utiliz- vascular hospitalization, and HF hospitalization. ing an entire supply of adenosine triphosphate every To assess modifiability of the prognostic metabo- 13 s; to accomplish this, it primarily uses free fatty lites with mechanical treatment for HF, we studied 41 acids (FAs) as energy substrates, and switches to consecutive patients, ages 18 years and older, who favor glucose metabolism during states of stress. were deemed to have end-stage HF and required During the progression of HF, glycolysis rises as mechanical circulatory support with continuous-flow an adaptation to the reduced oxidative metabolism LVAD as a bridge to transplantation or destination that is uncoupled from glucose oxidation. This is therapy at Duke University Medical Center between further exacerbated by an increase in circulating FAs January 1, 2011, and October 30, 2012. Details of the that including cohort and methodology are described elsewhere (2). reduced update and mitochondrial oxidative meta- These patients had agreed to collection and banking bolism (4). of peripheral blood samples for the purpose of occurs via several mechanisms, Metabolomics, the study of small-molecule me- research. Patients had blood samples collected prior tabolites, aims to uncover the underlying patho- to LVAD and had paired long-term samples available physiological processes of the body with regard to post-LVAD placement (median time: >136 days energy homeostasis and metabolism; however, the [range: 94 to 180 days]). prognostic or therapeutic implications of metabolic Approved by the Duke University Medical Center profile derangements in HF remain unclear (5–7). Institutional Review Board, these studies were per- With the availability of novel compounds that stabi- formed in accordance with the ethical guidelines of lize or reverse mitochondrial dysfunction, human the Declaration of Helsinki; all patients provided studies to delineate the circulating profile of this written informed consent. derangement, and establishing potential for revers- Using a targeted, quantitative tandem flow injection ibility of metabolic derangements in HF, could lead mass spectrometry-based approach, we determined to a better understanding of whether they might be levels of 45 acylcarnitines and 15 amino acids in both efficacious in this disease state (8,9). study populations. Proteins were first removed by We therefore sought to characterize circulating precipitation with methanol; aliquoted supernatants metabolites associated with poor outcomes in chronic were dried and esterified with hot acidic methanol systolic HF patients and assess whether these prog- (acylcarnitines) and n-butanol (amino acids). For the nostic analysis, we used tandem mass spectrometry with a profiles are modifiable with mechanical Ahmad et al. JACC VOL. 67, NO. 3, 2016 JANUARY 26, 2016:291–9 Quattro Micro instrument (Waters Corp., Milford, Massachusetts), and the addition of internal standards 293 Metabolite Profiles of Systolic Heart Failure T A B L E 1 Baseline Characteristics enabled quantitative assessment of metabolites. Testing for all of the assays was done in random batch HF-ACTION Chronic HF (n ¼ 453) LVAD Study End-Stage HF (n ¼ 41) p Value 0.029 order by the Metabolomics/Biomarker Core Laboratory Age, yrs 59 (51–68) 68 (54–74) of the Duke Molecular Physiology Institute at Duke Male 324 (71.5) 29 (70.7) 0.91 University; testing personnel were blinded to the BMI, kg/m2 30.4 (26.3–35.8) 28.7 (24.3–34.9) 0.22 clinical status of patients, and samples were randomly Caucasian 302 (66.7) 29 (70.7) 0.60 distributed without knowledge of event status. Hypertension 282 (62.5) 28 (68.3) 0.46 Hyperlipidemia 306 (67.5) 25 (61.0) 0.39 Diabetes 147 (32.5) 25 (61.0) 139 (137–141) 135 (131–138) In the HF-ACTION cohort, the associations of metabolite component factors with peak VO 2 and with Sodium, mmol/l 0.0002 <0.0001 clinical outcomes were assessed. Paralleling out- Blood urea nitrogen, mg/dl 20 (15–28) 28 (17–41) 0.0098 comes from the main clinical trial, the primary clinical Creatinine, mg/dl 1.2 (1.0–1.5) 1.4 (1.2–1.9) 0.0013 outcome was the composite variable of all-cause mor- Total cholesterol, mg/dl 162 (139–192) 119 (95–138) <0.0001 tality or all-cause hospitalization; secondary clinical LDL, mg/dl 89 (70–117) 61 (46–76) <0.0001 0.0061 outcomes included all-cause hospitalization, cardiovascular death or cardiovascular hospitalization, or cardiovascular death or heart failure exacerbation. STATISTICAL ANALYSIS. In the HF-ACTION cohort, 38 (32–48) 31 (23–40) HbA1c, % HDL, mg/dl 6.8 (6.0–8.1) 6.9 (5.8–7.6) LVEF, % 25 (20–30) 20 (15–20) <0.0001 0.027 Peak VO2, ml/kg/min 14.3 (11.3–17.3) 12.5 (9.2–13.7) Ve-VCO2 slope 32.3 (27.9–38.1) 42.1 (36.4–47.6) 823.3 (323.0–2,088.0) 3,108.0 (2,160.5–7,417.0) NT-proBNP, pg/ml 0.33 0.0003 <0.0001 principal components analysis (PCA) with varimax rotation was used to reduce the large number of correlated metabolites into uncorrelated factors, as we have done previously (6,11). Multiple regressions were used to evaluate the association of baseline Values are median (interquartile range) or n (%). BMI ¼ body mass index; HbA1c ¼ hemoglobin A1c; HDL ¼ high-density lipoprotein cholesterol; HF ¼ heart failure; HF-ACTION ¼ Heart Failure: A Controlled Trial Investigating Outcomes of Exercise Training; LDL ¼ lowdensity lipoprotein cholesterol; LVAD ¼ left ventricular assist device; LVEF ¼ left ventricular ejection fraction; NT-proBNP ¼ N-terminal pro–B-type natriuretic peptide; Ve-VCO2 slope ¼ ventilation vs. CO2 production. PCA-derived factor levels with baseline peak VO2 , and Cox proportional hazards regression modeling was for the integrity of the data. All analyses were per- used to assess the relation between factors and clin- formed with SAS version 9.2 (SAS Institute Inc., Cary, ical outcomes. Models were conducted in 3 stages: 1) North Carolina) and R 2.15.3 (R Development Core unadjusted; 2) adjusted for age, sex, and body mass Team, Vienna, Austria). A p value #0.05 was consid- index (BMI); and 3) adjusted for all known predictors ered statistically significant for all analyses. of each outcome, which had been previously identi- RESULTS fied in the full HF-ACTION cohort (12). In each model, we considered all factors simultaneously and used BASELINE stepwise selection to select the set of significant fac- baseline PATIENT CHARACTERISTICS. Per tors. For the peak V O2 model, covariates were age, sex, (HF-ACTION) and end-stage HF (LVAD) patient pop- race, region, BMI, diabetes, peripheral vascular dis- ulations (Table 1), the median age was 59 years in the ease, New York Heart Association functional class, chronic HF group and 68 years in the end-stage HF LVEF, ventricular conduction, and test modality. group. The patients were similarly distributed with Covariates included in clinical outcomes models were regard to sex, race, BMI, and comorbidities (including age, sex, race, geographic region, LVEF, blood urea hypertension, hyperlipidemia, and diabetes), as well nitrogen, presence of severe mitral regurgitation, as objective laboratory measures. The groups differed characteristics of the chronic the HF medications, symptom scores, and measures from the in objective measures of cardiovascular fitness: peak baseline cardiopulmonary exercise testing. The pro- VO2 of 14.3 ml/kg/min in chronic HF compared with portional hazards assumption was checked for sig- 12.5 ml/kg/min in end-stage HF; Ve-VCO 2 slope of 32.3 nificant factors in each Cox model, and was found to in chronic HF compared with 42.1 in end-stage HF; be met in all cases. Kaplan-Meier methods were used and N-terminal pro–B-type natriuretic peptide of to generate time-to-event curves for significant 823 ng/l in chronic HF and 3,108 ng/l in end-stage HF. metabolite factors. Baseline characteristics and indi- LVEF, as ascertained by echocardiogram, was not vidual metabolites were compared between the statistically significantly different: 25% in chronic HF HF-ACTION and end-stage HF LVAD groups using versus 20% in end-stage HF. Baseline characteristics Pearson chi-square tests for categorical variables and of the subset of HF-ACTION patients used for this Wilcoxon rank sum tests for continuous variables. The study were not substantially different from the authors had full access to and take full responsibility overall cohort (Online Table 1). Ahmad et al. 294 JACC VOL. 67, NO. 3, 2016 JANUARY 26, 2016:291–9 Metabolite Profiles of Systolic Heart Failure T A B L E 2 Association of Principal Component Factors With Baseline Peak V O 2 in the Chronic HF Cohort Outcome: Peak VO2 (ml/kg/min) Unadjusted Adjusted* Factor‡ Estimate (SE) Wald Chi-Square Test 1 1.28 (0.33) 15.38 2 0.94 (0.32) 8.33 3 0.57 (0.20) 7.93 Adjusted for Full Prediction Model† p Value Estimate (SE) Wald Chi-Square Test Estimate (SE) Wald Chi-Square Test <0.0001 0.79 (0.30) 6.90 <0.01 0.69 (0.26) 6.88 <0.01 0.94 (0.30) 0.01 0.40 (0.19) 9.73 <0.001 0.88 (0.27) 10.56 0.001 4.65 0.03 p Value p Value <0.01 4 0.42 (0.20) 4.59 0.03 0.50 (0.18) 7.42 0.006 0.58 (0.16) 12.71 <0.001 5 0.47 (0.23) 4.23 0.04 0.68 (0.21) 10.39 0.001 0.38 (0.19) 4.10 0.04 7 0.71 (0.20) 12.41 <0.001 0.37 (0.19) 4.01 0.05 *Adjusted for age, sex, and BMI. †Adjusted for age, sex, race, region, BMI, diabetes, peripheral vascular disease, New York Heart Association functional class, LVEF, ventricular conduction, and test modality. ‡Any factor not listed was not significant at p # 0.05. Factors consistently associated with the outcome after all adjustments are indicated in italics. Other factors were not significant at p # 0.05 in that model. SE ¼ standard error; other abbreviations as in Table 1. BASELINE METABOLOMIC FACTORS. PCA identi- (long-chain dicarboxylacylcarnitines), factor (branched amino acids and related catabolites), factor 5 consistent pathways (Online Table 2) similar to our (long-chain acylcarnitines), and factor 8 (short-chain previous studies (5,13). In the original HF-ACTION dicarboxylacylcarnitines) were all associated with trial, baseline peak V O2 was the most significant pre- peak VO 2 in the fully adjusted model. dictor of mortality in this population (chi-square ¼ 153) Table 3 shows the association of principal compo- and we sought to determine if there was a similar nent factors with clinical outcomes. For the fully association with metabolite profiles. As shown in adjusted model, factors 5 (long-chain acylcarnitines) Table 2, factor 1 (medium-chain acylcarnitines), factor 2 and 7 (medium-chain acylcarnitines) were associated T A B L E 3 Association of Principal Component Factors With Clinical Outcomes in Chronic HF Cohort (HF-ACTION) Unadjusted Factor HR (95% CI) Wald Chi-Ssquare Test Adjusted* p Value Adjusted for Full Prediction Model† HR (95% CI) Wald Chi-Square Test p Value HR (95% CI) Wald Chi-Square Test p Value Outcome: all-cause mortality or all-cause hospitalizations 3 1.13 (1.01–1.27) 4.33 0.04 5 1.31 (1.16–1.48) 17.72 <0.0001 1.30 (1.14–1.48) 15.51 <0.0001 1.24 (1.09–1.42) 10.49 0.001 7 1.18 (1.06–1.32) 8.88 <0.01 1.15 (1.02–1.29) 5.33 0.02 1.16 (1.02–1.31) 5.46 0.02 0.85 (0.75–0.97) 6.15 0.01 0.88 (0.78–0.99) 4.00 <0.05 1.48 (1.08–2.05) 5.81 0.01 <0.05 1.42 (1.16–1.74) 11.35 <0.001 1.22 (1.06–1.39) 8.14 <0.01 1.28 (1.09–1.51) 8.61 <0.01 9 0.82 (0.73–0.93) 10.61 <0.01 13 0.89 (0.79–1.00) 3.85 <0.05 1.67 (1.25–2.22) 11.95 <0.001 1.56 (1.15–2.14) 7.91 5 1.61 (1.30–1.99) 19.61 <0.0001 1.59 (1.28–1.97) 18.02 8 1.57 (1.21–2.04) 11.64 <0.001 1.43 (1.08–1.90) 6.23 1.31 (1.15–1.50) 15.66 <.0001 1.22 (1.01–1.47) 4.30 0.038 Outcome: all–cause hospitalization 1 2 4 fied 13 metabolite factors grouping in biologically <.0001 <0.01 Outcome: cardiovascular death or cardiovascular hospitalization 5 1.33 (1.16–1.52) 17.74 8 1.26 (1.06–1.50) 6.66 <0.01 <.0001 9 0.86 (0.76–0.98) 5.51 0.01 Outcome: cardiovascular death or HF hospitalization 2 1.37 (1.08–1.73) 6.97 <0.01 1.35 (1.06–1.73) 5.87 0.02 5 1.41 (1.20–1.67) 16.82 <0.0001 1.41 (1.19–1.67) 15.95 <0.0001 8 1.55 (1.27–1.91) 17.88 <0.0001 1.51 (1.22–1.88) 14.13 <0.001 Hazard ratios indicate 1-unit increase in factor. *Adjusted for age, sex, and BMI. †Adjusted for age, sex, race, geographic region, LVEF, blood urea nitrogen, presence of severe mitral regurgitation, medications, symptom scores, and measures from the baseline cardiopulmonary exercise test. Any factor not listed was not significant at p # 0.05. Factors consistently associated with the outcome after all adjustments are indicated in italics. Other factors were not significant at p # 0.05 in that model. CI ¼ confidence interval; HR ¼ hazard ratio; other abbreviations as in Table 1. Ahmad et al. JACC VOL. 67, NO. 3, 2016 JANUARY 26, 2016:291–9 with an increase in risk of the primary outcome of all-cause mortality or hospitalization (hazard ratio F I G U R E 1 Primary Outcome: Factor 5 and Time to Event [HR]: 1.24; 95% confidence interval [CI]: 1.09 to 1.42 and HR: 1.16; 95% CI: 1.02 to 1.31, respectively). risk of the primary outcome (HR: 0.88; 95% CI: 0.78 to was associated with a greater risk of all-causehospitalization (HR: 1.42; 95% CI: 1.16 to 1.74), and cardiovascular death or cardiovascular hospitalization (HR: 1.22; 95% CI: 1.06 to 1.39). Of note, neither factor 7 nor 9 has associations with risk of hospitalization or cardiovascular death. Figure 1 shows Kaplan-Meier curves of the association between tertiles of factor 5 for the primary endpoint of the trial; the highest tertile showed a greater rate of death or hospitalization compared with the middle and lowest tertiles. the independent association 0.7 Death or Hospitalization 0.99). With regard to secondary outcomes, factor 5 Primary endpoint (death or hospitalization) by tertile of factor 5 0.8 Factor 9 (amino acids) was associated with decreased Given Highest tertile Middle tertile Lowest tertile 0.6 0.5 0.4 0.3 0.2 0.1 0 0 0.5 between 1 Years 1.5 factor 5 and peak V O2 as well as all clinical outcomes, we examined whether its constituent metabolites As the adjusted Kaplan-Meier curve demonstrated, patients with heart failure in the (i.e., metabolites with the highest factor load in the highest tertile of long-chain acylcarnitine factor (factor 5) had an increased risk of the factor) changed significantly with LVAD support primary outcome of all-cause mortality and hospitalizations compared with patients in (Figure 2). As shown, C16, C18:1, and C18:2 were lower tertiles. Risk increased for all patients over time. significantly higher at baseline in patients with endstage HF prior to LVAD placement and decreased after support. The other major components of factor 5—arginine, C18, and C20:4—did not change significantly with LVAD support. The mammalian heart has a unique ability to switch between fuel sources to adapt to changing physiological or dietary conditions—so-called metabolic flexibility. Healthy myocardium primarily meets DISCUSSION its requirements for energy through the oxidation of long-chain fatty acids (LCFA), where carnitine plays a This study examined the association of baseline key role as a carrier (15) (Central Illustration). LCFAs metabolomic profiles with measures of cardiorespi- are activated by esterification to coenzyme A (CoA) at ratory fitness and clinical outcomes in 453 ambula- the outer mitochondrial membrane. The inner mito- tory patients with chronic systolic HF. We found a chondrial membrane is impermeable to the acyl-CoA metabolite factor (factor 5), composed mostly of long- esters. The “carnitine shuttle,” which regulates the chain acylcarnitines, that was independently associ- flux of acyl-CoA esters into the mitochondria requires ated with lower peak V O 2 as well as both the primary the use of 3 major proteins: carnitine palmitoyl- and secondary clinical endpoints of the parent trial transferase (CPT) I, carnitine-acylcarnitine translo- (HF-ACTION). Of the major components of this factor, case (CACT), and CPT II. levels of C16, C18:1, and C18:2 acylcarnitines were Many clinical and experimental studies have significantly higher in patients with end-stage HF demonstrated that the failing heart undergoes meta- prior to LVAD implantation but decreased with cir- bolic remodeling and develops a metabolic inflexi- culatory support. The direction of this improvement bility, switching to glucose utilization at the expense would have predicted better outcomes in HF- of FA oxidation (3). Although the molecular changes ACTION. This pattern of metabolite abnormalities underlying the change in fuel utilization are complex suggested impaired mitochondrial FA oxidation, a and still incompletely understood, mitochondrial finding previously described in HF. This further dysfunction remains a common pathological theme confirms that HF is characterized by dysfunction in a (16). In our study, we observed that elevated plasma central pathway of energy utilization by the heart and levels of key long-chain acylcarnitines (C16 and C18) peripheral musculature, that the measured abnor- were independently associated with impaired cardio- malities possess prognostic importance, and that use respiratory capacity and also increased risk of all of adverse clinical outcomes. These C16 and C18 acyl- mitochondrial-based 295 Metabolite Profiles of Systolic Heart Failure therapeutics might promise in treating chronic systolic HF (3,14). hold carnitines are derivatives of the most abundant dietary 2 Ahmad et al. 296 JACC VOL. 67, NO. 3, 2016 JANUARY 26, 2016:291–9 Metabolite Profiles of Systolic Heart Failure plasma levels of these molecules are characteristic of F I G U R E 2 Factor 5 and LVAD Support disorders of the carnitine shuttle, specifically, CPT II and CACT deficiencies, both of which are associated Metabolite levels in chronic HF patients and changes with LVAD placement in end-stage HF with skeletal and cardiac myopathy (17). CPT II defiMetabolite Levels 80 60 40 20 0 Chronic HF End Stage HF Arginine Post LVAD † 0.1 0.05 0 Chronic HF End Stage HF * † 0.1 demise from skeletal muscle damage and cardiomy- defects, that the degree of these defects worsen in * patients with more advanced disease, and that they † may be reversible with cardiac support. In addition to defects in the carnitine shuttle leading to mitochondrial dysfunction and impaired FA oxidation, the accumulation of circulating long-chain 0.05 acylcarnitines may reflect a shift toward increased myocardial glucose oxidation with down-regulation Post LVAD C18:1 of fatty acid oxidation that has been described in the 0.04 Metabolite Levels 0.04 0.03 0.02 0.01 0 Chronic HF End Stage HF recapitulates a milder form of these basic metabolic 0.1 C18:2 Metabolite Levels opathy. Our findings suggest that chronic systolic HF Post LVAD 0.15 0 Chronic HF End Stage HF 0.05 progression of HF. To support this hypothesis, a † recent study in murine models of HF demonstrated 0.03 progressive down-regulation of genes involved in 0.02 myocyte FA oxidation and transport and a coordinated increase in myocardial long-chain acylcarnitine 0.01 species that marked the transition from compensated 0 Chronic HF End Stage HF Post LVAD ability, with muscle weakness and cardiomyopathy thal disease, CACT deficiency results in early infant 0.05 0.2 Post LVAD ciency can present with extreme phenotypic varibeing common associated findings. A far rarer and le- 0 Chronic HF End Stage HF C16 * 0.15 Metabolite Levels 0.15 † Metabolite Levels Metabolite Levels 100 C18 hypertrophy to HF (18). Moreover, increased rates of Post LVAD FA oxidation may produce a “bottleneck” of substrate C20:4 flux into the Krebs cycle, leading to the accumulation * Significant pre-post LVAD of oxidative intermediates such as acylcarnitines, † Significant between chronic HF and pre-LVAD mitochondrial dysfunction, and the depletion of adenosine triphosphate needed for contractile function (19). The possibility also exists that these changes Although levels of the factor 5 metabolites C16, C18:1, and C18:2 were significantly higher at baseline in patients with end-stage heart failure (HF) prior to left ventricular assist device in plasma metabolites reflect impaired peripheral (LVAD) placement, those levels decreased after support. The other major components of factor metabolism. Another recent animal study demon- 5—arginine, C18, and C20:4—did not change significantly with LVAD support. *Metabolite strated that increased skeletal muscle mitochondrial levels differed significantly pre- and post-LVAD implantation. †Metabolite levels differed efficiency, as reflected by more complete FA oxida- significantly between patients with chronic HF and those with end-stage HF pre-LVAD. tion, may underlie changes in exercise capacity (peak V O2) (20). Thus, the changes in plasma metabolites fatty acids, palmitate and oleate, respectively. Patients observed in our study may indicate impaired periph- with end-stage as compared with chronic systolic HF eral fatty acid oxidation and thus impaired peak VO 2, demonstrated significantly higher levels of C16 and which is a major predictor of adverse outcomes in C18, which decreased with LVAD support. These find- individuals with heart failure. Overall, more work ings are consistent with the notion that the syndrome utilizing metabolic flux techniques in cellular and of HF may be characterized by a general state of animal models may help to unravel the mechanisms metabolic inflexibility and mitochondrial inefficiency that link elevated circulating acylcarnitines and HF. that leads to accumulation of metabolic intermediates Although our primary hypothesis was that eleva- of FA oxidation such as the long-chain acylcarnitines. tions in long-chain acylcarnitine levels simply signal Moreover, our findings indicate that these metabolic mitochondrial dysfunction, there is a distinct possi- changes have distinct prognostic implications. bility that they may also contribute to disease proas- gression. Studies have demonstrated that cardiac sociation of these long-chain acylcarnitines is unclear. myocytes exposed to hypoxia exhibit rapid accumu- One explanation may derive from abnormalities lations seen in rare Mendelian disorders. Irregularities in amphiphilic molecules have been shown to inhibit The precise mechanisms underlying the in long-chain acylcarnitines, and these Ahmad et al. JACC VOL. 67, NO. 3, 2016 JANUARY 26, 2016:291–9 CENTRAL I LLU ST RAT ION Metabolite Profiles of Systolic Heart Failure Metabolite Profiles of Systolic Heart Failure: Free Fatty Acid Metabolism •Elevations associated with adverse outcomes in heart failure Arrhythmias Insulin Resistance Inflammation Adverse Remodeling Serum Levels of Long Chain Acylcarnitines •Modifiable with ventricular support C14-C18 Fatty Acids Long Chain Fatty Acid Transporter Plasma Membrane ACYL-CoA Carnitine Glucose Carnitine Transporter Carnitine GLUT CPT I Glycolysis CoASH AcylCarnitine Carnitine Worsening Heart Failure Outer Mitochondrial Membrane CACT AcylCarnitine Carnitine CPT II CoASH Acyl-CoA Carnitine Inner Mitochondrial Membrane ACETYL-CoA Ketogenesis TCA Cycle ENERGY Energy Production Ahmad, T. et al. J Am Coll Cardiol. 2016; 67(3):291–9. The primary energy source for the normally functioning human heart is free fatty acids, which are broken down by b-oxidation and entered into the Krebs cycle for eventual conversion into adenosine triphosphate (ATP). Long-chain fatty acids are converted to their respective acyl-coenzyme A (CoA) ester by the ATP-dependent acyl-CoA synthetases. These acyl-CoA esters are then converted into acylcarnitine and free CoA by carnitine palmitoyltransferase (CPT)-I at the outer mitochondrial membrane. The resulting acylcarnitine is then transported across the inner mitochondrial membrane by the carnitine:acylcarnitine translocase in exchange for free carnitine. Once inside the mitochondrial matrix, the acyl-CoA ester is reformed by CPT-II, and carnitine is released for further exchange by the carnitine:acylcarnitine translocase. In the failing heart, dysfunction in these key enzymes may lead to inadequate substrate utilization, which would be hypothesized to be reflected in serum elevations of long-chain fatty acid intermediate metabolites such as long-chain acylcarnitines. These can contribute to worsening heart failure via promotion of arrhythmias, insulin resistance, adverse remodeling, and decreased energy production. Targeting these pathways might lead to novel therapeutics for heart failure. CACT ¼ carnitine-acylcarnitine translocase; TCA ¼ tricarboxylic acid. excitatory sodium currents in vitro (21). Furthermore, Long-chain acylcarnitines are also associated with long-chain acylcarnitines increase calcium efflux in a insulin resistance; these reside in cell membranes concentration-dependent manner in isolated cardiac where they can potentially interfere with insulin sarcoplasmic reticulum vesicles (21). This might pre- signaling directly within the cell membrane (13). This dispose HF patients to malignant arrhythmias (22). might explain the noted state of insulin resistance 297 298 Ahmad et al. JACC VOL. 67, NO. 3, 2016 JANUARY 26, 2016:291–9 Metabolite Profiles of Systolic Heart Failure seen in patients with HF (23). If true, these data will metabolite profiles and therefore cannot assume that provide additional support for developing therapeutic they represent myocardial metabolism, but rather strategies to improve these functions in the myocar- reflect global changes in metabolism. dium. Furthermore, there may be a role for testing the efficacy of currently available mitochondrial-based therapies such as mitoprotective agents and L-carnitine supplementation in HF (24). The improvement with LVAD support we noted indicates that these molecules may also play a role in monitoring the efficacy of current and novel therapeutics in HF (25). Several strategies have been proposed for specific molecular targets for modifying mitochondrial function: micronutrient supplementation, increasing mitochondrial biogenesis, decreasing production of reactive oxygen species, and improvement of cellular iron homeostasis (24,26,27). Of particular interest: a novel class of compounds that selectively target cardiolipin on the inner mitochondrial membrane to optimize efficiency of the electron transport chain and thereby restore cellular bioenergetics (9,28). A key next step might be to evaluate changes in cardiorespiratory capacity and metabolite profiles as biomarkers of response during trials of these and other agents in clinical trials. STUDY LIMITATIONS. Our patient population was a subset of the total HF-ACTION study; however, there were no meaningful differences in key baseline characteristics between these patients and the overall trial. The LVAD patient population was relatively small and was from a single center. These results should be confirmed in another similar population. Our current study should therefore be considered hypothesis-generating; further studies are required to CONCLUSIONS We found that greater circulating levels of longchain acylcarnitines were independently predictive of functional status and mortality in patients with chronic systolic HF. The abnormalities were modifiable with LVAD support in end-stage HF patients. These findings suggest a potentially novel way to prognosticate and manage HF patients in clinical practice while providing an impetus for pharmacological targeting of the mitochondria for treatment of HF. REPRINT REQUESTS AND CORRESPONDENCE: Dr. Tariq Ahmad, cine, Yale Cedar Section University Street, New of Cardiovascular School Haven, of Medi- Medicine, Connecticut 330 06510. E-mail: [email protected]. 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