CARDIOVASCULAR JOURNAL OF AFRICA • Vol 22, No 4, July/August 2011
AFRICA
175
Cardiovascular Topics
Proteomic analysis of mitochondrial proteins in a mouse
model of type 2 diabetes
MF ESSOP, WA CHAN, S HATTINGH
Abstract
Objective:
Impaired mitochondrial function may contribute
to the onset of contractile dysfunction with insulin resistance/
type 2 diabetes. Our aim was therefore to determine altera-
tions in the mitochondrial proteome of a mouse model of
obesity/type 2 diabetes.
Methods
: Mitochondrial proteins were isolated from hearts
collected from 18- to 20-week-old female db/db mice and
compared to matched controls. We performed two-dimen-
sional polyacrylamide gel electrophoresis to determine differ-
entially expressed proteins. Peptides of interest were further
analysed by mass spectrometry and Mascot software was
employed to identify protein matches.
Results
: Our data showed that ATP synthase D chain,
ubiquinol cytochrome-C reductase core protein 1 and elec-
tron transfer flavoprotein subunit alpha peptide levels were
altered with obesity. Moreover, we found coordinate down-
regulation of contractile proteins in the obese heart, i.e.
α
-smooth muscle actin,
α
-cardiac actin, myosin heavy-chain
α
and myosin-binding protein C.
Conclusion
: We propose that decreased contractile protein
levels may contribute to contractile dysfunction of hearts
from diabetic mice.
Keywords:
heart, proteomics, obesity, diabetes, contractile
proteins
Submitted 9/4/10, accepted 12/7/10
Published online 11/9/10
Cardiovasc J Afr
2011;
22
: 175–178
DOI: 10.5830/CVJA–2010–058
Type 2 diabetes is characterised by metabolic perturbations
that may contribute to cardiac contractile dysfunction in the
absence of atherosclerosis or hypertension, i.e. diabetic cardio-
myopathy.
1
For example, previous studies reported mitochondrial
dysfunction in the hearts of ob/ob and db/db transgenic mice,
well-described rodent models of obesity and type 2 diabetes.
2,3
Here it was proposed that reduced peptide levels of mitochon-
drial respiratory chain complexes I, III and V in ob/ob mice, and
lower expression of the F1
α
-subunit of ATP synthase in db/db
mice may contribute to decreased mitochondrial oxidative phos-
phorylation capacity. In addition, increased myocardial oxygen
consumption (MVO
2
) of diabetic hearts resulted in reduced
cardiac efficiency in diabetic mice, proposed to occur as a result
of fatty acid-induced uncoupling of myocardial mitochondrial
oxidative phosphorylation.
4
We also recently found that obese
rats displayed increased myocardial damage and attenuated
respiratory capacity in response to acute oxygen deprivation.
5
Together, these studies show that impaired mitochondrial
function plays a pivotal role in the onset of contractile dysfunction
associated with insulin resistance and type 2 diabetes. However,
the complete identity of mitochondrial peptides involved in this
process remains unclear. The aim of this study was therefore to
determine alterations in the mitochondrial proteome in a trans-
genic mouse model of obesity and type 2 diabetes, investigating
the hypothesis that db/db mouse hearts display lower expres-
sion of contractile and mitochondrial energy metabolic proteins
compared to wild types.
Methods
To investigate our hypothesis we employed 18- to 20-week-old
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, Maine) and exposed for one
week to a reverse 12-hour light 12-hour dark cycle with free
access to standard mouse chow and water. The reverse cycle
was employed since mice are most metabolically active during
the night period, and it allowed us to easily sacrifice mice in the
middle of their night phase during our normal laboratory work-
ing hours.
All animal experiments were approved by the University
of Cape Town’s Animal Research Ethics Committee (approval
number 03/030) 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).
Mitochondrial isolation procedure
On the day of the experiment, mice were anesthetised using
sodium pentobarbital (50 mg/kg intra-peritoneally) and heparin-
Cardio-Metabolic Research Group (CMRG), Department
of Physiological Sciences, Stellenbosch University,
Stellenbosch, South Africa
MF ESSOP, PhD,
Hatter Institute for Cardiovascular Research, Faculty of
Health Sciences, University of Cape Town, South Africa
WA CHAN, MSc
Department of Medical Physiology, Stellenbosch University
Faculty of Health Sciences, Tygerberg, South Africa
S HATTINGH, MSc
