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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.
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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.
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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.
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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.
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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
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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].
PERSPECTIVES
COMPETENCY IN MEDICAL KNOWLEDGE:
Circulating levels of long-chain acylcarnitine metabolite in patients with HF are associated with adverse
clinical outcomes.
TRANSLATIONAL OUTLOOK: The metabolic processes that regulate blood levels of mitochondrial FA
metabolites are potential targets for diagnostic or
therapeutic modalities in patients with worsening HF.
confirm the findings. Last, we report on peripheral
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A PP END IX For supplemental tables,
please see the online version of this article.
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