Possible mechanism of increasing resistance of the myocardium during combination of post infarction remodeling and diabetes mellitus

doi:10.4236/ns.2011.34038

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Vol.3, No.4, 295-300 (2011) Natural Science

http://dx.doi.org/10.4236/ns.2011.34038

Possible mechanism of increasing resistance of the

myocardium during combination of post infarction

remodeling and diabetes mellitus

Margarita V. Egorova*, Sergey A. Afanasiev, Dina S. Kondratyeva, Boris N. Kozlov, Sergey V. Popov

Laboratory of Molecular Cell Pathology and Genetic Diagnosis, Research Institute of Cardiology, Siberian Branch of Russian Academy

of Medical Science, Tomsk, Russia; *Corresponding Author: [email protected]

Received 2 February 2011; revised 21 February 2011; accepted 13 March 2011.

ABSTRACT

It was shown that the energy metabolism of the

heart mitochondria of experimental animals and

patients is more resistant to damage at com-

bined postinfarction cardiosclerosis and dia-

betes in comparison with the individual pathol-

ogy. We found that the changes of free fatty acid

content and conjugation of the processes of

oxidation and phosphorylation in heart mito-

chondria are components of the metabolic sta-

bility of myocardium at the combined develop-

ment of postinfarction cardiosclerosis and dia-

betes mellitus. Our data demonstrate a direct

link between the violations of the processes of

oxidative phosphorylation and accumulation of

free fatty acids owing to change in activity of

endogenous phospholipases, in particularly, mi-

tochondrial phospholipase A2. Similar results

were obtained for intraoperative biopsy speci-

mens of patients’ hearts, and of adult Wistar

rats’ hearts. We hypothesized that the preserva-

tion of energy metabolism is a manifestation of

summing up of compensatory processes at de-

velopment of nonspecific response of cells to

damage at the early stages of pathological pro-

cess.

Keywords: Postinfarction Remodeling; Diabetes;

Heart Mitochondria; Fatty Acids; Phospholipase A2

1. INTRODUCTION

It is well known that many pathologies of the cardio-

vascular system are accompanied by increased activity

of endogenous phospholipases and, as a consequence, by

accumulation of free fatty acids [1] which, in turn, pro-

vokes uncoupling of the processes of oxidation and

phosphorylation in mitochondria [2]. A decrease of insu-

lin action on adipose tissue at diabetes mellitus (DM)

leads to the increased content of fatty acids in blood and

their intake into the myocardial cells. Myocardial con-

sequences of DM even in conditions of adequate oxygen

supply of myocardium resemble metabolic imbalance in

myocardium of patients with severe coronary heart dis-

ease (CHD) [3,4]. At DM, as at CHD, the corresponding

complex of pathophysiological changes appears either

because of the inability of mitochondria to oxidize the

entire volume of incoming fatty acids, or because of

critical lowering of coronary blood flow (at CHD), or

because of the enhanced transport of fatty acids in the

cytosol (at DM) [3,4]. It is logical to suppose that

hemodynamic changes on the DM background can in-

crease the probability of lethal outcome that is confirmed

by clinical studies [4,5].

However, there is data of manifestation of myocar-

dium resistance to ischemia (in vivo and in vitro) of

animals with short term of induced diabetes [6-8]: the

induction of diabetes on the background of postinfarc-

tion cardiosclerosis preserves, paradoxically, myocar-

dium contractile properties. Animals with combined pa-

thology are characterized by less pronounced changes in

glucose levels, body weight and heart [9,10].

The purpose of this study was evaluation of influence

of diabetes mellitus and postinfarction remodeling on

ability to oxidative phosphorylation of isolated heart mi-

tochondria of animals and humans both in the case of in-

dividual pathologies and in the case of their combination.

2. EXPERIMENTAL PART

2.1. Materials

The work was accomplished on mature male rats of

Wistar and intraoperative biopsy samples of patients.

2.1.1. Animals

5 groups for 8 rats were formed from animals:

M. V. Egorova et al. / Natural Science 3 (2011) 295-300

296

Group I - control animals,

Groups II and V – animals with induced DM,

Group III - rats after coronary occlusion,

Group IV - rats with combined pathology: DM was

induced after coronary occlusion after 2 weeks.

2.1.2. Patients

Studies were carried out on intraoperative atrial biop-

sies of male patients aged 52 - 69 years with underlying

diagnosis of coronary heart disease (CHD). The biopsy

specimens were divided into two groups:

The first group – CHD: patients with a diagnosis of

coronary heart disease (12 samples).

Underlying disease: CHD, exertional angina, func-

tional class III (FC). Multivascular atherosclerosis of

coronary arteries. Postinfarction cardiosclerosis (4 - 6

years after acute myocardial infarction). Chronic heart

failure (HF) by classifying the New York Heart Associa-

tion (NYHA) II, with preserved LV systolic function:

left ventricular ejection fraction (LVEF) > 45% (59% -

65%). Background pathology: arterial hypertension, de-

gree III, Risk 4, dislipidemia.

The second group – CHD + DII: patients with a diag-

nosis of CHD with a combination of diabetes mellitus

type 2 (6 samples).

Underlying disease: CHD, exertional angina FC III.

Postinfarction cardiosclerosis (after acute myocardial in-

farction – 4 - 6 years old). HF NYHA II, LVEF > 45%

(59% - 65%). Background pathology: arterial hyperten-

sion III, Risk, 4, dislipidemia. Concomitant disease: Dia-

betes mellitus type 2, moderate, subcompensated (Hb A1C

7.5%; fasting glucose 10 - 13 mmol/L, in urine – 40 - 45

g/day; cholesterol total 5.2 - 6.5 mmol/l, low density

lipoprotein (LDL) cholesterol > 3.0 mmol/l, high density

lipoprotein HDL < 0.9 mmol/l). Body mass index (BMI)

of 35 - 39 kg/m2, abdominal obesity 2.

The duration of the underlying disease from the time

of registration of 8 - 10 years, concominant disease—at

least 5 years.

Standard treatment: antianginal and antihypertensive

medicines, hypolipidemic agents (statin or/and fibrate),

and, for diabetes - hypoglycemic agent (metformin 1500 -

2000 mg/d).

Myocardial tissue samples of patients were frozen in

liquid nitrogen, prior to withstanding them at least 1

hour in cold Krebs-Henseleit buffer containing 20% di-

methyl sulfoxide (DMSO) [2]. Before the experiment,

biopsies thawed in warm Krebs-Henseleit buffer and

then used as freshly isolated tissue [2].

2.2. Methods

2.2.1. Simulation of Diabetes Mellitus

The development of diabetes was induced by a single

injection of streptozotocin (Sigma, USA) in a dose of 60

mg/kg, intraperitoneally, diluted ex tempera in 0,01

mol/L citrate buffer (pH 4.5) [6]. Diabetes mellitus was

verified by an increase of glucose concentration in rat

blood by 4,5 times and decrease of body weight of 56%

(p < 0.05) in comparison with the animals injected with

citrate buffer. Glucose concentration in blood serum was

determined with help of enzymatic – colorimetric test

(“Biocon Diagnostic”, Germany). The animals in Groups

II and IV were included into the study in 4 weeks after

the induction of diabetes mellitus, the animals in Group

V – in 6 weeks after.

2.2.2. Modeling of Postinfarction

Cardiosclerosis

Modeling of postinfarction cardiosclerosis in animals

was performed under deep ether anesthesia. The thoracic

cage of animals was opened and dissected into the two

ribs. After pericardiotomy, the coronary occlusion was

performed by ligation in the upper third of the left de-

scending coronary artery. Then, after removing air from

the thoracic cage cavity, the wound was sutured in layers.

After 40 days myocardial infarction was formed in these

animals: a morphological control of changes in the

structure of myocardial tissue was performed by means

of histological study, as previously described [1]. The

animals in this group had myocardial hypertrophy (Fig-

ure 1) (the heart size of the operated animals exceeded

the heart size of control rats, an average of 80%), and the

necrosis zone was about 12% of the total mass of the

hypertrophied left ventricle (Table 1). For the experi-

ment we used animals in 6 weeks after coronary occlu-

sion. Mitochondria obtained from control animal hearts

(sham-operated animals) were used as a control.

2.2.3. Measurementes

Heart mitochondria of patients and animals were re-

Figure 1. The typical form of rat heart. Note: A – the heart of

the control animal, B - the heart of the animal after 40 days

after coronary occlusion; 1 - place ligation, 2 - zone scar.

M. V. Egorova et al. / Natural Science 3 (2011) 295-300

297

Table 1. Weights of rats after experimental coronary occlusion.

Parameter sham-operated

animals

40 days after

coronary occlusion

Body weight (g) 287 ± 23.07 236.6 ± 3.07

Heart weight (mg) 955.9 ± 44.68 1491.2 ± 20.96 *

LV weight (mg) 620.5 ± 35.37 962.2 ±13.09*

Weight of the

damaged area (mg) 0 109.1 ± 1.44*

ceived using standard method of differential centrifuga-

tion in sucrose medium containing (mM) 300 of sucrose,

10 EDTA, 8 Tris, pH 7.4 [8]. For storage of mitochon-

dria, we used 250 mM sucrose solution.

The rate of oxygen uptake by mitochondria was de-

termined polarographically by Clark electrode. Meas-

urements were carried out in medium (pH 7.4) contain-

ing (mM): sucrose (300), KCl (10), KH2PO4 (5), succi-

nate (5), EGTA (1), MgCl2 (1,2), Tris (5). We used the

following additives: ADP - 100 uM, p-bromophenacyl

bromide (BPB) - 15 uM, arachidonic acid (AA) - 45 μM.

We used Sigma and ICN reagents.

The respiratory control (RC) was defined as the ratio

of the respiration rate at the maximum ATP synthesis to

respiration rate in the absence of ATP synthesis [9].

The rate of oxygen consumption is given in nM O2 per

minute per 1 mg of protein. The protein concentration in

the sample was determined by the standard Lowry me-

thod.

The content of fatty acids was determined in serum,

homogenates and mitochondrial suspension by enzy-

matic endpoint method (“DiaSys Diagnostic Systems”,

Germany) and calculated on 1 mg of protein.

2.3. Statistical Methods

All data are presented as a mean ± standard error of

the mean. The critical level of significance when testing

statistical hypotheses (p) was taken 0.05. In connection

with the fact that the distribution law of the studied pa-

rameters does not correspond to normal (Shapiro-Wilk

test, p> 0.05) law, we used nonparametric criteria to re-

veal differences in the groups. For independent data (one

index in various groups) used rank Mann-Whitney test.

For dependent data (various parameters in one group)

used Wilcoxon test.

3. RESULTS AND DISCUSSION

3.1. Change of the Content of Fatty Acids in

Serum and Myocardium

It is known that fatty acids are involved in the main-

tenance of membrane gomeostasis. Reorganization of

membrane lipid composition was modulated by “remod-

eling” of membrane phospholipids with phospholipase

A2 and arachidonic acid—this is a quick and subtle

regulation of membrane lipid composition in response to

changing concentrations and ratios of fatty acids [11].

The change in FFA concentration leads to a change in

the permeability of cardiomyocyte membrane, that, in

turn, influences functional activity of cardiomyocytes

[12,13]. We carried out research of fatty acid content in

serum, homogenates of the myocardium and in the mi-

tochondria suspension of animals and humans in all

groups studied (Table 2). It was found that fatty acid

content in blood serum of experimental animals was

reliably higher in all experimental groups in relation to

control, but in comparison between the experimental

groups no reliable differences were observed (Table 2).

There was no significant difference between control and

experimental animals in the homogenate. The most in-

teresting result was found at comparison of the data of

fatty acids content in mitochondrial suspension: a reli-

able difference in fatty acids content was observed not

only in relation of experimental animals to control ones,

but also between the groups (Table 2). The comparison

of data showed that the smallest difference in fatty acid

content in the mitochondrial suspension in relation to

control was observed at combination of pathologies.

Similarly, patients with CHD and CHD + DII did not

reveal statistically significant differences in fatty acid

content in the blood serum and myocardium homogenate,

but in a mitochondrial suspension with combination of

pathologies much less fatty acid content was observed

(Table 2).

3.2. Mitochondrial Respiration in Studied

Groups

Comparing the initial rate of mitochondrial respiration

in the studied groups of animals we found that in all ex-

perimental groups, this figure was significantly higher

than in the group of control animals (Table 3). In Group

II it increased by 4 times, in Group III –more than by 3

times, in Group IV with the combined pathology—only

by 2 times. Reducing the RC value in II-IV groups

demonstrated decrease in the conjugation degree of oxi-

dation and phosphorylation at these pathologies, how-

ever, in Group IV the uncoupling degree is also less

pronounced in comparison with individual pathologies

(Table 3).

The initial rate of oxygen uptake by human heart mi-

tochondria at CHD is almost by 2 times higher than at

combination CHD + DII (Table 3). Low RC indicates

uncoupling of oxidation and phosphorylation in both

groups, but lower rate of oxygen consumption (along

with higher RC) shows that this uncoupling is less pro-

M. V. Egorova et al. / Natural Science 3 (2011) 295-300

298

Table 2. The content of fatty acids in serum and myocardium of animals and humans.

The content of fatty acids (nM per mg protein)

Experimental groups

serum homogenate mitochondria

animals

Group I 0.38 ± 0.08 1.02 ± 0.14 0.83 ± 0.12

Group II 1.68 ± 0.21* 1.51 ± 0.17 5.83 ± 1.31*#^

Group III 0.83 ± 0.14* 1.19 ± 0.14 2.86 ± 1.15*#^

Group IV 1.45 ± 0.35* 1.35 ± 0.15 1.88 ± 0.78*#

patients

CHD 7.35 ± 0.93 9.33±1.62 7.2 ± 1.44#

CHD+DII 9.63 ± 0.81 8.15 ± 1.34 4.2 ± 1.36^#

Note. The experimental conditions and groups are described in the “Experimental part”. * - differences of the results in the column are statis-

tically significant as compared with Group I; # - differences of the results in each group are statistically significant as compared with each

other; ^ - differences between groups are statistically significant.

Table 3. Rate of oxygen consumption and respiratory control of heart mitochondria of animals and humans.

The rate of oxygen consumption, nM O2 per minute per mg of protein.

Experimental groups

initially +BPB

animals

Group I 10.5 ± 1.8 10.6 ± 1.4 3.4 ± 0.09

Group II 44.7 ± 2.8* 38.0 ± 1.7*#^ 2.0 ± 0.01

Group III 35.2 ± 3.5* 21.1 ± 2.4*#^ 1.9 ± 0.02

Group IV 20.9 ± 1.5* 11.6 ± 1.5#^ 2.3 ± 0.05

patients

CHD 33.2 ± 1.5 18.7 ± 2.4#^ 2.0 ± 0.01

CHD+DII 17.6 ± 2.1 7.5 ± 1.2#^ 2.4 ± 0.03

Note. The experimental conditions and groups are described in the “Experimental part”. * - differences of the results in the column are statis-

tically significant as compared with Group I; # - differences of the results in each group are statistically significant when comparing between

indexes “initially” and “+ BPB”; ^ - differences between groups are statistically significant.

nounced in the case of a CHD and DII combination (Ta-

ble 3). Presented data and our earlier observations [10]

allow us to assert that at combination of pathologies car-

diomyocytes are accompanied by less pronounced viola-

tion of the energy metabolism both in animals and in

humans.

We have previously suggested and confirmed the as-

sumption that the violation of energy metabolism in rat

cardiomyocytes may be related to changes in the accu-

mulation of fatty acids and in the activity of endogenous

phospholipases [14]. Inhibition of phospholipase A2 by

p-bromophenacyl bromide (BPB) in postinfarction rat

cardiomyocytes resulted in normalization of cellular

respiration to the level of normal cardiomyocytes. Acti-

vation of phospholipase A2 by arachidonic acid or melit-

tin in cardiomyocytes of control rats significantly in-

creased the need of cells in oxygen [14].

In this study, in the presence of BPB, we observed a

pronounced decrease of the oxygen uptake rate by ani-

mals cardiomyocytes mitochondria of the III-IV Groups,

while in Group IV this figure was equal to that in control

group (Group I) (Table 3). Continuation of this trend is

observed for the human heart mitochondria: in the pres-

ence of BPB a oxygen uptake rate in patients with CHD

and CHD+DII decreased by 44% and 56% respectively

relative to the initial oxygen consumption rate (Table 3).

A significant reduction in the oxygen consumption

rate by mitochondria of animals and humans with a

combination of pathologies in the presence of BPB

demonstrates lability of mitochondrial phospholipase A2.

M. V. Egorova et al. / Natural Science 3 (2011) 295-300

299

It is the evidence of the greater stability of mitochondrial

membrane to damages and, consequently, gives hope

that violation of energy metabolism (closely associated

with membrane processes) is not irreversible.

3.3. Mitochondrial Respiration in Groups

with Different Terms of Diabetes

A comparative analysis of oxygen consumption rate

by rat heart mitochondria at different stages of strepto-

zotocin-induced diabetes showed that in animals of the

Group V a sharp difference of indices is observed not

only in comparison with control animals (Group I), but

also in comparison with diabetes at an earlier stage

(Group II) (Figure 2). Initial oxygen consumption rate

in Group V differs from one of control animals more

than by 10 times and more than 3 times greater than this

figure in comparison with Group II. The degree of un-

coupling of oxidation and phosphorylation becomes dan-

gerously high (RC - 1,5), indicating a critical violation

of energy in cardiomyocytes. Inhibition phospholipase

A2 by BPB does not lead to normalization of respiration

(Figure 2). When studying the influence of arachidonic

acid we observed significant stimulation of oxygen con-

sumption rate of heart mitochondria in Group I and, al-

beit to a much lesser extent, in Group II (Figure 2). We

are prone to consider effect as modulation of phospholi-

pase A2 activity [2]. Lack of stimulating effect of ara-

chidonic acid on oxygen consumption rate in the Group

V (Figure 2), in such a manner, confirms violation of

Figure 2. Effect of BPB and arachidonic acid (AA) on the rate

of oxygen consumption by mitochondria hearts of rats at dif-

ferent stages of diabetes. Note: The experimental conditions

and groups are described in the “Experimental part”. * - dif-

ferences of the results in the column are statistically significant

as compared with Group I; # - differences of the results in each

group are statistically significant when comparing between

indexes “initially” and “+BPB”; ^ - differences between groups

are statistically significant.

membrane remodeling and irreversibility of their dam

age. In these circumstances, even slight changes in myo-

cardial perfusion inevitably lead to cell death.

Our results and some literature data [6-10], at first

glance, are inconsistent with clinical observations about

rapidly growing degree of lethal outcome in such com-

bination of pathologies [10,5]. However, it is well

known that any disturbing factors action start the process

of nonspecific reaction in cells, developing in certain

consequence [15,16]. Early stages of pathological proc-

esses are connected with active start and use of compen-

satory processes to restore functional activity of a cell,

on the later stages this process becomes irreversible

[15,16]. It is possible that in our case cooperative effect

is observed: it is necessary to reveal what specific proc-

esses take part in it. Only the fact raises no doubt that

these processes are connected with activation of mem-

brane enzymes and reorganization of membrane. It is

also possible that along with this, the oxidation proc-

esses are switching to alternatives variances, as it occurs,

for example, during hypoxia (rapid oxidation of succinic

acid) [17].

One of the most powerful natural endogenous mecha-

nisms of adaptation during prolonged ischemia is a

“preconditioning phenomenon”. A lot of data testifying

to multiple levels of organization of this mechanism (see,

for example, reviews [18,19]) has been obtained in last

decades. Although our experimental conditions do not

meet the requirements of manifestation of the precondi-

tioning phenomenon (alternation of short episodes of

sublethal ischaemia and reperfusion), it is possible to

draw parallels. At preconditioning, in addition to short-

term adaptive reaction within 1-2 hours, developing de-

layed, less powerful but more prolonged (72 hours) reac-

tion, which was called “second window” [20]. Is it pos-

sible that combination of pathologies opens the “third

one”?

4. CONCLUSIONS

Thus, the results obtained testify to the fact that dis-

turbance of myocardial energy is the expression of non-

specific reaction of myocardial cells to injury both in

case of combined CHD + DII and in case of separate

pathologies. Our data demonstrate a direct link between

violations of the processes of oxidative phosphorylation

and accumulation of free fatty acids due to changes in

the activity of endogenous phospholipases, in particular,

mitochondrial phospholipase A2.

This article was prepared based on the research

funded by the Ministry of Education and Science under

the Federal Program “Research and development of prior

directions of scientific-technological complex of Russia

for 2007-2012” (HA No. 02.527.11.0007) and the grant

M. V. Egorova et al. / Natural Science 3 (2011) 295-300

300

of the 7th Framework Programme of Russia-EU (No.

241558).

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