Metabolic gene switching in the murine female heart
parallels enhanced mitochondrial respiratory function in
response to oxidative stress
M. Faadiel Essop
1,2
, W. Y. A. Chan
2
and Heinrich Taegtmeyer
3
1 Department of Physiological Sciences, Stellenbosch University, South Africa
2 Hatter Heart Research Institute, Faculty of Health Sciences, University of Cape Town, South Africa
3 Department of Internal Medicine, Division of Cardiology, University of Texas, Houston Medical School, TX, USA
Premenopausal women have a lower risk for develop-
ing cardiovascular disease as compared to age-matched
males [1]. Moreover, experimental studies show increa-
sed resistance to ischemia⁄ reperfusion injury in female
versus male hearts [2,3]. The molecular regulatory
mechanisms underlying such gender-based differences
are unclear. However, estrogen may play a key role in
this process [4], and is thought to signal its cardio-
protective effects via the prosurvival serine-threonine
protein kinase, Akt (also known as protein kinase B)
[2]. In agreement with this, elevated levels of activated
Akt in female hearts are linked to improved cardiac
cell survival [5], and a recent study implicated the
PI3-K ⁄ Akt signaling pathway in estrogen-mediated
cardioprotection [6].
Adaptive metabolic remodeling is considered to be
an important component of cardioprotective mecha-
nisms in response to decreased oxygen supply. For
example, enhanced glucose utilization is proposed to

over, females displayed improved recovery of cardiac mitochondrial respi-
ratory function and higher ATP levels versus males in response to acute
oxygen deprivation. All these changes were reversed in female db ⁄ db
hearts. However, we found no significant gender-based differences in levels
of Akt, suggesting that Akt-independent signaling mechanisms are respon-
sible for the resilient mitochondrial phenotype observed in female mouse
hearts. As glucose is a more energetically efficient fuel substrate when oxy-
gen is limiting, this gene program may be a crucial component that
enhances tolerance to oxygen deprivation in female hearts.
Abbreviations
Akt, serine-threonine protein kinase (protein kinase B); GLUT, glucose transporter; MCAD, medium-chain acyl-CoA dehydrogenase; mCPT1,
muscle-type carnitine palmitoyltransferase 1; PDK-4, pyruvate dehydrogenase kinase 4; PGC-1, peroxisome proliferator-activated receptor-
gamma coactivator-1; PPARa, peroxisome proliferator-activated receptor-alpha; UCP3, uncoupling protein 3.
5278 FEBS Journal 274 (2007) 5278–5284 ª 2007 The Authors Journal compilation ª 2007 FEBS
function [8]. Likewise, high fatty acid oxidation rates
in the diabetic heart results in reduced cardiac effi-
ciency [9].
In addition to its cytoplasmic role, Akt can also
translocate to the nucleus, where it has transcriptional
effects. For example, constitutively activated Akt
resulted in reduced myocardial gene expression
of peroxisome proliferator-activated receptor-alpha
(PPARa) and peroxisome proliferator-activated recep-
tor-gamma coactivator-1 (PGC-1), pivotal nuclear
regulators of numerous fatty acid metabolic genes
[10]. In light of this, we hypothesized that Akt trig-
gers a metabolic gene switch from fatty acids to
increased glucose metabolism in female hearts,
thereby conferring protection against acute oxygen
deprivation. Moreover, we propose that this selective

male controls) (Fig. 2A). In parallel, expression of the
genes encoding muscle-type carnitine palmitoyltransfer-
ase 1 (mCPT1) and medium-chain acyl-CoA dehydro-
genase (MCAD) (both PPARa target genes) was
Table 1. Baseline characterization of male and female obese mice.
Values are expressed for 18–20-week-old male and female db ⁄ +
versus db ⁄ db mice (mean ± SEM, n ¼ 10 animals). ** P < 0.001
compared with age-matched db ⁄ +mice.
Body weight (g)
Heart ⁄ body
weight ratio
(· 1000)
Fasting blood
glucose
(mmolÆL
)1
)
Male
db ⁄ + 28.0 ± 0.7 4.0 ± 0.1 5.5 ± 0.4
db ⁄ db 47.2 ± 1.7** 2.1 ± 0.1** 26.8 ± 0.9**
Female
db ⁄ + 20.8 ± 0.5 3.6 ± 0.1 4.2 ± 0.2
db ⁄ db 44.7 ± 1.2** 2.2 ± 0.1** 25.9 ± 1.2**
A B
C D
Fig. 1. Immunohistochemical analysis of
phospho-Akt in male and female control
(db ⁄ +) versus obese (db ⁄ db) mice. (A) Male
control, (B) male obese, (C) female control
and (D) female obese mice. Phospho-Akt is

findings, we evaluated mitochondrial respiratory
function at baseline and in response to acute oxygen
AB
CD
EF
Fig. 2. Cardiac metabolic gene expression in male and female control versus obese mice. (A) PPARa, (B) mCPT1, (C) MCAD, (D) UCP3,
(E) GLUT4 and (F) PDK-4 in male and female obese versus control mice. Data are expressed as mean ± SEM. *P < 0.001 versus male
control mice; **P < 0.01 versus male obese mice;
#
P < 0.01 versus female control mice;
##
P < 0.001 versus female control mice;
§
P < 0.001 versus male control mice.
Metabolic gene switching in murine female heart M. F. Essop et al.
5280 FEBS Journal 274 (2007) 5278–5284 ª 2007 The Authors Journal compilation ª 2007 FEBS
deprivation. Here, females displayed increased mito-
chondrial respiratory function at baseline as compared
to males (Table 2). The efficiency of respiration
(ADP ⁄ O) and ADP phosphorylation rate were similar
between male and female mitochondria at baseline. In
contrast to what was expected, our data suggest that
male obese mice coped well when challenged by oxi-
dant stress; that is, respiratory function and myo-
cardial ATP levels were not significantly altered
(Fig. 3A,B). We are unsure why this occurred, and
propose that the stress applied was not severe
enough or that some adaptive mechanisms were initi-
ated in the male obese mice. However, female controls
exhibited enhanced recovery of state 3 mitochondrial

sis may also be implicated in this process. In agree-
ment, recent studies reported that the orphan nuclear
receptor estrogen receptor-alpha, proposed to mediate
estrogen signaling, may play a transcriptional role in
mitochondrial biogenesis [13,14]. Further studies are,
however, required to investigate this possibility.
Limitations
Although the gene data in this study support a fuel
substrate switch away from fatty acids, further studies
measuring actual cardiac fuel substrate utilization are
Table 2. Cardiac mitochondrial respiration for male and female obese mice. Heart mitochondria were isolated from 18–20-week-old mice as
described. Values are expressed as mean ± SEM (n ¼ 7 animals). * P < 0.05 compared with male db ⁄ +mice. ** P < 0.05 compared with
male obese db ⁄ db mice.
Male Female
db ⁄ +db⁄ db db ⁄ +db⁄ db
State 2 respiration (nmolÆmin
)1
Æmg
)1
protein) 29.4 ± 1.6 32.6 ± 1.9 36.6 ± 1.7* 35.0 ± 2.0
State 3 respiration (nmolÆmin
)1
Æmg
)1
protein) 145.0 ± 9.6 165.8 ± 9.8 175.6 ± 7.1 177.8 ± 12.9
State 4 respiration (nmolÆmin
)1
Æmg
)1
protein) 33.5 ± 2.0 30.7 ± 2.4 38.8 ± 1.03 36.8 ± 1.6

are therefore required to identify the precise signaling
mechanisms that control the metabolic remodeling
that we observed in the female murine heart at base-
line. As glucose is a more energetically efficient fuel
substrate than fatty acids when oxygen is limiting,
we believe that this mechanism may represent a
crucial component underlying enhanced recovery
in younger female hearts in response to oxygen
deprivation.
Experimental procedures
Animals
To investigate our hypothesis, we employed 18–20-week-
old male and female leptin-receptor deficient (db ⁄ db)
(BKS.Cg-m+ ⁄ +Lepr
db
⁄ J strain) and heterozygous (db ⁄ +)
mice. Mice were obtained from Jackson Laboratory (Bar
Harbor, ME) and exposed to a reverse 12 h light ⁄ 12 h
dark cycle with free access to standard mouse chow and
water. Two weeks before mice were killed, blood glucose
levels were measured using a glucose meter (ACCU-
CHECK Active Meter; Roche, Basel, Switzerland) after a
6 h fast. All animal experiments were approved by the
University of Cape Town’s Animal Research Ethics
Committee, and the investigation conforms to 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).
Immunohistochemistry
Heart tissues were fixed with paraformaldehyde and embed-

rates were polarographically measured at 25 °Cas
described previously [20], with modifications. Isolated mito-
chondria were added to the electrode chamber containing
incubation medium (25 mm Tris ⁄ HCl, 250 mm sucrose,
8.5 mm KH
2
PO
4
, pH 7.4). We employed a mixture of
5mm malate and 25 lm palmitoyl-l-carnitine as oxidative
substrates. State 2 respiration (resting) was measured after
addition of oxidative substrates, and state 3 respiration
after the addition of 300 lm ADP to the electrode
chamber.
To test the ability of mitochondria to withstand oxidative
stress, 3 mm ADP was added after state 4 respiration, and
the chamber was closed and sealed for a 20 min period. As
a result, oxygen in the closed chamber would be used to
convert ADP to ATP, and an anaerobic condition estab-
lished. After 20 min of oxygen lack, the chamber was
reoxygenated for 6 min, and the percentage recovery of
state 3 respiration was calculated as the ratio of oxygen
consumption before and after oxygen lack. All mitochon-
drial polarographic studies were normalized to total mito-
chondrial protein content [21].
Mitochondrial ATP levels
Postanoxic mitochondrial ATP concentration was assayed
using a luciferin ⁄ luciferase luminometry luminescence
method [22] with modifications. Freshly isolated mitochon-
dria were placed in boiling water (3 · sample volume) for

Medical Research Council and National Research
Foundation for financial support. The work of HT
was supported in part by grants from the NHLBI
(RO1-HL073162-01 and T32-HL07591).
References
1 Kannel WB & Wilson PW (1995) Risk factors that
attenuate the female coronary disease advantage. Arch
Intern Med 155, 57–61.
2 Bae S & Zhang L (2005) Gender differences in cardio-
protection against ischemia ⁄ reperfusion injury in adult
rat hearts: focus on Akt and protein kinase C signaling.
J Pharmacol Exp Ther 315, 1125–1135.
3 Gabel SA, Walker VR, London RE, Steenbergen C,
Korach KS & Murphy E (2005) Estrogen receptor beta
mediates gender differences in ischemia ⁄ reperfusion
injury. J Mol Cell Cardiol 38, 289–297.
4 Mendelsohn ME & Karas RH (1999) The protective
effects of estrogen on the cardiovascular system. N Engl
J Med 340, 1801–1811.
5 Shiraishi I, Melendez J, Ahn Y, Skavdahl M, Murphy E,
Welch S, Schaefer E, Walsh K, Rosenzweig A, Torella D
et al. (2004) Nuclear targeting of Akt enhances kinase
activity and survival of cardiomyocytes. Circ Res 94,
884–891.
6 Sovershaev MA, Egorina EM, Andreasen TV, Jonassen
AK & Ytrehus K (2006) Preconditioning by 17beta-
estradiol in isolated rat heart depends on PI3-K ⁄ PKB
pathway, PKC, and ROS. Am J Physiol Heart Circ
Physiol 291, H1554–H1562.
7 Opie LH & Sack MN (2002) Metabolic plasticity and

S, Daniels TG, Szustakowski J, Nirmala NR, Wu Z &
Stevenson SC (2007) Estrogen-related receptor alpha is
essential for the expression of antioxidant protection
genes and mitochondrial function. Biochem Biophys Res
Commun 357, 231–236.
15 Depre C, Shipley GL, Chen W, Han Q, Doenst T,
Moore ML, Stepkowski S, Davies PJ & Taegtmeyer H
(1998) Unloaded heart in vivo replicates fetal gene
expression of cardiac hypertrophy. Nat Med 4
, 1269–
1275.
16 Young ME, Patil S, Ying J, Depre C, Ahuja HS, Ship-
ley GL, Stepkowski SM, Davies PJ & Taegtmeyer H
(2001) Uncoupling protein 3 transcription is regulated
by peroxisome proliferator-activated receptor (alpha) in
the adult rodent heart. FASEB J 15, 833–845.
17 Belke DD, Betuing S, Tuttle MJ, Graveleau C, Young
ME, Pham M, Zhang D, Cooksey RC, McClain DA,
Litwin SE et al. (2002) Insulin signaling coordinately
M. F. Essop et al. Metabolic gene switching in murine female heart
FEBS Journal 274 (2007) 5278–5284 ª 2007 The Authors Journal compilation ª 2007 FEBS 5283
regulates cardiac size, metabolism, and contractile pro-
tein isoform expression. J Clin Invest 109, 629–639.
18 Young ME, Razeghi P, Cedars AM, Guthrie PH &
Taegtmeyer H (2001) Intrinsic diurnal variations in car-
diac metabolism and contractile function. Circ Res 89,
1199–1208.
19 Sordahl LA, Besch HR Jr, Allen JC, Crow C, Linden-
mayer GE & Schwartz A (1971) Enzymatic aspects of
the cardiac muscle cell: mitochondria, sarcoplasmic