CARDIOVASCULAR JOURNAL OF AFRICA • Vol 22, No 4, July/August 2011
AFRICA
177
Data from the mass spectrometry analysis were processed and
employed to create PKL files, which were used to search against
the MASCOT software program (
. The
latter searched the SwissProt database to identify peptide protein
matches.
Statistical analysis
Due to the small heart sizes, we pooled (for each experi-
ment) two hearts for control and obese mice, respectively, for
mitochondrial isolation. For this study we performed three
independent mitochondrial isolation experiments as described
above. Subsequently, three independent gels were performed per
mitochondrial isolation experiment for db/db and db/
+
mouse
hearts, and only changes found in two or more experiments were
selected for further analysis. Data are presented as increased or
decreased fold change versus matched controls. Statistical differ-
ences between groups were calculated using the Student’s
t
-test.
Statistical significance was considered when
p
<
0.05.
Results
Two-dimensional PAGE analysis highlighted several differences
between obese db/db mouse hearts versus matched db/
+
controls
(Fig. 1). Proteins of interest were further analysed by mass
spectrometry and peptides were identified using software as
described in the methods section. We divided altered mitochon-
drial protein expression into two broad groupings, i.e. regulating
mitochondrial energy metabolism (Table 1), and forming part of
the myocardial contractile apparatus (Table 2).
Seven proteins regulating mitochondrial energy metabolism
were differentially expressed (statistically significant) between
obese and control hearts. These data show both up- and down-
regulation of key regulators of electron transfer in the electron
transport chain (ETC) and mitochondrial ATP production.
However, expression of only three proteins, i.e. ATP synthase
D chain (increased 2.5-fold), ubiquinol cytochrome-C reductase
core protein 1 (decreased 2.1-fold) and electron transfer flavo-
protein subunit alpha (increased 5.9-fold) were altered in at least
two independent experiments.
Interestingly, we also found differentiated expression for
several contractile/cytoskeletal proteins (Table 2), although only
four displayed decreased expression in at least two independ-
ent experiments. Here
α
-smooth muscle actin,
α
-cardiac actin,
myosin heavy-chain (MHC)
α
and myosin-binding protein C
(MyBP-C) protein levels were coordinately down-regulated in
obese versus control hearts.
TABLE 1. PROTEINS IDENTIFIED IN OBESE FEMALE MICE: MITOCHONDRIAL ENERGY METABOLISM
Spot SSP Mr (Da) pI
Mascot
score Protein name
Swiss prot acces-
sion number
% Sequence
coverage
No of
peptides
Fold increase
or decrease
Functional group or
function
1 2305 56265 5.19 873 ATP synthase subunit
beta,
P56480
44
18
2.7 ATP synthesis,
(catalytic subunit)
2* 5002 18664 5.52 273 ATP synthase D chain
Q9DCX2
70
11
2.5 ATP synthesis, (rota-
tional mechanism)
3* 6003 53420 5.75 280 Ubiquinol cytochrome-C
reductase core protein 1
Q9CZ13
21
7
–2.1 Electron transport
4 8406 59830 9.22 693 ATP synthase subunit
alpha
P56757
30
15
14.9 ATP synthesis (regu-
latory subunit)
5 3101 27640 5.19 297 NADH dehydrogenase
flavoprotein 2
Q9D6J6
45
9
–1.8 Electron transfer
6 2903 80724 5.51 563 NADH ubiquinone oxido-
reductase 75 kDa subunit
Q91VD9
26
18
–2
Electron transfer
7* 9302 35360 8.62 623 Electron transfer flavopro-
tein subunit alpha
Q99LC5
51
15
5.9 Electron transfer
Data represent differentially expressed proteins between control and obese female mice. Only proteins that were significantly different are
reported (
p
<
0.05). The SSP number is assigned by the PDQuest software program (Bio-Rad, Hercules CA). *Proteins identified in two or more
independent experiments.
TABLE 2. PROTEINS IDENTIFIED IN OBESE FEMALES: CONTRACTION/CYTOSKELETAL
Spot SSP Mr (Da) pI
Mascot
score Protein name
Swiss prot acces-
sion number
% Sequence
coverage
No of
peptides
Fold increase
or decrease
Functional group or
function
8 0203 32718 4.69 429 Tropomyosin 1, alpha
chain
P58771
34
11
2.5 Striated muscle
contraction
9* 3201 42381 5.23 268 Actin, smooth muscle
P62737
22
7
–2.3 Structural constitu-
ent of cytoskeleton
10* 3203 42334 5.23 318 Actin, alpha cardiac
P68033
35
10
–4.0 Structural constitu-
ent of cytoskeleton
11* 7002 224349 5.57 225 Myosin heavy chain
QO2566
2
6
–3.8 Muscle contraction
12* 5401 141799 6.06 179 Myosin binding protein C,
cardiac
O70468
6
9
–1.9 Muscle contraction
13 0206 22390 5.03 283 Myosin light chain
P05977
33
8
1.7 Muscle contraction
Data represent differentially expressed proteins between control and obese female mice. Only proteins that were significantly different are report-
ed (
p
<
0.05). The SSP number is assigned by the PDQuest software program (Bio-Rad). *Proteins identified in two or more independent experi-
ments.
