Delta-6-desaturase Links PUFA Metabolism with Phospholipid

Transcription

Delta-6-desaturase Links PUFA Metabolism with Phospholipid Remodeling
and Disease Progression in Heart Failure
Le et al: Phospholipid Remodeling in Heart Failure
Catherine H. Le, PhD1,2*; Christopher M. Mulligan, MS1,3*; Melissa A. Routh, MS1,2;
Gerrit J. Bouma, PhD4; Melinda A. Frye, PhD4; Kimberly M. Jeckel, PhD4;
Genevieve C. Sparagna, PhD6; Joshua M. Lynch, MS6; Russell L. Moore, PhD5;
Downloaded from http://circheartfailure.ahajournals.org/ by guest on November 17, 2016
Sylvia A. McCune, PhD5; Michael Bristow, MD, PhD7; Simona Zarini, PhD8;
Robert C. Murphy, PhD8; Adam J. Chicco, PhD1-5
1
Integrative Cardiac Biology Laboratory, 2Program in Cell and Molecular
Moleecu
cula
larr Bi
la
Biol
Biology,
oloog
ol
og Departments
of 3Food Science and
nd H
Human
uman
um
a N
an
Nutrition,
utrition, 4Biomedic
Biomedical
Bi
diiccal
al Sciences,
S i
aand
n 5Health and Exercise
nd
Science, Colorado State
tate Univers
University,
r itty,
rs
y Fort
For
ort Collins,
Coll
Co
l ins,
ll
s, CO;
CO
O; 6De
Department
Depa
partme
ment
ntt ooff In
Inte
Integrative
tegr
te
graattiv Physiology,
gr
University of Colorado,
ado
ado
o, Bo
Boul
Boulder,
ulddeer,
r C
CO;
O 7Di
O;
Division
Divi
visi
vi
sion
si
on of
of Cardiology
Card
Ca
rdio
rd
iolo
io
logy
lo
gy and
anndd 8De
Department
Depa
part
pa
rtme
rt
mennt of
me
Pharmacology,, University
v
versity
of Colorad
Colorado
ad
do at
at D
Denver,
enve
en
v r, D
Denver,
enve
en
ver,
ve
r C
CO
O
*These authors contributed equally
Correspondence to
Adam J. Chicco, PhD
Colorado State University
200 West Lake Street, Mailstop 1582
Phone: 970-491-3605
Fax: 970-491-0445
Email: [email protected]
DOI: 10.1161/CIRCHEARTFAILURE.113.000744
Journal Subject Codes: Myocardial biology:[107] Biochemistry and metabolism, Heart
failure:[110] Congestive, Hypertension:[16] Myocardial cardiomyopathy disease, Basic science
research:[130] Animal models of human disease
Abstract
Background—Remodeling of myocardial phospholipids has been reported in various forms of
heart failure for decades, but the mechanism and pathophysiological relevance of this phenomenon
have remained unclear. We examined the hypothesis that delta-6 desaturase (D6D), the rate
limiting enzyme in long-chain polyunsaturated fatty acid (PUFA) biosynthesis, mediates the
signature pattern of fatty acid redistribution observed in myocardial phospholipids following
chronic pressure overload, and explored plausible links between this process and disease
pathogenesis.
Methods and Results—Compositional analysis of phospholipids from hearts explanted from
patients with dilated cardiomyopathy revealed elevated PUFA product/precursor ratios reflective
of D6D hyperactivity, manifesting primarily as lower levels of linoleic acid with reciprocally
tte
tern
rn
n ooff re
remo
mode
mo
dell
de
higher levels of arachidonic and docosahexaenoic acids. This pattern
remodeling
was
icula
laar as
aassist
sist
si
st ddevice.
e
ev
attenuated in failing hearts chronically unloaded with a left ventricular
Chronic
n vvivo
ivo rev
iv
everrsed similar patterns of
ev
of myocardial
myocaard
r iaal PUFA redistrib
b
inhibition of D6D in
reversed
redistribution
in rat
ove
ove
verload and
an
nd hypertensive
hypeertensive
hy
vee heart
heartt dise
seaase, aand
se
ndd ssignificantly
ig
gnifica
canntly aattenuated
ca
ttt
models of pressure overload
disease,
cardiac
dysfunctio
dy
ionn inn both
io
botth models.
m de
mo
d ls. D6D inhibition
n also attenuated
hypertrophy, fibrosiss andd contractile dysfunction
ns in ppathogenic
atho
at
hoge
ho
g ni
ge
nicc ei
eico
cosa
co
sano
sa
noid
no
id sspecies,
p ci
pe
cies
es,, li
es
lipi
p d pe
pi
pperoxidation,
rox
oxid
idat
id
atio
at
ionn, aand
io
nd E
myocardial elevations
eicosanoid
lipid
ERK1/2 activation;
normalized cardiolipin composition in mitochondria; reduced circulating levels of inflammatory
cytokines; and elicited model-specific effects on cardiac mitochondrial respiratory efficiency,
NFțB activation and caspase activities.
Conclusions—These studies demonstrate a pivotal role of essential fatty acid metabolism though
D6D in myocardial phospholipid remodeling induced by hemodynamic stress, and reveal novel
links between this phenomenon and the propagation of multiple pathogenic systems involved in
maladaptive cardiac remodeling and contractile dysfunction.
Key Words: heart failure, hypertension, fatty acid, metabolism, inflammation
1
Heart failure is a complex, multifactorial syndrome characterized by progressive cardiac
remodeling and contractile dysfunction that leads to an impaired matching of blood supply to
tissue demands. Antecedent hypertension is present in the majority of cases1, supporting a
paradigm by which chronic hemodynamic overload of the myocardium results in initially
compensatory adaptations that ultimately become maladaptive, leading to pathologic
hypertrophy, fibrosis and mechanical failure. Several well-established pathogenic systems have
been implicated in the transition from adaptive cardiac hypertrophy to maladaptive remodeling
and failure, including chronic inflammation2, oxidative stress3, impaired myocardial energetics4
and apoptosis5. However, the molecular triggers and precise contribution
ntribu
buti
bu
tion
ti
on of
of these
thees
th
es processes
nse investigation
ns
inves
e tiigation and debate.
es
remain areas of intense
Alterations inn the
the
h fatty
fattty
t acid
acidd composition
co
omp
mpos
osittio
os
ionn of
of myocardial
myoocar
my
ardiial phospholipids
phosp
sphooli
sp
lipi
pidds hhave
pi
av
v been reported
6, 7
c
pathologyy fo
forr ov
ove
er 25
2 yyears,
eaars
rs, in
ncl
c ud
udin
ing human cardiom
in
m
in various forms of cardiac
over
including
cardiomyopathies
and animal models of pressure overload hypertrophy 77, 8, hypertensive heart disease 7, 9, postinfarct remodeling10, diabetic cardiomyopathy11, and senescence 12. Interestingly, despite
distinct etiologies, genetic backgrounds, and biochemical techniques, the pattern of phospholipid
remodeling has consistently manifested as a proportional loss of the essential polyunsaturated
fatty acid (PUFA) linoleic acid (18:2n6; LA), often paralleled by reciprocal increases in long
chain highly unsaturated fatty acids such as arachidonic acid (20:4n6, AA) and/or
docosahexaenoic acid (22:6n3, DHA). While several hypotheses have been proposed, the
mechanism and pathophysiological relevance of this phenomenon have remained areas of
speculation.
2
The present investigation explored the hypothesis that the redistribution of phospholipid
PUFAs in the pressure overloaded heart results from increased activity of delta-6 desaturase
(D6D), the rate-limiting enzyme in the production of long-chain PUFAs such as AA and DHA
from LA and D-linolenic acid (18:3n3, ALA), respectively13 (Figure 1). Serum PUFA ratios
reflective of systemic D6D hyperactivity have been reported in patients with hypertension14, 15,
and are highly predictive of overall cardiovascular mortality in humans16. However, no
experimental studies have investigated the role of this enzyme in myocardial phospholipid
remodeling or the pathogenesis of heart disease. Herein, we present the first comprehensive
with
hou
o mechanical
analysis of phospholipid composition in the failing human heart with and without
unloading support, and investigated the effects of chronic pharmacological
acolo
lo
ogi
gica
call D6
ca
D6D
D inhibition in
ess
ess
ssure overload
ovver
erlo
l ad hypertrophy
hyp
yper
ertr
er
trop
tr
o hyy and
op
an
nd hypertensive
hype
hy
pert
pe
r en
ensive heart
ensi
hea
eart
rt ddisease.
isea
is
ease
ea
se. These studies
se
rodent models of pressure
a ro
al
role
le ooff D6
D6D
D in
in m
yoo rd
yocard
rdia
iall ph
ia
phos
osph
osph
phol
olip
ol
ipid
ip
id rremodeling
emod
em
oddel
elin
ingg re
in
rresulting
sult
su
lt
demonstrate a central
myocardial
phospholipid
from
nd rreveal
ev
vea
eall no
nove
vell li
ve
link
nkss be
nk
betw
twee
tw
eenn th
ee
this
iss pprocess
roce
ro
cess
ce
ss aand
nd ddisease
isea
is
ease
ea
se pprogression
ro
hemodynamic stresss aand
novel
links
between
at the
molecular, organ, and systemic levels.
Methods
Human Heart Tissue
Left ventricular tissue was obtained by an Institutional Review Board-approved protocol
maintained by the University of Colorado Denver Cardiac Tissue Bank. Hearts donated for
research purposes were obtained under written consent from family members of organ donors or
by direct written consent from patients undergoing cardiac transplantation. Patient characteristics
are presented in Table S1.
3
Animal Models
Lean male spontaneously hypertensive heart failure (Mccfacp-/-; SHHF) rats were obtained from a
colony maintained at the University of Colorado. SHHF rats were selected for these studies
based on their well-characterized development of progressive hypertensive cardiomyopathy that
shares many of the hallmark biochemical and pathophysiological features of DCM in humans17.
Two cohorts of animals were studied: 1) at 21-22 months of age when rats exhibit pathologic
), and 2) follo
ow
cardiac hypertrophy progressing toward dilated heart failure (HF),
following
thoracic
path
thol
th
olog
ol
ogic
og
ic hhypertrophy in
aortic banding at 3 months of age to induce hemodynamic stress andd pa
pathologic
ela
lated pathology
paath
hol
o og
ogyy (TAC)
(T
TAC
AC))7. Cohorts
Coh
o or
ortss of
of HF and
and TAC
AC (2
(2 weeks
week
we
ekss post-surgery)
ek
the absence of age-related
divided
vide
vi
dedd aan
de
and
nd ma
matc
matched
tcheed on eechocardiography
tc
choc
ch
ocar
oc
arrdi
d og
ogra
raph
ra
phyy pa
ph
para
parameters
raame
mete
terrs bbefore
te
e or
ef
oree being semianimals were each divi
randomly assigned to
o rreceive
ecei
ec
eive
ei
ve tthe
he D
D6D
6D iinhibitor
nhib
nh
ibit
ib
itor
it
or ((SC)
SC)) oorr no ddrug
rugg fo
ru
forr 4 we
week
weeks.
ekss. T
ek
The
h effect of the
D6D inhibition was also examined in 3 month old SHHF rats exposed to a sham TAC surgery
(Sham) that were followed along with TAC groups for 4 weeks as an experimental control. All
animals were provided Purina 5001 chow and water ad libitum for the duration of the study. At
the conclusion of the study, animals were sacrificed with a lethal dose of sodium pentobarbital
(150 mg/kg i.p.) followed by midline thoracotomy and removal of the heart. All procedures
were approved by the Animal Care and Use Committee at Colorado State University and/or
University of Colorado Boulder in strict compliance with the Guide for the Care and Use of
Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 8523, revised 1996).
4
Inhibition of D6D in vivo
Rats were administered the potent, orally active D6D inhibitor SC-26196 (a gift from Dr. Mark
Obukowicz, Pfizer Corporation), at a dose previously reported to selectively inhibit D6D enzyme
activity with no effect on other desaturase enzymes in rodents in vivo (100 mg/kg/d mixed in
chow for 4 weeks), based on daily food consumption records taken over 3-4 weeks prior to
treatment18.
Echocardiography and Blood Pressure
urane
ne aanesthesia
nest
ne
sthe
st
hessi
he
sia prior to and
Transthoracic echocardiography was performed under light isoflurane
k eexperimental
xperim
im
men
enta
tall pe
ta
peri
riod
ri
od uusing
s ng a 12
si
12 M
Hz ppediatric
e ia
ed
iatr
ttrricc ttransducer
rans
ra
n du
duce
cerr connected
ce
c
following the 4 week
period
MHz
to a
n s 55
nos
55500
500
00 U
l ras
lt
raasou
o nd aass pr
prev
evio
ev
ious
io
usly
us
ly
y ddescribed
escr
es
crib
cr
ibed
ib
ed1199. Tail
Tai
aill cuff
cuff
ff blood
blo
lo pressure
Hewlett Packard Sonos
Ultrasound
previously
measurements were obtained
obta
ob
tain
ta
ined
in
ed in
in the
the Sham
Sham and
and HF
HF groups
gro
roup
up
ps using
usin
us
ingg the
in
the Kent
Kent Coda
Cod
odaa 6 system (Kent
Scientific, Torrinton, CT) in lightly isoflurane-anesthetized rats.
Lipid analyses
See the Expanded Methods on the online supplement for a detailed description of lipid analyses.
Briefly, phospholipids were extracted by thin layer chromatography (total) or liquid
chromatography (individual species) for compositional analysis by gas chromatography (fatty
acid composition) or electrospray ionization mass spectrometry (cardiolipin molecular species).9
Myocardial contents of free AA and eicosanoid species were quantified in lipid extracts obtained
from 50 mg of LV tissue by LC/MS/MS methods using deuterated standards as previously
described 20.
5
Mitochondrial isolation and respiratory function
Mitochondria were freshly isolated from ~300 mg of left ventricular (LV) tissue by differential
centrifugation in the absence of proteases and assayed for respiratory function using a Clark-type
electrode system (Strathkelvin) with pyruvate + malate as substrates as previously described19 .
Biochemical analyses
Immunoblotting was performed by standard methods using commercially available antibodies.
tric assays (Biovision).
(Bio
iov
io
Serum glucose and free fatty acids were determined by colorimetric
Caspase
nesce
c nc
ce
ncee as
assa
sayy (Promega).
sa
activities were determined in 30 mg of LV homogenates by luminescence
assay
ypr
ypr
prooline wa
wass qu
quan
antiitat
an
ated
ed aass a ma
arker
er ooff co
ccollagen
oll
llag
ll
agen
ag
n (f
(fib
ibro
ib
rosi
ro
sis)
si
s) iin
n 30-40 mg of
Myocardial hydroxyproline
quantitated
marker
(fibrosis)
2
collor
col
orim
imet
im
e riic assay
et
ass
ssay
a of
of Sw
Swit
itze
it
zerr an
ze
nd Su
Summ
mmer
mm
er 21
. Serum
Ser
erum
um ccytokines
y ok
yt
okin
in were
septal tissue by the colorimetric
Switzer
and
Summer
determined in 80 ȝL
L of ssample
ampl
am
p e by
pl
yE
ELISA
LISA
LI
SA cytokine
cyt
y ok
okin
inee array
in
arra
ar
rayy (Raybiotech).
ra
(Ray
(R
ay
ybi
biot
otec
ot
ech)
ec
h)). qRT-PCR
qRT
RT-P
-PC
-P
C was
performed using 2X SYBR Green qPCR Master Mix and validated gene specific primers (listed
in Table S2) with resulting data normalized to 18S rRNA and analyzed according to the
comparative (ǻǻCt) Ct method.
Statistical analyses
All data are presented as group means r standard error. Human heart data were compared by
one-way ANOVA with Tukey HSD tests post hoc when appropriate. Data from the animal
studies were analyzed by separate 2 (condition) X 2 (drug) ANOVAs for determination of main
and interaction effects of SC treatment and TAC or HF vs. Sham cohorts, followed by Dunnett’s
test for comparison of group means vs. Sham control. Mean differences between SC-treated and
6
untreated groups were compared by Independent sample t-tests to examine a priori hypothesized
effects of D6D inhibition within each condition. Pre- to post-treatment differences in
echocardiography parameters were examined by paired t-tests for each condition cohort. Pearson
correlations were calculated to determine the associations between phospholipid fatty acid
composition and lipid peroxidation adducts. All reported p-values are two sided. Statistical
significance was established at P < 0.05, using SPSS 21 software (IBM) for all analyses.
Results
rtt aand are reversed
Phospholipid indices of D6D activity are elevated in the failing human heart
by mechanical unloading
etar
et
a y supplementation,
supp
ppple
leme
ment
me
ntat
ntat
nt
atio
atio
ion,
n, desaturation
des
e at
atuuraatiionn aand
n eelongation
nd
l ng
lo
ngatio
ionn of
io
of L
A aand
ndd ALA provide the
In the absence of dietary
LA
a n PUFAs
ain
PUF
UFAs
As present
preese
sentt in
in mammalian
mamm
ma
mmaaliian
mm
n tissues.
tis
issu
sues
su
es. The
es
The most
most abundant
abu
bund
ndan
nd
an
n of these are AA
majority of long-chain
ong with
wit
ithh LA account
acc
ccou
ount
ou
nt for
for over
ove
verr 90%
90% of PUFAs
PUF
UFAs
As in
in membrane
memb
me
mbra
mb
rane
ra
ne phospholipids,
p
and DHA, which along
their
primary site of incorporation. D6D catalyzes rate-limiting steps in this pathway (Figure 1),
therefore, changes in specific tissue phospholipid PUFA product/precursor ratios (e.g., AA/LA,
20:3n6/LA and DHA/22:5n3) reflect chronic changes in D6D activity in vivo16 . Reduced levels of
cardiolipin molecular species normally enriched with LA acyl chains have been reported in hearts
explanted from patients with dilated and ischemic cardiomyopathies6 7. However, whether these
changes occur in other phospholipid classes or are reflective of a global redistribution of PUFAs in
the total myocardial phospholipid pool was not addressed. Therefore, we performed a detailed
compositional analysis of myocardial phospholipids from patients with dilated cardiomyopathy
(DCM; n = 8) with or without mechanical unloading with a left ventricular assist device for at least
4 months prior to explant (LVAD; n = 4) (Figure 2).
7
Patient characteristics are presented in Table S1. Hearts from DCM patients exhibited
markedly lower proportions of total phospholipid LA, with correspondingly higher levels of AA,
DHA and PUFA product/precursor ratios reflective of D6D hyperactivity compared to donor
hearts from age-matched individuals with no cardiac pathology (NF; n = 8). Comparatively minor
differences were seen in saturated and monounsaturated fatty acid species (Figure S1). Chronic
LVAD support was associated with significantly higher phospholipid LA levels and reduced AA,
DHA and D6D product/precursor ratios compared to DCM. Compositional analysis of the three
major phospholipid species present in the mammalian heart, phosphatidylcholine (PC),
ns of fatty acid
phosphatiydylethanolamine (PE) and cardiolipin (CL), revealed similar pattern
patterns
tudee ooff ch
han
ange
gess and relative
ge
redistribution observed in total phospholipids, though the magnitude
changes
dua
u l fatty
fattty acids
ac ds varied
var
arie
iedd considerably
ie
cons
co
nssid
i errably
y between
bet
e weeen classes
c as
cl
asse
sess (Figure
se
(F
Fig
igur
ure S1).
ur
abundance of individual
e se
erse
er
sess ph
phos
osph
os
p ol
ph
olip
ipid
ip
id P
UFA
UF
A remodeling
remo
re
mode
mo
deli
de
ling
li
ngg in
in TAC
TAC and
and HF
D6D inhibition reverses
phospholipid
PUFA
As seen in DCM patients, hearts from both TAC and HF animals exhibited markedly lower
phospholipid LA content, with corresponding elevations in AA, DHA and D6D
product/precursor ratios in total myocardial phospholipids compared to Sham controls (Figure
3A,B). Chronic administration of the D6D inhibitor ameliorated these effects of TAC and HF,
bringing total phospholipid LA, AA, DHA and D6D activity indices to near Sham control levels.
Despite intrinsic differences in baseline fatty acid composition, similar patterns of PUFA
redistribution were seen within individual phospholipid classes in both models that were reversed
by D6D inhibition (Figure S2). Interestingly, only minor effects of D6D inhibition were seen on
phospholipid PUFA composition in the Sham rats, suggesting that “basal” levels of PUFAs in
membranes are highly defended, perhaps aided by an incomplete inhibition of D6D enzymatic
8
activity in vivo (over a 24 hour period) and/or trace amounts of AA and DHA present in rodent
chow (Purina 5001).
D6D inhibition normalizes the cardiolipin molecular species profile in mitochondria
Alterations in the highly regulated fatty acid composition of cardiolipin may have particular
relevance in cardiac pathologies22. Therefore, cardiolipin molecular species were also examined
in cardiac mitochondria isolated from animals in this study. As previously reported 7, a marked
loss of the predominant tetra-linoleoyl species (L4CL) was seen in TAC and HF mitochondria,
or DHA (highly
(highl
hlly unsaturated CL;
which corresponded to elevations in species containing AA and/or
HUFA(CL)), without any appreciable effect on total CL content (Figure
(Figu
gure
gu
re 3C
3C and
and S4). D6D
inhibition completely
ly normalized
ly
normal
a izzed the
al
the CL
CL molecular
mole
mo
l cu
cularr species
sppec
ecie
ies profile
ie
prof
pr
offil
ile in TAC
TAC aand
nd H
HF, restoring
L4CL and reducing HUFA(CL)
HUFA
HU
FA(C
(CL)
(C
L) species
spe
peci
c ess to
to Sham
Shham control
con
o tr
trol
ol llevels
evel
ev
e s in
el
n bboth
othh gr
ot
ggroups.
oups
ou
p .T
ps
These
h
data
provide novel evidence
nce for
for a central
cen
entr
tral
tr
al role
rol
olee of LA
LA desaturation
desa
de
satu
sa
tura
tu
rati
ra
tion
ti
on and/or
and
nd/o
/orr en
/o
endo
endogenous
doge
do
g no
ge
nous
us H
HUFA
production in modulating CL composition in the hypertrophied and failing heart.
Myocardial free arachidonic acid and eicosanoid contents
Once liberated from phospholipids by phospholipase enzymes, AA can serve as a substrate for
multiple oxygenase enzymes and non-enzymatic oxidation pathways capable of generating a host
of bioactive eicosanoid species with complex effects on inflammatory, cardiovascular and
transcriptional regulation 23, 24 . Both TAC and HF elicited significant increases in free AA and
several eicosanoid species in the heart, all of which were reduced to near control levels with
D6D inhibition (Figure 3D). Particularly significant changes were seen in 12- and 15hydroxyeicosatetraenoic acid (12- and 15-HETE), thromboxane A2 (TXA2), and isoprostanes
9
(IPs), which are formed via 12/15-LO, COX-2, and the non-enzymatic peroxidation of AA by
ROS, respectively.
Serum and hepatic phospholipids, circulating cytokines and animal characteristics
Analysis of total serum and liver phospholipids revealed similar patterns of PUFA distribution
seen in cardiac phospholipids in TAC and HF (Figure S2), suggesting that myocardial changes
may be reflective of elevated systemic (e.g., hepatic) D6D activity. No significant effects of
D6D inhibition were observed on body weight, serum glucose, free fatty acids, or blood pressure
ron
o
in any of the groups (Table). However, significant elevations in serum interfer
interferon-gamma
(IFNg)
d HF
F groups
grou
gr
ou
ups w
e attenuated
er
and serum monocyte chemotactic protein-1 (MCP-1) in TAC and
were
18
tment, consistent
tm
con
on
nsi
s stten
entt with
with an
an anti-inflammatory
an
nti
ti-inf
nflaamm
mmat
ator
at
oryy effect
or
e ffec
ef
ecct of D
6D iinhibition
n
.
with SC-26196 treatment,
D6D
nua
uate
tess maladaptive
te
mala
ma
lada
la
dapt
da
ptiv
pt
ivee cardiac
iv
card
ca
rdia
rd
iacc remodeling
ia
remo
re
mode
mo
deli
de
ling
li
ngg aand
nd ccontractile
ontr
on
trac
tr
acti
ac
tile
ti
le ddysfunction
ysf
ys
sff
D6D inhibition attenuates
Echocardiography performed before and after the 4-week experimental period revealed
significant progression of left ventricular dilatation and contractile dysfunction in the untreated
TAC and HF animals that were significantly attenuated or reversed by D6D inhibition (Figure
4A, B). These improvements corresponded to lower final heart weights in the treated TAC and
HF rats compared to untreated animals, and a trend for lower wet lung weights suggestive of
reduced pulmonary congestion (Figure 4C, Table S3). Notably, a mild degree of cardiac
hypertrophy and reduced fractional shortening is evident in the Sham SHHF rats compared to
other rat strains at this age 7, 17 that was unaffected by D6D inhibition, indicating that this
treatment attenuates the progression of more marked pathologic remodeling and dysfunction
seen following chronic hemodynamic stress. Myocardial fibrosis, assessed by tissue
10
hydroxyproline content, was also reduced by D6D inhibition in TAC and HF (Figure 5B), which
paralleled histological evidence of reduced interstitial and perivascular collagen deposition
(Figure 5B, S7). TAC and HF significantly increased mRNA expression of atrial natriuretic
peptide (ANP) and myocardial extracellular-regulated activated kinase 1/2 (ERK1/2)
phosphorylation compared to Sham controls, which was largely prevented by D6D inhibition
treatment in both models (Figure 5C, D). TAC, and to a lesser extent HF, were both associated
with myocardial activation of nuclear factor kappa B (NFƸB), indicated by degradation of its
endogenous inhibitory regulator I kappaB alpha (IƸBD), which was significantly attenuated in
TAC, but not HF (Figure 5E).
ria
ial respiration
respir
irrat
atio
on and
and caspase
casp
ca
spas
sp
asse activities
acctiiviiti
ties
es
Cardiac mitochondrial
n /o
nd/o
/orr ef
ffi
f ciien
ncy
c ooff ox
ooxidative
i attiv
id
ivee ph
phos
ossph
phor
oryl
or
ylat
yl
atiion iin
at
n ca
card
rdiaac mitochondria
rd
m tto
mi
o
Reduced capacity and/or
efficiency
phosphorylation
cardiac
may
bbioenergetics
ioe
oene
nerg
ne
rg
get
etic
icss an
ic
andd co
cont
ntri
nt
ribu
ri
butte th
bu
thee de
deve
veelo
lopm
p en
pm
entt an
and/
d/or
d/
or pprogression
rogr
ro
gres
gr
esss
es
impair myocardial bi
contribute
development
and/or
of heart
failure4. However, TAC had no effect on mitochondrial state 3 (ADP phosphorylating) or state 4
(uncoupled) respiration in the presence or absence of D6D inhibition (Figure 6A). Conversely,
HF was associated with depressed state 3 respiration, respiratory control (RCR) and oxidative
phosphorylation efficiency (ADP/O ratio) compared to TAC and Sham. D6D inhibition
decreased state 4 respiration and restored RCR and ADP/O to near Sham control levels in HF
mitochondria, but had no effect on state 3 respiration. Immunoblotting of mitochondrial
proteins for respiratory complex subunits revealed a deficiency in complexes 1 and 2 in HF
consistent with reduced respiratory capacity in this group (Figure 6B), which was similarly
unaffected by D6D inhibition. Activities of capase-9 and caspase-3/7 were elevated in
11
myocardial tissue from TAC and HF rats compared to Sham controls (Figure 6C). Treatment
with SC-26196 significantly attenuated caspase activities in TAC, but had no effect in HF.
D6D inhibition reduces cardiac lipoxidative stress
Aldehyde products of PUFA peroxidation such as malondialdehyde (MDA) and 4hydroxynonenal (HNE) are common markers of oxidative stress that correlate closely with the
incidence and severity of heart failure in humans25. The peroxidizability of PUFAs increases
exponentially with their double bond content 26, therefore the calculated cardiac membrane
nd was normalized
normali
liiz to Sham
peroxidizability index increased significantly in TAC and HF, and
levels by D6D inhibition (Figure 7A). Consistent with this observation,
rvati
tion
on,, MDAon
MDA- and
a HNE-protein
adducts were elevated
ed in
ed
in TAC
AC and
andd HF
HF hearts,
hear
he
arts
ar
ts, and
and were
w ree significantly
we
sig
igni
nifi
ni
f ca
fi
cant
an ly
y reduced
red
educ
uced
uc
ed by
by D6D inhibition
(Figure 7B). Myocardial
a di
ardi
dial
al MDA
MDA levels
lev
evel
e s correlated
c rr
co
r ellat
ateed positively
poosi
s ti
tive
veely w
with
ithh th
it
thee re
rela
relative
lati
la
t ve pproportion
ti
ro
op
of DHA
in myocardial phospholipids
p ol
phol
ph
olip
ipid
ip
idss across
id
acro
ac
ross
ro
ss the
the experimental
expper
eriime
ment
ntal
nt
al groups,
gro
roup
upps,, whereas
whe
here
reas
re
as a strong
str
tron
ongg ne
on
negative
e
correlation was seen between HNE and phospholipid LA (Figure 7C). D6D inhibition also
tended to decrease myocardial superoxide dismutase (SOD) and glutathione peroxidase (GSHPx)
enzyme contents (Figure 7D), collectively suggesting a decrease in myocardial oxidative stress.
Cardiac and hepatic D6D expression
While our lipid analyses support a central role of D6D in myocardial phospholipid remodeling
associated with pressure overload, the mechanism driving D6D activity in response to
hemodynamic stress is less clear. Expression of D6D is very low in the heart, and while
detectable, we found no evidence of upregulation at the protein or mRNA levels, nor any
significant changes in expression of downstream elongation/desaturation enzymes in TAC, HF or
12
human DCM (Figure S5). Interestingly, LVAD treatment significantly reduced myocardial D6D
mRNA expression compared to DCM and NF controls, despite no detectable changes in enzyme
protein content, suggesting potential regulatory role of hemodynamic unloading on at least
enzyme expression in the human heart. Hepatic D6D might have influenced myocardial
membrane composition by altering the distribution of PUFA supplied to the heart from the
circulation (Figure S2), however liver D6D expression was also unaffected by TAC or HF
(Figure S5). Little is known regarding posttranslational regulation of D6D activity, but putative
mechanisms are currently being investigated in our laboratory.
Discussion
em nstrat
emo
a es a pivotal
at
pivo
pi
vota
vo
tall role
ta
role
l of
of essential
esse
senttia
iall fatty
fatt
fa
t y acid
tt
ac metabolism
met
etab
ab
abol
bol
olis
ism
is
m through
t
The present study demonstrates
D6D in
t re ppattern
ture
atte
at
tern
te
rn off pphospholipid
hoosp
s ho
holiipi
pidd PU
PUFA
FA rredistribution
edis
ed
istr
is
trib
tr
ibuuttiion
ib
n aassociated
s oc
ss
ocia
i te
ia
t d with cardiac
generating the signature
le ex
expe
peri
pe
rime
ri
ment
me
ntal
nt
al m
odel
od
els,
el
s, aand
nd sshow
how
ho
w th
that
at iitt is cclosely
lose
lo
sely
se
ly
y llinked
inke
in
kedd to hhemodynamic
ke
pathology in multiple
experimental
models,
stress in the failing human heart. The remarkable phenotypic effects of D6D inhibition in TAC
and HF animals highlight the pathophysiological importance of this process, and provide novel
insight into the mechanisms responsible for maladaptive remodeling and contractile dysfunction
in the pressure overloaded myocardium. While previous studies have associated serum markers
of D6D activity with coronary artery disease risk and inflammation 16, 27, the present study is the
first to demonstrate a role for this enzyme in regulating myocardial membrane composition and
responses to pathologic stress.
The proportional loss of phospholipid LA is the most marked and consistent
manifestation of membrane remodeling associated with cardiac overload across species, tissues,
and phospholipid classes observed herein and in previous studies. The reversibility of this effect
13
by D6D inhibition implicates the conversion of LA into downstream PUFA
desaturation/elongation products, the most prominent being AA. However, reciprocal increases
in phospholipid AA were not seen to the same magnitude as LA losses in the present study.
Importantly, PUFAs must be hydrolyzed from phospholipids by phospholipase A2 (PLA2)
enzymes in order to react with desaturation/elongation enzymes, and are subsequently reesterifed
into phospholipids by acyltransferase enzymes with varying substrate specificities 28. Similarly,
AA is cleaved from myocardial phospholipids by PLA2 enzymes for subsequent metabolism as
“free” AA by cyclooxygenase (COX), lipoxygenase (LO), or cytochrome P450 monooxygenase
effects
feec 24. Myocardial
enzymes 23, generating a host of eicosanoid species with diverse biological effe
PLA2 activity is elevated in states of pathologic stress, including heart
heaart
r ffailure
a lu
ai
lure
re 29, and
1, 32
upregulation of myocardial
ocar
oc
a dial C
COX
OX 30 aand
OX
nd 112/15-LO
2 15
2/
1 -L
-LO
O31,
ppathways
athw
at
hway
hw
ayss have
ay
h ve bbeen
ha
eenn im
ee
impl
implicated
p
in the
d omy
dio
myop
oppat
opat
athy
hy. Th
hy
T
erref
efor
ore, D
or
6D-d
6D
-d
depe
pend
nden
nd
entt in
en
incr
crea
cr
e se
sess in m
yooccaa
yoca
pathogenesis of cardiomyopathy.
Therefore,
D6D-dependent
increases
myocardial
levels of
free AA and its eicosanoid
sano
sa
noid
no
id dderivatives
eriv
er
ivaati
iv
tive
vess ob
ve
obse
observed
serv
se
rved
rv
ed iin
n TA
TAC
C an
andd HF aanimals
nim
imal
alss ma
al
mayy ha
have
a contributed to
maladaptive remodeling in these cohorts.
Several lines of evidence implicate myocardial AA-derived eicosanoids in the
development of cardiac fibrosis and failure. Among these, the pathogenic roles of TXA2 and
12/15-HETE have been established through the development of transgenic mice with
cardiomyocyte-specific overexpression of COX-2 30 and 12/15-LO31, respectively; both of which
develop marked cardiac fibrosis, hypertrophy and contractile dysfunction. Myocardial AA
promotes extracellular receptor kinase (ERK) signaling and cardiac hypertrophy via activation of
specific intracellular G protein-coupled receptors by TXA2 and ROS-derived isoprostanes in
cardiomyocytes33, 34. Overproduction of 12-HETE also increases ERK activity, hypertrophy and
fibronectin content in cardiac fibroblasts32, suggesting a potential role in myocardial fibrosis.
14
Therefore, D6D inhibition might have attenuated maladaptive remodeling by reducing ERK
signaling by multiple AA-derived eicosanoid species. Eicosanoids also serve as potent initiators
and propagators of inflammatory signaling, many of which converge on the NFƸB pathway 35.
Cardiomyocyte NFƸB signaling has been implicated in the pathogenesis of cardiac hypertrophy
and failure 36 and has been associated with COX-2 induction in the failing human heart 37. D6D
inhibition tended to attenuate NFƸB activation in TAC in the present study, but had no effect in
HF. This is consistent with clinical evidence for a greater degree of inflammation associated
with compensatory hypertrophy during early aortic stenosis compared to decompensated HF38.
ffects thatt are ddifferentially
i
Therefore, NFƸB may mediate temporal and/or model-specific effects
modulated by altered myocardial eicosanoid levels and/or membrane
ranee P
PUFA
UFA
UF
A co
com
composition.
m
In addition to
o it
its
ts supp
suppression
pre
ress
ssio
ss
ionn of ffree
io
reee AA
re
A aand
ndd eic
eicosanoid
icos
ic
osan
os
anoi
an
o d le
oi
leve
levels,
els
ls, D6
D6D
D in
inhi
inhibition
hi
reversed
several significant changes
han
han
nggees in the
the
h fatty
fat
a ty
y acid
aciid composition
comp
co
mpos
mp
ossittio
on of individual
ind
ndiv
nd
ivid
iv
id
duaal ph
pho
phospholipid
osppholi
hooli
l p classes that
could have altered functional
u ct
unct
un
ctio
iona
io
nall pr
na
pproperties
oper
op
erti
er
ties
ti
es ooff me
memb
membranes
mbra
mb
rane
ra
ness in w
ne
which
hich
hi
ch tthey
heyy re
he
resi
reside.
side
si
de. W
de
While
h a
comprehensive evaluation of these is beyond the scope of this investigation, the reversal of
PUFA remodeling in cardiolipin is particularly relevant and warrants further discussion.
Cardiolipin is a dimeric tetra-acyl phospholipid found exclusively in mitochondria, where
it provides critical structural and functional support to proteins involved in oxidative
phosphorylation, and regulates apoptotic signaling by binding cytochrome c to the inner
mitochondrial membrane 22, 39. The majority of cardiolipin molecular species in the healthy
mammalian heart contain four LA acyl chains (L4CL). Several studies report that this LA
enrichment is lost in states of cardiac pathology, which has been suggested as a potential
contributor to mitochondrial dysfunction and disease progression 22. It is well established that
the compositional uniformity of cardiolipin results from a series of deacylation/ reacylation
15
reactions following de novo biosynthesis in mitochondria. At least three enzymes have been
identified that are capable of catalyzing these reactions; however, none have exhibited the
anticipated specificity for LA acyl chains that predominate in the mammalian heart 39 . It was
recently suggested that the LA enrichment may arise from an enzymatic equilibrium distribution
of fatty acids exchanged between multiple phospholipid species40. Our data are consistent with
this hypothesis, demonstrating that changes in cardiolipin composition coincide with changes in
PC, PE and total phospholipid fractions reflective of a global redistribution of membrane
PUFAs. Notwithstanding changes in specific acyltransferase enzymes implicated in aberrant
pressuure overloaded and
cardiolipin remodeling 41, our findings indicate that loss of L4CL in the pressure
failing heart results from increased flux of PUFAs through D6D andd ddownstream
owns
ow
nstr
ns
trea
tr
eam
ea
m
elongation/desaturation
enzymes
and/or
liver.
reduces
tion
tio
io
on enzy
yme
mes in tthe
he hheart
eart
ea
r an
rt
nd/
d orr liv
ver
er.. Th
This
i uultimately
is
l im
lt
mat
atel
elyy re
el
redu
duce
du
ce the
bioavailability of phospholipid
h sp
hosp
spho
h li
ho
lipi
ipi
p d LA for
forr cardiolipin
carrdiol
olip
ipin
ip
in re
rremodeling
mode
mo
deli
de
ling
li
ng iin
n th
thee he
hear
heart,
a t,
t ffavoring
av
avor
voorri an exchange
of LA for long chain
(i.e.,
AA
DHA).
n PUFA
PUF
UFA
A products
p od
pr
oduc
ucts
uc
ts ooff tthis
hiss pa
hi
ppathway
thwa
th
wayy (i
wa
.e.,
e A
A an
andd DH
DHA)
A)).
Reduced capacity and/or efficiency of oxidative phosphorylation in cardiac mitochondria
may contribute to the development and/or progression of heart failure, and aberrant cardiolipin
remodeling has been postulated as a mechanism for these defects7, 19. However, despite a
significant loss of L4CL, TAC had no effect on mitochondrial respiratory function in the
presence or absence of D6D inhibition. Impaired oxidative phosphorylation capacity was seen in
HF mitochondria, but this was unaffected by D6D inhibition and may have resulted from the loss
of complexes 1 and 2 that deliver reducing equivalents to respiratory chain. Reductions in
oxidative phosphorylation efficiency may also contribute to cardiac dysfunction in heart failure
42
. Therefore, increases in RCR and ADP/O with D6D inhibition, while relatively modest, could
have contributed to improvements in cardiac function observed in HF. Taken together, these
16
findings indicate that the LA enrichment of cardiolipin, at least within the range observed in
these studies, does not significantly influence the oxidative phosphorylation capacity of cardiac
mitochondria, but may support efficient respiratory coupling. The observed dissociation of
mitochondrial respiratory parameters from changes in cardiolipin composition and cardiac
function in TAC and HF groups further argues against their direct relationship in these models.
Apoptotic loss of cardiomyocytes during maladaptive cardiac remodeling may hasten the
progression of pathologic hypertrophy to decompensated failure5 . Myocardial activities of
capase-9 and caspase-3/7 were elevated in both TAC and HF, but were only attenuated by D6D
ntt effect
e
inhibition in TAC. This model-specific effect of argues against ann independent
of PUFA
ocarrdi
dial
a aapoptotic
al
popt
po
ptot
pt
o signaling in
metabolism or phospholipid (i.e., cardiolipin) remodeling on myocardial
he ppattern
he
atternn of m
yoca
yo
card
ca
rdia
rd
iall NFƸ
ia
NF
FƸB activation
actiiva
ac
vati
tionn seen
ti
see
een
en inn these
the
hesee animals.
ani
nima
ma This is
vivo, but parallels the
myocardial
NFƸB
ence linking
en
lin
nki
k ng NFƸB
NFƸB ac
cti
t vi
vity
ty aand
nd aapoptotic
popt
po
ptot
pt
oticc ssignaling
ot
ig
gnnaali
ling
ng iin
n th
he ppr
re
consistent with evidence
activity
the
pressure
eart
ea
rt43. Ho
Howe
However,
weve
we
ver,
ve
r, tthe
he eextent
xten
xt
entt to w
en
which
hich
hi
ch ccaspase
aspa
as
p se aactivities
pa
ctiv
ct
ivit
iv
itie
it
iess re
ie
refl
reflect
flee cumulative
fl
overloaded failing heart
apoptotic myocyte loss that contributed to the observed changes in cardiac structure and function
is unclear. Nevertheless, the absence of changes in caspase activities in HF despite marked
improvements in cardiac structure and function with D6D inhibition suggests that mechanisms
other than apoptotic signaling mediate the pathogenic effects of phospholipid remodeling in this
model.
Membrane PUFAs are a primary target of ROS, which react with hydrogens in methylene
groups adjacent to their double bonds, triggering an autocatalytic series of oxidation events
leading to chain breaks and reactive aldehyde formation. Serum levels of lipid peroxidation
products such as MDA are positively associated with NYHA clinical class in HF patients25,
suggesting that this process may be of prognostic importance. The susceptibility of PUFAs to
17
peroxidation increases exponentially with double bond content 26, therefore modification of
membrane PUFA composition could influence the extent of aldehyde formation in states of
oxidative stress. Consistent with this hypothesis, D6D inhibition significantly attenuated
increases in myocardial MDA- and HNE-protein adducts in TAC and HF, which correlated
closely with changes in phospholipid DHA and LA levels, respectively. Interestingly, while
D6D inhibition completely abolished increases in myocardial MDA in both models, it led to
significant, but less robust reductions in HNE. MDA and HNE are typically used
interchangeably as markers of oxidative stress in tissues, however they differ with regard to their
e
origin and cellular toxicities. MDA is formed primarily from the autocatalytic pperoxidation
of
marilly by fforming
ormi
or
ming
mi
n mutagenic
DHA and during TXA2 synthesis, and is thought to be toxic primarily
DNA-adducts 44. HNE
N is de
NE
derived
eri
rive
vedd fr
ve
from
om tthe
he pperoxidation
errox
o id
dattio
on of nn6
6 PU
PUFA
PUFAs,
As,
s pprimarily
riima
mari
riily AA, and exerts
rily
c s bby
cts
y cr
cros
osss--liink
nkin
ng pr
rotei
tei
eins
ns aand
nd ppromoting
romo
ro
motiing iinflammatory
mo
nnfflaamm
mmat
attorry aan
n fibrogenic
its pathological effects
cross-linking
proteins
and
signaling 45. Relatively
vel
elyy minor
mino
mi
norr changes
no
chan
ch
ange
an
gess in phospholipid
ge
pho
hosp
spho
sp
holi
ho
lipi
li
p d AA
pi
AA and/or
and/
an
d/or
d/
or the
the large
lar
argge increases
incr
in
cree
cr
in LA
might explain the less pronounced decreases in HNE with D6D inhibition, whereas dramatic
changes in DHA and TXA2 parallel similar changes in MDA. Therefore, modulation of
membrane PUFA content and composition appears to strongly influence the extent and pattern of
lipid aldehyde species that accumulate in the heart during states of oxidative stress.
In summary, our studies demonstrate a central role of essential fatty acid metabolism
through D6D in generating the signature pattern of PUFA redistribution in myocardial
phospholipids widely reported in cardiac pathologies, and suggests important pathophysiological
consequences of this phenomenon in heart failure. While precisely defining how cardiac and
hepatic lipid metabolism interact to influence myocardial membrane composition and disease
progression in hypertensive heart disease will require further investigation, a primary pathogenic
18
effect of this process may be an increase in myocardial free AA, favoring production of multiple
eicosanoid species implicated in hypertrophic and fibrotic remodeling. An exchange of
membrane LA for highly unsaturated AA and DHA may also promote lipid peroxidation and
alter critical membrane-dependent processes such as the efficiency of oxidative phosphorylation
and perhaps others not evaluated herein. The observed model-specific effects of D6D inhibition
on mitochondrial respiration, NF-țB and apoptotic signaling, despite eliciting consistent benefits
on cardiac structure and function, argue against primary roles of these systems in mediating the
pathogenic effects of phospholipid remodeling in heart failure. Similar patterns of PUFA
pot
po
redistribution observed in myocardial and serum phospholipids highlight the potential
ypert
rtten
ensi
s on aand
si
nd ccardiovascular
prognostic value of serum D6D activity ratios associated with hypertension
emiologiica
em
call literature
liite
tera
ratu
ra
ture
tu
re14, 16. In
In co
conclusion,
oncclu
lusi
sion
si
on, ta
on
targeting
arg
rgetin
ingg the
in
the D6D
D6D pathway may
morality in the epidemiological
tive
vee new
new
w approach
app
ppro
ro
oac
a h to the
the study
stu
tudy
dy and
dy
and treatment
tre
reat
atme
at
ment
me
nt of
of he
hear
heart
art fa
ar
fail
failure
i ur
uree and perhaps
represent an integrative
other pathologies associated
s ci
soci
so
ciat
ated
at
ed with
wit
ithh th
this
is ssignature
ig
gna
natu
ture
tu
re ppattern
atte
at
tern
te
rn ooff pphospholipid
hosp
ho
sppho
holi
lipi
li
p d PU
pi
PUFA
FA rremodeling.
e
Acknowledgements
The authors thank Allen Medway and Dr. Brian Stauffer for assistance with obtaining human
heart samples and patient information at the University of Colorado Denver, and Juliano Silveria
for assistance with the qRT-PCR.
Sources of Funding
Funding for this project was provided by a Scientist Development Grant from the American
Heart Association (0835545N) and NIH grant HL094890-01A1 to AJC.
19
Disclosures
None.
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23
Table. Animal Characteristics and Serum Analyses
Sham
Sham+SC
TAC
TAC+SC
HF
HF+SC
Body Weight, g
285 ± 8
289 ± 10
260 ± 8
259 ± 10
394 ± 10
381 ± 11
Systolic BP, mmHg
151 ± 10
153 ± 11
ND
ND
175 ± 15
188 ± 11
Glucose, mM
6.2 ± 0.9
6.1 ± 0.3
6.6 ± 0.5
6.8 ± 1.1
6.8 ± 0.5
5.5 ± 3
FFA, ȝM
314 ± 15
306 ± 20
308 ± 28
287 ± 25
283 ± 13
296 ± 25
IFNg, pg/mL
28 ± 18
ND
89 ± 9*
51 ± 15†
135 ± 12*
87 ± 19*†
IL-1ȕ, pg/mL
107 ± 50
ND
161 ± 15*
163
63 ± 47
152
152 ± 50
92 ± 21
IL-10, pg/mL
549
549 ± 289
289
9
ND
ND
241 ± 41*
41
337
3337 ± 41
309
309
09 ± 45
430 ± 49
MCP-1, pg/mL
1143
114
143
3 ± 645
645
ND
2267
226
267
7 ± 28
285*
5* 1573
1573
73 ± 261†
261
61†
† 2175
217
75 ± 151* 1674 ± 241†
TIMP-1, pg/mL
4405
4405 ± 1745
174
745
5
ND
4436
443
436
6 ± 43
437
7
Serum Analyses
5086
508
508
086
6 ± 473
473
4773
477
477
773
3 ± 418
5122 ± 680
Data are means ± SEM. Abbreviations: BP, blood pressure; FFA, free fatty acids; IFNg, interferon
gamma. IL, interleukin; MCP-1, macrophage chemotactic protein-1 TIMP-1, TIMP metallopeptidase. *P
< 0.05 vs. Sham; †P < 0.05 vs. untreated.
24
Figure Legends
Figure 1. Central role of D6D in PUFA metabolism. A) Delta-6 desaturase (D6D) catalyzes
rate limiting steps in the production of long chain PUFAs from linoleic (18:2n6, LA) and
linolenic (18:3n3) acids obtained in the diet. Fatty acid nomenclature: C:XnY where C = number
of carbons, X = number of double bonds, and Y = location of first double bond from the omega
carbon. Double arrows indicate additional reactions catalyzed by elongase enzymes (E), delta-5
desaturase (D5D) and peroxisomal fatty acid E-oxidation (EOx). LA, AA and DHA (shaded) are
otal
ot
a fatty acids in
the only PUFAs that readily accumulate in cardiac phospholipidss to >2% off tot
total
ubstrrat
ates
es routinely
rou
outi
tine
ti
n used to
humans and rats. Underscored fatty acids are D6D products or substrates
zym
zym
yme activity
acti
tivi
ti
vity
vi
ty in
in tissues.
tiss
ti
ssue
ss
ues.
ue
s. AA
A serves
serv
rvess as
as the
th
he substrate
suubs
b trrat
atee for
f r multiple
fo
mult
mu
l i lipoxygenase
estimate chronic enzyme
g as
genas
asee (COX)
(COX
(C
OX)) enzymes,
OX
en
nzyme
mes, or
me
or may
may be
b nnon-enzymatically
on-eenz
nzym
ym
mat
a ic
ical
ally
al
l oxidized
ly
oxi
x di
d ze by reactive
(LO) and cyclooxygenase
S), generating
S)
gene
ge
neera
rati
ting
ti
ng
g an
an array
arra
ar
rayy of bioactive
ra
bio
ioac
acti
ac
tive
ti
ve eicosanoid
eic
icos
osan
os
anoi
an
oidd species
oi
sppec
ecie
iess (s
ie
(see
ee ttext for
oxygen species (ROS),
abbreviations).
Figure 2. Phospholipid PUFA desaturation in human heart failure. Gas chromatographic
analysis of total phospholipid fatty acids extracted from human left ventricular tissue revealed a
loss of phospholipid LA paralleled by elevations in AA, DHA and PUFA product/precursor
ratios in hearts explanted from patients with dilated cardiomyopathy (DCM; n = 8) compared to
non-failing donor hearts (NF; n = 8), which was partially reversed in patients implanted with a
left ventricular assist device for 4-10 months (LVAD; n = 4). * P < 0.05 vs. NF. † P < 0.05 vs.
DCM.
25
Figure 3. Myocardial phospholipid composition and eicosanoids. Treatment of TAC and HF
animals with the selective D6D inhibitor SC-26196 for 4 weeks (black bars) normalized relative
proportions of LA, AA and DHA (A), and reversed D6D product/precursor indices (B) in the
global myocardial phospholipid pool (n= 8-10/group). C) Mass spectrometry of cardiolipin
molecular species revealed a restoration of L4CL and CL species containing highly unsaturated
fatty acids (HUFAs) to control levels in cardiac mitochondria with D6D inhibition (n = 46/group). D) SC-26196 treatment significantly attenuated elevations in myocardial unesterifed
(“free”) AA and several of its pathogenic eicosanoid derivatives (n = 4-6/group). Significant
ts leading to si
ig
group X SC interaction effects were seen for TAC and HF cohorts
significant
effects
GE2 iinn (D),
(D),
) therefore
th
only
of SC treatment on all outcomes except 16:0 and 18:0 in (A, and PG
PGE
significant group differences
fferrencess vs
ff
vs. Sh
Sham
am ccontrol
ontr
on
trol
tr
o aatt P < 00.05
.055 (*
.0
(*)
*) ar
are
re indi
indicated
dica
di
cate
ca
t d fo
te
forr cl
clarity.
l
See text
for abbreviations.
Figure 4. D6D inhibition attenuates contractile dysfunction and pathologic hypertrophy in
TAC and HF. Serial echocardiography revealed marked left ventricular dilatation (LV internal
diameter in diastole, LVIDd), systolic dysfunction (decreased fractional shortening) and diastolic
dysfunction (decreased E/A ratio) in TAC animals (n=8), which was significantly attenuated by
D6D inhibition (SC; n=8) beginning 2 weeks following surgery (Tx) (A). LV dilatation and
systolic dysfunction in aged SHHF rats (n = 12) during the 4 week experimental period was
attenuated or reversed by D6D inhibition (n = 10) (B). SC treatment resulted in significantly
lower heart weights in TAC and HF animals, and decreased pulmonary congestion in HF rats
(C). * P < 0.05 vs. baseline (A), Pre-Tx (B) or Sham (C). † P < 0.05 vs. untreated cohort.
26
Figure 5. Myocardial fibrosis, hypertrophic and inflammatory signaling
Masson’s trichrome staining of LV tissue from TAC and HF animals revealed interstitial and
perivascular fibrosis that was markedly reduced with D6D inhibition (A), which paralleled a
significant attenuation of elevated myocardial hydroxyproline (collagen) content in TAC and HF
animals (n = 6/group) (B). SC treatment prevented significant elevations in ANP expression (C)
and ERK phosphorylation (D) in TAC and HF, and attenuated loss of the endogenous NFțB
inhibitor IțBĮ (E) in TAC (n = 4-6/group). * P < 0.05 vs. Sham. † P < 0.05 vs. untreated cohort.
(phosp
sph
sp
Figure 6. Mitochondrial respiration and caspase activities. A)) State 3 (phosphorylating)
and
tate3
e3
3/4
4) an
andd ph
pho
State 4 (uncoupled) respiration, respiratory control ratio (RCR, state3/4)
phosphorylation
osphoryllatted
os
e pper
er O2 consumed,
con
onsu
s me
su
m d, A
ADP/O)
DP/O
DP
/O)) in
/O
in ccardiac
a diiac m
ar
mitochondria
ittoc
ocho
honn
ho
isolated
efficiency (ADP phosphorylated
p rim
per
imen
en
enta
nta
tall groups
grou
gr
oups
ou
p (n
(n =4-8/group).
=4-8
=4
-8/g
-8
/gro
/g
roup
ro
up)). B) Protein
up
Pro
ote
tein
in
n expression
exp
xpre
resssio
re
i n of ssubunits from
from each of the experimental
each of the five respiratory
p ra
pira
pi
rato
tory
to
ry complexes
com
ompl
p ex
pl
exees (CI-V)
( I(C
I-V)
V) obtained
obt
btai
aine
ai
nedd byy iimmunoblotting
ne
mmun
mm
unob
un
oblo
ob
lott
lo
ttin
tt
ingg 10 uug
in
g of
mitochondrial protein (n = 4/group). C) Caspase activities in myocardial homogenates assayed
by luminescence assay (n = 4-6/group). * P < 0.05 vs. Sham. † P < 0.05 vs. untreated.
Figure 7. Myocardial lipoxidative stress. A) Membrane peroxidizability index of total
myocardial phospholipids calculated from fatty acid analyses in Figure S1 as: (%monoenoic X
0.025)+ (%dienoic X 1)+(%trienoic X 2)+(%tetraenoic X 4)+(%pentaenoic X 6)+(%hexaenoic X
8). B) Relative contents of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) protein
adducts in myocardial homogenates (n = 4-6/group). C) Scatterplots of data from tissue for
which both total phospholipid fatty acid profiles and lipid aldehyde-adduct data reveal significant
correlations between MDA and membrane DHA, and between 4-HNE and LA. D) Myocardial
27
contents of Mn and Cu/Zn superoxide dismutase (SOD) isozymes and glutathione peroxidase
(GSHPx) (n = 4-6/group). A significant main effect of D6D inhibition was seen in all three
enzymes by ANOVA (P < 0.05).* P < 0.05 vs. Sham. † P < 0.05 vs. untreated.
28
Delta-6-desaturase Links PUFA Metabolism with Phospholipid Remodeling and Disease
Progression in Heart Failure
Catherine H. Le, Christopher M. Mulligan, Melissa A. Routh, Gerrit J. Bouma, Melinda A. Frye,
Kimberly M. Jeckel, Genevieve C. Sparagna, Joshua M. Lynch, Russell L. Moore, Sylvia A. McCune,
Michael Bristow, Simona Zarini, Robert C. Murphy and Adam J. Chicco
Circ Heart Fail. published online November 27, 2013;
Circulation: Heart Failure is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2013 American Heart Association, Inc. All rights reserved.
Print ISSN: 1941-3289. Online ISSN: 1941-3297
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World Wide Web at:
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CIRCHF/2013/000744/R1 Supplemental Material for
Delta-6-desaturase links PUFA metabolism with phospholipid remodeling
and disease progression in heart failure
Catherine H. Le, BS1,2*, Christopher M. Mulligan, MS1,3*, Melissa A. Routh, MS1,2,
Gerrit J. Bouma, PhD4, Melinda A. Frye, PhD4, Kimberly M. Jeckel, PhD4,
Genevieve C. Sparagna, PhD6, Joshua M. Lynch, MS6, Russell L. Moore, PhD5,
Sylvia A. McCune, PhD5, Michael Bristow, MD, PhD7, Simona Zarini, PhD8,
Robert C. Murphy, PhD8, Adam J. Chicco, PhD1-5†
Supplemental Methods
Human Heart Tissue
Left ventricular tissue from previously frozen human hearts was obtained by an Institutional Review
Board-approved protocol maintained by the University of Colorado Denver Cardiac Tissue Bank. Hearts
donated for research purposes were obtained under written consent from family members of organ
donors or by direct written consent from end-stage DCM patients undergoing cardiac transplantation.
Patient characteristics are presented in Table S1.
Animal Models
Lean male spontaneously hypertensive heart failure (Mccfacp-/-; SHHF) rats were obtained from a colony
maintained at the University of Colorado by Dr. Sylvia McCune. SHHF rats were selected for these
studies based on their well-characterized development of progressive hypertensive heart disease leading
to pathologic hypertrophy and terminal heart failure by 22-24 months of age, sharing many of the
hallmark biochemical and pathophysiological features of DCM in humans 1, including phospholipid
remodeling 2. Two cohorts of animals were studied: 1) at 21-22 months of age when animals exhibit
early signs of dilated heart failure (HF), and 2) following rapid induction of pathologic left ventricular
hypertrophy by thoracic aortic banding at 3 months of age (TAC; see Supplemental Methods). HF rats
were matched on echocardiographic parameters at 21 months of age prior to being semi-randomly
assigned to treatment or control groups for the 4 week treatment period. TAC surgery was performed as
previously described 2 in 3 month old rats. Two weeks following confirmation of successful TAC (see
Figure S3), two cohorts of animals were matched on aortic Doppler and M-mode echocardiography
parameters to ensure uniform responses and baseline pathology before being randomly assigned to
receive the D6D inhibitor or no drug for 4 weeks. The effect of the D6D inhibition was also examined in 3
month old SHHF rats exposed to a sham TAC surgery (Sham) that were followed along with TAC groups
for 4 weeks as an experimental control. All animals were provided Purina 5001 chow and water ad
libitum for the duration of the study. At the conclusion of the study, animals were sacrificed with a lethal
dose of sodium pentobarbital (150 mg/kg i.p.) followed by midline thoracotomy and removal of the heart.
All procedures were approved by the Animal Care and Use Committee at Colorado State University
and/or University of Colorado Boulder in strict compliance with the Guide for the Care and Use of
Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised
1996).
Inhibition of D6D in vivo
Rats were administered the potent, orally active D6D inhibitor SC-26196 (2,2-diphenyl-5-(4-[[(1 E)pyridin-3-yl-methylidene]amino]piperazin-1-yl)pentanenitrile a generous gift from Dr. Mark Obukowicz,
Pfizer Corporation; at 100 mg/kg/d mixed in chow based on daily food consumption records taken over
1 CIRCHF/2013/000744/R1 3-4 weeks, A detailed description of the pharmacokinetics and selectivity of this compound for D6D over
the other desaturase enzymes (D5D and stearoyl-CoA desaturase, D9D) has been reported elsewhere 3.
.The 100 mpk dose for 4 weeks was selected based on previous studies demonstrating full inhibition of
D6D (LA to AA conversion) and expected changes in tissue PUFA levels in rodent models 3-5 and pilot
studies in our lab demonstrating less inhibition with 50 mpk and similar effects of 200 mpk in aged SHHF
rats (data not shown).
Echocardiography and Blood Pressure
Transthoracic echocardiography was performed in isoflurane anesthetized rats prior to and following the
4 week experimental period using a 12 MHz pediatric transducer connected to a Hewlett Packard Sonos
5500 Ultrasound as previously described 6. Tail cuff blood pressure measurements were obtained in the
Sham and HF groups using the Kent Coda 6 system (Kent Scientific, Torrinton, CT) in lightly isofluraneanesthetized rats.
Lipid analyses
To determine the global fatty acid profile of tissue phospholipids, lipids were extracted from ~40
mg of tissue or 100 μL serum using 4 ml of 2:1 Chloroform:Methanol and 1ml of water. The aqueous
layer was removed and the lipid containing fraction was dried down using nitrogen and re-suspended in
0.5ml hexane. Thin layer chromatography was then used to separate out the phospholipid fraction using
a 20cm x 20cm silica gel TLC plate in a 70:30:1 hexane:ethyl ether:acetic acid solution. The band
associated with the phospholipid fraction was scraped and dissolved in 0.5ml hexane and 0.5ml 0.5N
KOH. 2ml of 14% BF3-methanol was then added and samples were heated at 100 °C for 30 minutes to
obtain methyl esters for subsequent determination of fatty acid composition by gas chromatography
(GC). Separation of individual phospholipid classes was performed by normal phase liquid
chromatography (Agilent Zorbax Rx-Sil column, 4.6 X 250mm, 5-micron) using a
Hexane:Isopropanol:Potassium Acetate mobile phase gradient optimized for separation of PE, PC and
CL by UV detection (206 nm). Fractions were collected based on elution time of known standards,
evaporated under a nitrogen stream, and resuspended in hexane for GC analysis as described above.
GC analysis was performed using an Agilent Technologies DB-225 30m x 0.250mm x 0.25µm
column (model 122-2232, J&W Scientific) on an Agilent 6890 Series Gas Chromatographer. The initial
temperature of the oven was 120 °C with an initial ramp temperature of 10°C/min for 8 minutes, then
2.5°C/min for 4 minutes and held at 210°C for the remaining 6 minutes for a total run time of 20 min. The
inlet split ratio was 15:1 with the column at constant flow and an initial flow, pressure, and velocity at
1.8ml/min, 23.59 psi, and 42 cm/sec, respectively.
Tissue PC and PE contents were determined by colorimetric phosphorus assay on LC fractions
obtained from 20-30mg tissue as described above. Briefly, evaporated fractions were heated in
perchloric acid overnight at 160˚C and cooled, followed by the sequential addition of 275 µl of H2O, 41.7
µl of 20.2 mM Ammonium molybdate, and 41.7 µl of 0.568 M Ascorbic Acid, vortexing each for 20
seconds. The assay mix was then heated for 5 minutes at 100˚C, cooled and read on a
spectrophotometer at 800 nm for quantitation of total phosphorus based on a KH2PO4 standard curve.
Cardiolipin molecular species were determined in lipid extracts from 0.25 mg of mitochondrial
protein isolated from rat left ventricle or 40 mg of LV protein (humans) by electrospray ionization mass
spectrometry previously described in detail 7. “Total” cardiolipin represents the m/z sum of the 10 most
prevalent CL species detected. Myocardial contents of free AA and eicosanoid species were quantified
in lipid extracts obtained from 50 mg of LV tissue by LC/MS/MS methods developed in Dr. Murphy’s
laboratory using deuterated standards as previously described 8.
Mitochondrial isolation and respiratory function
Mitochondria were freshly isolated from ~300 mg of left ventricular (LV) tissue by differential
centrifugation in the absence of proteases as described previously 6. State 3 (ADP-stimulated) and state
2 CIRCHF/2013/000744/R1 4 (ADP-limited) mitochondrial respiratory function was measured in isolated mitochondria (0.25 mg
protein) using a Clark-type electrode system (Strathkelvin) at 30C with pyruvate + malate as substrates
as previously described 6.
Biochemical analyses
Immunoblotting for D6D, (p)ERK, IκBα, MDA and HNE-adducts, and antioxidant enzymes were
performed in 40-50 mg LV homogenates by standard methods using commercially available antibodies
and chemiluminescent detection. Blot densities were normalized to total lane protein content (Coomassie
or ponceau staining) to control for any differences in loading on each membrane, then expressed relative
to at least 2 Sham samples on each gel to allow for comparisons between blotting experiments. The
source, catalog number and blotting conditions used for each of the antibodies used are listed below.
GSHPX1 (Abnova, #MAB0845) 1:5000 in 5%NFM O/N @4°C
MnSOD (Assay Designs, #SOD-111) 1:10,000 in 5%NFM O/N @4°C
CuZnSOD (Assay Designs, #SOD-101) 1:10,000 in 5%NFM O/N @4°C
MDA (Anti-Malondialdehyde) (Calbiochem, #442730) 1:5000 in 5%NFM O/N @4°C
HNE (Anti-HNE-Michael Adducts Reduced) (Calbiochem, # 393207) 1:5000 in 5%NFM O/N @4°C
pERK (Cell Signaling, #9101S) 1:1000 in 5%BSA O/N @4°C
ERK (Cell Signaling, #9102) 1:1000 in 5%BSA O/N @4°C
OXPHOS (Mitosciences, #MS604) 1:2000 in 5%NFM O/N @4°C
IkBalpha (Abcam, #ab32518) 1:10000 in 5%NFM O/N @4°C
Serum glucose and free fatty acids were determined by colorimetric assays according to the
manufacturer’s instructions (Biovision). Caspase activities were determined in 30 mg of LV
homogenates by luminescence assay (Promega). Myocardial hydroxyproline was quantitated as a
marker of collagen (fibrosis) in 30-40 mg of septal tissue by the colorimetric assay of Switzer and
Summer 9. Serum cytokines were determined in 80 μL of sample by ELISA cytokine array (Raybiotech).
qRT-PCR was performed using 2X SYBR Green qPCR Master Mix and the LightCycler480 PCR system
(Roche Applied Sciences). Total RNA (10-15mg tissue) was converted into quantifiable cDNA using
Trizol, followed by Qiagen RNeasy Mini kit according to the manufacturer’s instructions. RNA purity was
assessed using a NanoDrop ND1000 spectrophotometer, and only samples with a 260:280 ratio 2.0 or
greater were used. Gene specific primers (listed in Table S2) were designed and validated by
sequencing PCR products, and dissociation curve analysis. qRT-PCR data was normalized to 18S rRNA,
and analyzed according to the comparative (∆∆Ct) Ct method 10. Changes in relative transcript level was
compared to sham control and expressed as fold change.
Statistical analyses
All data are presented as group means  standard error. Human heart data were compared by one way
ANOVA with Tukey tests post hoc when appropriate. Rat data were analyzed by 3(condition) X 2(drug)
ANOVA to determine main and interaction effects with Tukey tests post hoc for determination of
significant group differences. Within-group differences in echocardiography data from pre- to posttreatment were compared by paired t-tests. Statistical significance was established at P < 0.05 for all
analyses.
3 CIRCHF/2013/000744/R1 Supplemental Tables
Table S1. Patient Characteristics
Lab
code
SEX
AGE
PATHOLOGY
EF
(%)
BP
(mmHg)
NF1
NF2
NF3
NF4
NF5
NF6
NF7
NF8
F
F
M
M
M
F
M
M
50
61
68
20
19
22
69
72
None
None
None
None
None
None
None
None
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
106/56
115/63
110/52
90/58
100/62
n/a
115/51
n/a
I1
I2
I3
I4
I5
I6
I7
F
F
M
M
M
M
M
46
53
65
48
57
63
39
DCM
DCM
DCM
DCM
DCM
DCM
DCM
<20
<25
24.00
10.00
<25
<20
12.00
n/a
86/64
115/63
105/63
109/68
98/66
99/61
L1
L2
L3
L4
M
M
M
M
47
34
52
56
LVAD 5 mo
LVAD 4 mo
LVAD 10 mo
LVAD 9 mo
<15
<25
8.45
10.00
n/a
n/a
161/79
136/50
4 CIRCHF/2013/000744/R1 Table S2. Primer sequences used for qRT-PCR
gene
species
primer
sequence
fads2
Rat
Forward
TGTCCACAAGTTTGTCATTGG
fads2
Rat
Reverse
ACACGTGCAGGCTCTTTATG
fads2
Human
Forward
ATCCCTTTCTACGGCATCCT
fads2
Human
Reverse
TAGGCCTCCTGGTCAATCTC
fads1
Rat
Forward
TGGAGAGCAACTGGTTTGTG
fads1
Rat
Reverse
GTTGAAGGCTGACTGGTGAA
fads1
Human
Forward
TTGGCCTGGATGATTACCTT
fads1
Human
Reverse
CTGTGTCACCCACACAAACC
Elovl5
Rat
Forward
TACCACCATGCCACTATGCT
Elovl5
Rat
Reverse
GACGTGGATGAAGCTGTTGA
Elovl5
Human
Forward
GTGCACATTCCCTCTTGGTT
Elovl5
Human
Reverse
TGGTCCTTCAGGTGGTCTTT
Elovl2
Rat
Forward
TTTGGCTGTCTCATCTTCCA
Elovl2
Rat
Reverse
GGGAAACCGTTCTTCACTTC
Elovl2
Human
Forward
CCCTTCGGTTGTCTCATCTT
Elovl2
Human
Reverse
CAGGTGGCTCTTGCATATCTT
5 CIRCHF/2013/000744/R1 Table S3. Animal Morphology
Body Weight, g
Sham
Sham+SC
TAC
TAC+SC
HF
HF+SC
285 ± 8
289 ± 10
260 ± 8
259 ± 10
394 ± 10*
381 ± 11*
Heart, gww
1.18 ± 0.03
1.19 ± 0.06 1.44 ± 0.06* 1.25 ± 0.03† 1.77 ± 0.04*
Lungs, gww
1.45 ± 0.04
1.50 ± 0.09 2.21 ± 0.25*
1.99 ± 0.21*
2.42 ± 0.10*
2.19 ± 0.07*†
Brain, gww
1.76 ± 0.03
1.74 ± 0.02
1.77 ± 0.01
1.79 ± 0.02
2.11 ± 0.02*
2.10 ± 0.04*
Heart/BW, g/kg
4.15 ± 0.10
4.12 ± 0.12 5.57 ± 0.25* 4.88 ± 0.20*† 4.39 ± 0.09*
4.17 ± 0.09†
Heart/Brain, g/g
0.67 ± 0.02
0.68 ± 0.03 0.81 ± 0.04* 0.70 ± 0.01† 0.83 ± 0.02*
0.77 ± 0.02*†
Lungs/BW, g/kg
4.75 ± 0.45
5.19 ± 0.60 8.59 ± 1.11*
7.71 ± 0.80*
6.09 ± 0.24*
5.74 ± 0.19*
Lungs/Brain, g/g
0.86 ± 0.04
0.86 ± 0.04 1.25 ± 0.14*
1.11 ± 0.11*
1.16 ± 0.05*
1.05 ± 0.04*
Data are means ± SEM. *P < 0.05 vs. Sham; †P < 0.05 vs. untreated.
6 1.60 ± 0.04*†
CIRCHF/2013/000744/R1 Supplemental Figures
Figure S1. Fatty acid composition of phospholipid extracts from human left ventricle.
Data are means ± SEM of %total fatty acids from phospholipids extracted from 30-50 mg of left
ventricular (LV) tissue by thin layer chromatography (total phospholipids) or HPLC (individual
phospholipid classes) as described in Methods (n = 4-8/group).
7 CIRCHF/2013/000744/R1 Figure S2. Phospholipid fatty acid composition of rat heart, liver and serum
Data are means ± SEM of %total fatty acids from phospholipids extracted from 30-50 mg of left
ventricular (LV) or liver tissue and30 µL serum, by TLC (total phospholipids) or HPLC
(individual phospholipid classes) as described in Methods (n = 4-8/group).
8 CIRCHF/2013/000744/R1 Figure S3. Aortic outflow Doppler on TAC mice.
Following successful TAC surgery, aortic outflow acceleration time (AT) decreases markedly,
while total ejection time (ET) increases, resulting in a dramatic elevation in ET/AT indicative of
severe aortic stenosis. Thus, the ET/AT ratio served as a basis for pre-treatment matching of
TAC and TAC+SC groups 2 weeks following surgery to ensure similar extents of aortic
constriction prior to beginning treatment. No change in AT or ET/AT from 12-16 weeks in TAC
or TAC+SC indicates a nearly identical maintenance of aortic constriction throughout the
experimental period in both groups. Data are means ± SEM, n = 4-8/group. **P < 0.01 vs.
Sham and Sham+SC. *P < 0.05 for TAC+SC vs. Sham.
9 CIRCHF/2013/000744/R1 Figure S4. Total CL content and representative mass spectra of CL molecular species
from cardiac mitochondria.
Quantitation of the 18 most abundant CL species revealed no significant change in the
amount of CL present in cardiac mitochondria (above left). Representative mass
spectra obtained via electrospray ionization mass spectrometry show the relative
abundance of CL molecular species obtained from cardiac mitochondria isolated from
TAC and HF rats with and without D6D inhibition (SC-26196). X axis unit is
mass/charge ratio (m/z), which is identical molecular weight ,thus the peak at m/z 1448
is L4CL, and peaks at higher m/z values contain various combinations of AA and/or
DHA (see Sparagna et al. J Lipid Res 46:1196, 2005). Y axis units are arbitrary relative
peak intensities representing the relative abundance of CL species for each spectra.
10 CIRCHF/2013/000744/R1 Figure S5. D6D pathway expression
(A) Relative mean (+/- SEM) data for qRT-PCR of mRNA encoding D6D (fads2), delta-5
destaurase (fads1), and elongase-5 (elovl5) in the rat and human heart.*P < 0.05 vs. NF and
DCM/HF; n = 4-6/group. (B) Representative blots and mean (+/- SEM) data for D6D protein by
immunoblotting in rat and human tissues (n = 6/group).
11 CIRCHF/2013/000744/R1 Figure S6. Representative blots from Figures 6B (top) and 7D (bottom).
Blots are representative chemiluminescent images from several blotting experiments comparing
all or some of the experimental groups. Data presented in Figures 6 and 7 were derived from
several blotting experiments with various combinations of samples normalized to coomassie or
ponceau staining for total protein, expressed relative to sham samples on each gel.
12 CIRCHF/2013/000744/R1 Figure S7. Semi-quantitative histological analysis of myocardial fibrosis
Semi-quantitative analysis of myocardial fibrosis was performed on Masson’s trichrome stained
sections (10 µm thick) derived from 20-35 mg paraffin-embedded sections of LV free wall tissue
fixed in 4% paraformaldehyde in PBS. The percent of interstitial collagen deposition was
determined by measuring the total stained (blue) area relative total tissue area in at least 4
separate 1 X 1.4 mm light microscopy images of sections obtained from 2-4 rats per group using
ImageJ software (NIH). Data are mean values of multiple sections from 2-4 rats per group 
SE. * P < 0.05 vs. Sham control. † P < 0.05 vs. untreated.
13 CIRCHF/2013/000744/R1 Supplemental References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Heyen JR, Blasi ER, Nikula K, Rocha R, Daust HA, Frierdich G, Van Vleet JF, De Ciechi
P, McMahon EG, Rudolph AE. Structural, functional, and molecular characterization of
the shhf model of heart failure. Am J Physiol Heart Circ Physiol. 2002;283:H1775-1784
Sparagna GC, Chicco AJ, Murphy RC, Bristow MR, Johnson CA, Rees ML, Maxey ML,
McCune SA, Moore RL. Loss of cardiac tetralinoleoyl cardiolipin in human and
experimental heart failure. J Lipid Res. 2007;48:1559-1570
Obukowicz MG, Welsch DJ, Salsgiver WJ, Martin-Berger CL, Chinn KS, Duffin KL, Raz
A, Needleman P. Novel, selective delta6 or delta5 fatty acid desaturase inhibitors as
antiinflammatory agents in mice. The Journal of pharmacology and experimental
therapeutics. 1998;287:157-166
Duffin KL, Obukowicz MG, Salsgiver WJ, Welsch DJ, Shieh C, Raz A, Needleman P.
Lipid remodeling in mouse liver and plasma resulting from delta6 fatty acid desaturase
inhibition. Lipids. 2001;36:1203-1208
Hansen-Petrik MB, McEntee MF, Johnson BT, Obukowicz MG, Masferrer J, Zweifel B,
Chiu CH, Whelan J. Selective inhibition of delta-6 desaturase impedes intestinal
tumorigenesis. Cancer letters. 2002;175:157-163
Mulligan CM, Sparagna GC, Le CH, De Mooy AB, Routh MA, Holmes MG, Hickson-Bick
DL, Zarini S, Murphy RC, Xu FY, Hatch GM, McCune SA, Moore RL, Chicco AJ. Dietary
linoleate preserves cardiolipin and attenuates mitochondrial dysfunction in the failing rat
heart. Cardiovasc Res.94:460-468
Sparagna GC, Johnson CA, McCune SA, Moore RL, Murphy RC. Quantitation of
cardiolipin molecular species in spontaneously hypertensive heart failure rats using
electrospray ionization mass spectrometry. Journal of lipid research. 2005;46:1196-1204
Zarini S, Gijon MA, Ransome AE, Murphy RC, Sala A. Transcellular biosynthesis of
cysteinyl leukotrienes in vivo during mouse peritoneal inflammation. Proc Natl Acad Sci U
S A. 2009;106:8296-8301
Switzer BR, Summer GK. Improved method for hydroxyproline analysis in tissue
hydrolyzates. Anal Biochem. 1971;39:487-491
Schmittgen TD, Livak KJ. Analyzing real-time pcr data by the comparative c(t) method.
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14

Delta-6-desaturase Links PUFA Metabolism with Phospholipid

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