Open Journal of Applied Sciences, 2012, 2, 248-256
doi:10.4236/ojapps.2012.24037 Published Online December 2012 (http://www.SciRP.org/journal/ojapps)
Myosin Heavy Chain Expre ssion and Oxid ative
Modifications in Diabetic Rat Hearts
Mai Kuratani1, Keita Kanzaki2, Noriyuki Yanaka3, Satoshi Matsunaga4, Masanobu Wada1
1Graduate School of Integrated Arts and Sciences, Hiroshima University, Hiroshima, Japan
2Faculty of Food Culture, Kurashiki-Sakuyo University, Okayama, Japan
3Graduate School of Biosphere Science, Hiroshima University, Hiroshima, Japan
4Faculty of Education and Culture, Miyazaki University, Miyazaki, Japan
Email: wada@hiroshima-u.ac.jp
Received August 27, 2012; revised September 29, 2012; accepted October 11, 2012
ABSTRACT
In this study, we tested the hypotheses that 1) diabetes-induced disturbances in cardiac my-ATPase activity would be
attributed to not only myosin heavy chain (MHC) isoform transitions, but also reduced amounts in MHC protein; and 2)
if diabetes results in declines in the MHC protein content, this change would relate to oxidative damage to MHC. Dia-
betes was induced by a single intraperitoneal injection of streptozotocin. After 6 weeks of injection, the left ventricles
were excised for mechanical and biochemical analyses. Peak twitch tension and the rate of force development in papil-
lary muscles were decreased by 23.4% and 34.1%, respectively. A 33.5% reduction in myofibrillar ATPase activity
occurred in conjunction with a 9.5% decrease in MHC protein as well as MHC isoform transitions towards a slower
phenotype. The decreased MHC content was not accompanied by elevations in carbonyl groups present in MHC. Whole
muscle analyses indicated that the contents of malondialdehyde and reduced glutathione were elevated. These results
suggest that decreases in the MHC content may be associated, at least in part, with a diabetes-related inactivation of
cardiac my-ATPase and may not be due to accumulation of oxidative damage to protein.
Keywords: Myofibrillar ATPase; Reactive Oxygen Species; Protein Degradation; Isomyosin
1. Introduction
The prevalence of diabetes mellitus is growing rapidly
from 170 million in 2000 to an estimated 366 million in
2030 [1]. Patients with diabetes are at increased risk for
cardiovascular diseases. Diabetes mellitus can induce ab-
normalities in ventricular muscle independent of changes
in blood pressure and coronary artery disease, a condition
called “diabetic cardiomyopathy”. Prominent defects of
diabetic cardiomyopathy include slowing of contraction
and relaxation, and reduced cardiac compliance [2].
It is well known that the prime determinant of the con-
tractile properties, particularly the maximum shortening
velocity, in the muscle cell is the catalytic activity of
myofibrillar ATPase (my-ATPase) which resides in the
head region of myosin heavy chain (MHC) [3]. On the
basis of these findings, an inactivation of cardiac my-
ATPase has been discussed as one of causes of diabetic
cardiomyopathy. Two functionally diverse MHC iso-
forms are expressed in mammalian cardiomyocytes, i.e.,
α-MHC and β-MHC. The two MHC isoforms display
93% amino acid identity [4], yet α-MHC exhibits more
than two fold higher my-ATPase activity [5]. Diabetic
cardiomyopathy has been demonstrated to be accompa
nied by the downregulation of α-MHC together with the
upregulation of β-MHC, resulting in the decreased
my-ATPase activity [6]. Therefore, the deteriorations in
shortening velocity and power output that occur in dia-
betic heart are explained, in part, by a shift in MHC to-
wards a slower phenotype. However, the maximum shor-
tening velocity in diabetic cardiomyocyte declines more
than would be expected from MHC isoform transitions
[6], thus suggesting that other mechanisms are involv-
ed.
Reactive oxygen species (ROS), such as the super-
oxide anion, the hydroxyl radical and the hydrogen per-
oxide, are continuously produced in most cells under
physiological conditions. Modest and transient increases
in ROS play essential roles in the normal cellular signal-
ing, whereas large and prolonged increases occur in pa-
thological conditions. Diabetes has been shown to bring
about ROS overproduction in cardiomyocytes due to
mitochondrial abnormality [7] and NADPH oxidase ac-
tivation [8]. These conditions are capable of activating a
number of secondary messenger pathways, leading to
cardiomyocyte apoptosis [9] and cardiac fibrosis [10]
which contributes to compromise the cardiac function.
Copyright © 2012 SciRes. OJAppS
M. KURATANI ET AL. 249
It is generally accepted that, in skeletal muscles, se-
vere oxidations of myofibrillar proteins (my-proteins)
exert the deleterious effect on the functional behavior of
the myofibril [11]. For instance, experiments on skinned
fibers have indicated that exposures to ROS result in re-
ductions in maximum force and/or the myofibrillar Ca2+
sensitivity [12,13]. The reductions seem likely to be ow-
ing, at least in part, to oxidation of cysteine residues, as
the effects are at least partially reversible with the disul-
fide reducing agent. The additional effect of oxidative
modification contributing to impaired myofibril function
appears to be a decrease in the my-protein content, given
that enhanced protein oxidation triggers protein degrada-
tion [14]. These findings on skeletal muscles raise the
plausible hypothesis that diabetic cardiomyopathy might
relate to not only MHC isoform transition, apoptosis and
fibrosis, but also oxidation of the existing my-proteins.
No published study presently exists, however, that ex-
amines a causal relationship among my-ATPase activity,
my-protein content and oxidative modifications of my-
proteins in diabetic heart.
In this study, we tested the two hypotheses that 1)
diabetes-induced disturbances in cardiac my-ATPase
activity would be attributed to not only MHC isoform
transitions, but also reductions in the MHC protein con-
tent (the first hypothesis); and 2) if diabetes results in
declines in the MHC content, this change would relate to
oxidative damage to MHC (the second hypothesis). To
this end, diabetic hearts from the rats were analyzed for
my-ATPase activity, and carbonyl and nitrotyrosine con-
tent. The present experiments conducted with the animals
subjected to streptozotocin (STZ) injection reveal that
depressions in my-ATPase activity and MHC content
occur without concomitant elevations in carbonyl group,
an indicator of protein oxidation, contained in MHC.
2. Materials and Methods
2.1. Induction of Diabetes
All procedures were approved by Animal Care Commit-
tee of Hiroshima University. The experiments were per-
formed on male Wistar rats weighing between 200 and
250 g. Throughout this study, the rats were housed at
22˚C with fixed 12-h light/dark cycles and provided
normal rat chow and water ad libitum. Diabetes was in-
duced by a single intraperitoneal injection of STZ (65
mg/kg body wt) according to the method previously de-
scribed [15]. Because STZ was dissolved in 0.1 M citrate
buffer (pH 4.5), control rats received citrate buffer only.
Three days later, blood glucose levels were determined
using Medisafe-Mini (GR0102, Terumo, Tokyo, Japan),
and STZ-treated rats with higher than 12 mM were con-
sidered diabetic according to Cai et al. [16]. Six weeks
after the injection, hearts from each group (n = 8 per each
group) were excised under anethesia using pentobarbital
sodium (50 mg/kg body wt). After papillary muscle ex-
periments (see below), the atria and aorta were cut off,
and the ventricular muscle tissues were supplied for bio-
chemical analyses.
2.2. Papillary Muscle Experiments
Left ventricular papillary muscles were removed under
the microscope and mounted between motionless lever
and force transducer. The muscle chamber was perfused
with Krebs-Ringer solution (in mM: 5 N,N-bis(2-hy-
droxyethl)-2-aminoethanesulfonic acid, 5 KHCO3, 115
NaCl, 20 NaHCO3, 1 MgCl2, 10 glucose, 0.3 glutamic
acid, 0.4 glutamine, and 2 CaCl2, 32˚C). The solution
was continuously bubbled with 95% O2 and 5% CO2,
which gives a bath pH 7.4. Papillary muscles were
stimulated with 0.2 Hz and voltage 30% above threshold
to contract for one hour equilibration period and set to
optimal length according to the procedure previously
described [17]. After steady-state conditions were ob-
tained, twitch tension was recorded with PowerLab
(ADInstruments, Tokyo, Japan). The mechanical proper-
ties evaluated in the experiment included peak twitch
tension, time to peak tension (TPT), time to half relaxa-
tion (TR50), and the mean rates of relaxation (–dT/dt) and
force development (+dT/dt).
2.3. Myofibrillar ATPase Activity
Ventricular muscle myofibrils were prepared on ice as
previously described [18]. Protein concentration was
determined according to Bradford [19] using bovine
serum albumin as a standard. My-ATPase activity was
spectrophotometrically determined at 37˚C according to
the procedure previously described [20]. The reaction
mixture was composed of 30 mM KCl, 30 mM Tris, 1
mM EGTA, 2 mM NaN3, 1 mM MgSO4, 1.1 mM CaCl2,
0.4 mM NADH, 10 mM phosphoenolpyruvate, 18 U·ml–1,
pyruvate kinase and 18 U·ml–1 lactate dehydrogenase.
The reaction was started by adding ATP to give a final
concentration of 1 mM. The oxidation of NADH was
monitored in a spectrophotometer for 3 min (340 nm).
2.4. Isomyosin Electrophoresis
The analytical procedure for pyrophosphate polyacryla-
mide gel electrophoresis (PAGE) has been described in
detail elsewhere [21]. Briefly, after pre-electrophoresis
for 30 min, 5 μg protein was loaded on slab gel. Pyro-
phosphate-PAGE was carried out at 0˚C with constant
voltage of 120 V for 72 h. The gels were silver-stained
according to Oakley et al. [22]. The relative contents
occupied by V1, V2 and V3 were densitometrically esti-
mated using Image J.
Copyright © 2012 SciRes. OJAppS
M. KURATANI ET AL.
250
2.5. Preperation of Myofibrillar Fraction
The fraction enriched in my-proteins was prepared in
ventricular muscles according to Thompson et al. [23].
The fractions were supplied for analyses of the MHC and
actin protein contents and the redox state of my-proteins.
Approximately 150 mg of tissues was homogenized on
ice in 8 volumes (mass vol–1) of a solution (solution 1)
composed of 20 mM imidazole and 0.25 mM phe-
nylmethyl sulfonyl fluoride (pH 7.4). After centri-
fugation at 12,000 g for 15 min at 4˚C, the pellet was
rehomogenized with solution 1 and the centrifugation
was repeated. The pellet was then homogenized with 4
volumes (mass vol–1) of a solution (solution 2) consisting
of 0.5% trifluoroacetic acid and 1 mM tris(2-carbox-
yethylphosphine)hydrochloride. After centrifugation, the
supernatant was collected and the remaining pellet was
rehomogenized with solution 2. After centrifugation, the
supernatant was collected again. The two collected su-
pernatants were combined and stored in –80˚C.
2.6. Myosin Heavy Chain and Actin Content
In order to separate my-proteins, sodium dodecyl sulfate
(SDS)-PAGE was performed using a 10% - 20% (mass
vol–1) gradient separating gel, as previously described
[24]. Ten μg of protein was subjected to electrophoresis
at 22˚C for 5 h, applying current of 20 mA. The gels
were stained with Coomassie blue R. The densitometri-
cally estimated amounts of MHC and actin were norma-
lized by reference to those in a standard my-protein.
2.7. Immunoblotting
Equal amounts of my-protein (7.5 μg for carbonyl; 20 μg
for nitrotyrosine) were separated by SDS-PAGE de-
scribed above. 2,4-dinitrophenylhydrazine (DNPH)-re-
active carbonyls were detected by using Oxiblot kit
(Chemicon international, Temecula, California, USA).
After SDS-PAGE, proteins were transferred onto poly-
vinylidene difluoride membranes. The membranes were
blocked in 1% (mass vol–1) bovine serum albumin, fol-
lowed by incubation with the primary (1:150) and the
secondary (1:300) antibodies for 1 h at 22˚C, respec-
tively.
For nitrotyrosine immunoblotting, electrophoretically
separated proteins were transferred onto nitrocellulose
sheet. The blots were blocked in 3% (mass vol–1) bovine
serum albumin, followed by incubation with the anti-
nitrotyrosine (1:1000; Cayman Chemical, Ann Arbor,
Michigan, USA) at 4˚C overnight. The blots were then
incubated with a horseradish peroxidase-conjugated sec-
ondary antibody (1:50,000; polyclonal rabbit anti-mouse
immunoglobulins, Dako, Glostrup, Denmark) at 22˚C for
1 h. Immunoreactive bands of carbonyl and nitrotyrosine
were visualized using the enhanced chemiluminescence
(ECL Western blotting detection reagents, Amersham
Biosciences, Rockford, Illinois, USA). The intensities of
bands were densitometrically evaluated. The carbonyl
contents of MHC and actin were normalized by each
protein content.
2.8. Sulfhydryl Group Content
The amounts of sulfhydryl (SH) group contained in my-
proteins were measured according to Favero et al. [25].
Fifty μg of proteins was incubated in a solution con-
sisting of 50 mM Tris-HCl (pH 7.0), 1% (mass vol1)
SDS, 1 mM EDTA and 1 mM 5,5’ dithiobis (2-nitro-
benzoic acid) (DTNB) for 1 h at 22˚C. The SH group
content was spectrophotometrically determined at a wave-
length of 412 nm.
2.9. Lipid Peroxidation
The method to measure products of lipid peroxidation
was based on thiobarbituric acid (TBA) assays to mea-
sure malondialdehyde (MDA) according to Ohkawa et al.
(1979) [26]. Approximately 100 mg of muscle pieces
was homogenized on ice in 5 volumes (mass vol–1) of
1.15% (mass vol–1) KCl. The homogenates were added to
a solution consisting of 20% (vol vol–1) acetic acid-
NaOH (pH 3.5), 0.37% (mass vol–1) SDS and 0.03%
(mass vol–1) TBA. This solution was mixed, heated for 1
h at 100˚C, incubated on ice for 10 min, and then cen-
trifuged at 16,000 g for 10 min at 4˚C. The MDA content
was spectrophotometrically determined in the superna-
tant at a wavelength of 532 nm.
2.10. Glutathione Content
The amounts of total (GSH + GSSG) and oxidized glu-
tathione (GSSG) were determined according to Baker et
al. [27]. Approximately 50 mg of muscle pieces was
minced, placed on ice in 9 volumes (mass vol–1) of 5%
(mass vol–1) 5-sulfosalicylic acid for 30 min, and then
centrifuged at 16,000 g for 10 min at 4˚C. For GSH +
GSSG measurement, 6% (vol vol–1; final concentration)
triethanolamine was added to the supernatant, and for
GSSG measurement, 2% (vol vol–1; final concentration)
2-vinylpyridine was additionally added. The assay buffer
contained 1.52 mM NaH2PO4, 7.6 mM Na2HPO4, 0.485
mM EDTA, 1 U ml–1 glutathione reductase and 0.1 mM
NADPH (pH 7.5). After the addition of an aliquot of the
sample, the assay mixture was incubated for 2 min. The
reaction was started by adding 5,5’-dithiobis-(2-nitro-
benzoic acid) to give a final concentration of 0.4 mM.
The glutathione content was spectrophotometrically de-
termined at a wavelength of 412 nm. The content of re-
duced glutathione (GSH) was calculated as the difference
Copyright © 2012 SciRes. OJAppS
M. KURATANI ET AL. 251
between GSH + GSSG and GSSG contents. All bioche-
mical measurements were performed in triplicate.
2.11. Statistical Analysis
Data are presented as the mean ± SE. Student’s unpaired
t-tests were used to establish significant differences be-
tween control and diabetic rats. All comparisons were
performed at a 95% confidence level.
3. Results
3.1. General Characteristics of Animals and in
Vitro Papillary Muscle Function
Treatment with STZ resulted in significant reductions in
the body and heart weights, which amounted to 65.0%
and 73.1% of control, respectively (Table 1). The blood
glucose level was 3.76-fold higher for diabetic than for
control rats. Papillary muscles from diabetic rats dis-
played an inability to generate force (Table 1). Twitch
tension, +dT/dt and –dT/dt were decreased to 76.6%,
65.9% and 63.9% of control, respectively. Changes in
+dT/dt were accompanied by significant increases in
TPT.
3.2. Myofibrillar ATPase Activity, Isomyosin
Distribution, and Myosin Heavy Chain and
Actin Contents
The catalytic activity of my-ATPase in diabetic
ventricles was depressed to 66.5% of control (Figure 1).
All three cardiac isomyosin, V1 (α-MHC homodimer). V2
(α-MHC/β-MHC heterodimer) and V3 (β-MHC homo-
dimer), were present in rat ventricles (Figure 2(a)).
Ventricles of control rats displayed V1 as prominent
isoforms (76.1%) together with relatively low amounts of
Table 1. General characteristics of animals and contractile
properties of left ventricular papillary muscle from 6-week
STZ-induced diabetic and age-matched control rats.
Control Diabetic
Body wt (g) 430.0 ± 6.0 279.4 ± 16.0#
Heart wt (mg) 1063.1 ± 19.8 777.0 ± 19.6#
Blood glucose level (mM) 6.6 ± 0.3 24.8 ± 1.6#
Twitch tension (mN/mm2) 12.4 ± 0.9 9.5 ± 0.9#
+dT/dt (mN/mm2/s) 91.3 ± 6.8 60.2 ± 5.1#
–dT/dt (mN/mm2/s) 48.5 ± 5.2 31.0 ± 2.6#
TPT (ms) 134.3 ± 5.5 160.3 ± 10.0#
TR50 (ms) 133.9 ± 13.1 154.5 ± 11.6
Values are means ± SE of n = 8 per group. STZ, streptozotocin; +dT/dt, rate
of force development; dT/dt, rate of relaxation; TPT, time to peak tension;
TR50, time to half relaxation. #P < 0.05, significantly different from control.
Con Dia
my-ATPase activity
(mol/min/g protein)
0
100
200
300
400
500
600
700
#
Figure 1. Myofibrillar ATPase (my-ATPase) activity in
ventricles from 6-week STZ-induced diabetic and age-mat-
ched control rats. The catalytic activity of my-ATPase was
measured in myofibril ex tracts. Values are means ± SE of n = 8
per group. Con, control; Dia, diabetic; STZ, streptozotocin.
#P < 0.05, significantly different from control.
V
3
V
2
V
1
Con Dia
(a)
Con Dia
Isomyosin distribution (%)
0
20
40
60
80
100 V1
V2
V3
#
#
(b)
Figure 2. Electrophoretic separation of isomyosins (a) and
percentage distribution of isomyosins (b) in ventricles from
6-week STZ-induced diabetic and age-matche d control rats.
Isomyosins were separated by electrophoresis under non-
denaturing conditions. The percentage distribution of the
three isomyosins was evaluated by the densitometry of sil-
ver-stained gels. Values are means ± SE of n = 8 per group.
V1, α-MHC homodimer; V2, α-MHC/β-MHC heterodimer;
V3, β-MHC homodimer; Con, control; Dia, diabetic; STZ,
streptozotocin; MHC, myosin heavy chain. #P < 0.05, sig-
nificantly different from control.
V2 (15.0%) and V3 (8.9%). In STZ-treated animals, the
isomyosin pattern was shifted towards the slowest V3,
which amounted to 78.0% of the total isomyosins (Fig-
ure 2(b)). V1 and V2 were found at relative concentra-
tions of 9.4% and 12.6%, respectively. STZ-treatment
produced a significant decrease to 90.5% in the MHC
content (Figure 3(b)). In contrast, an 8.4% decrease in
the actin content did not reach statistical significance
(Figure 3(c)).
Copyright © 2012 SciRes. OJAppS
M. KURATANI ET AL.
Copyright © 2012 SciRes. OJAppS
252
Con Dia
Actin content in myofibril
(% of control)
0
20
40
60
80
100
120
Con Dia
MHC content in myofibril
(% of control)
0
20
40
60
80
100
120
#
MHC
Actin
Con Dia
250
150
25
100
75
50
37
20
kDa
(a) (b) (c)
Figure 3. Electrophoresis of myofibrillar proteins (a) and myosin heavy chain (MHC) and actin protein contents ((b) and (c))
in ventricles from 6-w eek STZ-induced diabetic and age-matched control rats. Myofibrillar proteins were separated by poly-
acrylamide gradient (10% - 20%) gel electrophoresis. The contents of MHC and actin were evaluated by the densitometry of
Coomassie blue-stained gels. The results are expressed as a percentage of control value. Values are means ± SE of n = 8 per
group. Con, control; Dia, diabetic; ST Z, streptozotocin. #P < 0.05, significantly different from control.
3.3. Malondialdehyde and Glutathione Contents
in Whole Muscle
Con Dia
Malondialdehyde content
(nmol/g wet wt)
0
100
200
300
400
#
STZ-treatment resulted in a 31.0% increase in the MDA
content (Figure 4). GSH is mostly present in mM levels
in mammalian cells. A biological role has been suggested
for endogenous GSH in alleviating oxidative stress. The
GSH content in diabetic ventricles was increased to
129.7% of control (Figure 5(a)).
3.4. Sulfhydryl, Carbonyl and Nitrotyrosine
Contents in Myofibrillar Proteins Figure 4. Malondialdehyde content in whole ventricles from
6-week STZ-induced diabetic and age-matche d control rats.
The malondialdehyde content were measured by thiobar-
bituric acid assays. Values are means ± SE of n = 8 per
group. Con, control; Dia, diabetic; ST Z, streptozotocin. #P <
0.05, significantly different from control.
No changes in the SH group content were found in my-
proteins from diabetic ventricles (Figure 6). The car-
bonyl group content contained in total my-proteins was
markedly depressed in diabetic ventricles, which amount-
ed to 66.6% of control (Figure 7(b)). As described above,
density measures of the immunoblot were normalized to
the MHC or actin contents. Thus, the ratio values (the
density of immunoreaction/MHC or actin content) pro-
vides an estimate of the extent of oxidative modification.
The carbonyl group content was significantly reduced in
MHC, but not in actin (Figures 7(c) and (d)). In accor-
dance with previous studies [28], nitrotyrosine was not
detected in the MHC and actin bands (Figure 8(a)).
There was no diabetes-related alteration in the nitrotyro-
sine content in total my-proteins (Figure 8(b)).
subjects [29]. Numerous studies on both human and ro-
dent have provided evidence for systolic and diastolic
dysfunction in diabetic ventricles [30]. Our results from
mechanical analyses of papillary muscles agree with
these findings in past studies.
The major findings in the current study are that diabe-
tes-related decreases in my-ATPase activity were ac-
companied by reductions in the MHC content, but not by
increases in the carbonyl content contained in MHC. The
former confirms our first hypothesis, while the latter fails
to support our second hypothesis (see Introduction). As
evidenced by previous findings in animal studies and the
present data, diabetic cardiomyocyte displays a shift of
isomyosins from V1 with the highest my-ATPase activity
to V3 with the lowest activity, leading to a decay of
my-ATPase activity and eventually to systolic dysfunc-
tion [6]. However, the fact that the human heart primarily
4. Discussion
Diabetes mellitus is a well-recognized risk factor for de-
veloping heart failure. Indeed, the frequency of heart
failure has been shown to be twice in diabetic men and
five times in diabetic women, compared to age-matched
M. KURATANI ET AL. 253
Con Dia
GSH content
(mol/g wet wt)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
#
0
(a)
Con Dia
GSSG content
(nmol/g wet wt)
0
10
20
30
40
50
60
(b)
Figure 5. Glutathione content in whole ventricles from
6-week STZ-induced diabetic and age-matche d control rats.
Panels A and B show the contents of reduced glutathione
(GSH) and glutathione disulfide (GSSG), respectively. Val-
ues are means ± SE of n = 8 per group. Con, control; Dia,
diabetic; STZ, streptozotocin. #P < 0.05, significantly dif-
ferent from co ntrol.
Con Dia
Sulfhydryl content in myofibril
(% of control)
0
20
40
60
80
100
120
Figure 6. Sulfhydryl content in myofibrillar proteins from
6-week STZ-induced diabetic and age-matched control rat
ventricles. The results are expressed as a percentage of con-
trol value. Values are means ± SE of n = 8 per group. Con,
control; Dia, diabetic; STZ, streptozotocin.
expresses V3 makes it unlikely that the shift of MHC is
of physiological significance in diabetic cardiomyopathy
at least in human. In contrast to the intensive research
into MHC transitions, studies concerning the influence of
diabetes on the MHC content are sparse. The only study
by Zu et al. [31] revealed an approximately 80% reduc-
tion of the MHC content in mouse cardiac muscle 6
weeks after STZ injection. Although the magnitude of
changes differs markedly, the diabetes-related altertions
in the MHC content that we observed are qualitatively
MHC
Actin
Con Dia
250
150
25
100
75
50
37
20
kDa
(a)
Con Dia
Carbonyl content in myofibril
(% of control)
0
20
40
60
80
100
120
#
(b)
Con Dia
Carbonyl content in MHC
(% of control)
0
20
40
60
80
100
120
#
(c)
Con Dia
Carbonyl content in actin
(% of control)
0
20
40
60
80
100
120
(d)
Figure 7. Immonoblot analysis of carbonyls and carbonyl
content in myofibrillar proteins from 6-week STZ-induced
diabetic and age-matched control rat ventricles. Panel A
shows immunoblot labeled with anti-dinitrophe nyl antibody.
Panels B, C and D show the carbonyl contents in total myo-
fibrillar proteins, myosin heavy chain (MHC) and actin,
respectively. The carbonyl contents were evaluated by the
densitometry of immunoblots. The results are expressed as
a percentage of control value. Values are means ± SE of n =
8 per group. Con, control; Dia, diabetic; STZ, streptozoto-
cin. #P < 0.05, significantly different from control.
Copyright © 2012 SciRes. OJAppS
M. KURATANI ET AL.
254
Con Dia
Nitrotyrosine content in
myofibril (% of control)
0
20
40
60
80
100
120
250
150
25
100
75
50
37
20
kDa
MHC
Actin
Con Dia
(a) (b)
Figure 8. Immonoblot analysis of nitrotyrosines and nitro-
tyrosine content in myofibrillar proteins from 6-week STZ-
induced diabetic and age-matched control rat ventricles.
Panel A shows immunoblot labeled with anti-nitrotyrosine
antibody. Panel B shows the nitrotyrosine contents in total
myofibrillar proteins. The nitrotyrosine contents were eva-
luated by the densitometry of immunoblots. The results are
expressed as a percentage of control value. Values are
means ± SE of n = 8 per group. Con, control; Dia, diabetic;
STZ, streptzotocin.
similar to those of Zu et al. [31], suggesting that part of
the decreased my-ATPase activity may be caused by a
loss of MHC protein. The reasons for the quantitative
difference in the results between the study of Zu et al.
[31] and ours remain unclear, although differences in
diabetogenic protocols employed, assay procedures, and/
or species appear to be involved.
It is a well-known fact that diabetes mellitus leads to
increases in ROS production [8]. The elevated MDA
content indicates a diabetes-induced shift of the redox
state to the oxidized side in diabetic ventricles used in
this study. Generally, the amount of a given protein pre-
sent in muscle is regulated by the balance between pro-
tein synthesis and degradation. Enhanced protein oxida-
tion seems likely to trigger protein degradation. It is
proposed that since oxidized proteins are less thermosta-
ble than their native forms, they have to be eliminated,
and that carbonylation and tyrosine nitration are irre-
versible modifications that require the proteolytic re-
moval, thus accelerating protein degradation by the pro-
teasomal system [14]. On the basis of these findings, we
hypothesized that if diabetes results in declines in the
MHC content, oxidative damage to MHC would be a one
of the causes of the reduced MHC content (the second
hypothesis). However, as judged by immunoblot analy-
ses, our results do not provide evidence for oxidative
damage to my-proteins (Figures 7 and 8). Support for
the absence of oxidative modification also comes from
our results of unaltered SH group present in my-proteins
(Figure 6).
It is possible that the reduced MHC content could be
linked to alterations in the rate of MHC synthesis. Stu-
dies with focus on transcriptional factors indicated that
diabetes mellitus was responsible for nuclear factor-KB
activation [32]. The activation seemed to trigger a cas-
cade of signaling, which finally led to decreases and in-
creases in α- and β-MHC gene expression, respectively.
Although it is apparent that the nuclear factor-KB activa-
tion is capable of causing MHC isoform transitions men-
tioned above, further study will be required to elucidate
whether this correlates with the diabetes-induced decay
in MHC proteins.
An unexpected finding was the observation that STZ-
induced diabetes evoked reductions in the carbonyl con-
tent contained in MHC and my-proteins (Figure 7). This
finding is difficult to explain in light of ROS overpro-
duction in diabetic cardiomyocyte. Obviously, the re-
duced carbonyl is not caused by the reduced MHC, be-
cause our quantitative data shown in Figure 7 was nor-
malized to the MHC content (see Meterials and Meth-
ods). A possible explanation of the decreased carbonyl is
that alterations in the cellular redox state could result in
improvement of the ability of the cell to protect intracel-
lular organelles from the deleterious effect of ROS. GSH
is the most important antioxidant in the muscle cell and
serves as an antioxidant by reacting directly with ROS
and by providing substrate for glutathione peroxidase.
The literature on the impact of diabetic mellitus on the
glutathione content in heart is controversial. Reductions
in the ratio of GSH to GSSG [10], increases in GSH [6],
and no changes in GSH [33] have been reported in dia-
betic ventricle. Although the cause for the discrepancies
also remains unclear, the augmented amount of GSH
shown in Figure 5(a) reveal the improved antioxidant
ability, at least, in diabetic heart employed in this study.
This scenario, however, conflicts with our results of
the MDA content. The increased MDA might be ex-
plained by a high susceptibility of lipids to ROS and by
the site for ROS production. Superoxide generation has
been shown to be augmented to 2-fold in isolated mi-
tochondria from diabetic mouse heart [9]. Mitochondria
predominates in cardiac muscle and its inner membrane
of mitochondria is the most susceptible region [30].
Moreover, superoxide that has a negative charge does not
easily cross the mitochondrial membrane [34].
In summary, we show that diabetes-induced distur-
bances in my-ATPase activity occur in conjunction not
only with MHC transitions towards a slower phenotype,
but also with reductions in the MHC protein content. The
reduced MHC content is not accompanied by elevations
in DHNP-reactive carbonyls present in MHC. These re-
sults suggest that decreases in the MHC content may be
associated, at least in part, with a diabetes-related inacti-
vation of cardiac my-ATPase and may not be due to ac-
Copyright © 2012 SciRes. OJAppS
M. KURATANI ET AL. 255
cumulation of oxidative damage to protein. Future stu-
dies are needed to establish the mechanisms underlying
diabetes-induced loss of MHC proteins.
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