INTRODUCTION

The model of experimental, streptozotocin-induced diabetesmellitus (STZ-induced DM) (1, 2) has been shown to exhibitsigns of endogenous protection developing already during theacute phase and leading to unexpectedly high resistance of theheart to adverse external stimuli such us calcium overload andischemia. Further studies performed on this model led tofindings similar to those reported in research using animalmodels of ischemic preconditioning. It has been hypothesizedthat the heart, as a vital organ, has a number of compensatorymechanisms that become activated in an attempt toaccommodate energy demands in DM condition and maintaincardiac function in response to pathological stimulus representedby ischemic reperfusion (I/R) damage (3, 4). In its early stagesDM has all main attributes of metabolic preconditioning, thereby

providing the opportunity to monitor the initiation and course ofendogenous protection mechanisms in the acute phase of thedisease (5-8). The experimental model of STZ-induced DM hasbeen reported as representing type I diabetes where STZ istransported into the pancreatic beta cells - the islets ofLangerhans - through a glucose transporter GLUT-2, where itaccumulates and through a variety of mechanisms causes theirdestruction and the onset of type I diabetes (9, 10). This sort ofmetabolic preconditioning has been described in studies thatdocumented remodeling of subcellular membrane system in themyocardium with consequences which are not necessarilypathological, but rather they reflect a specific endogenous formof cardioprotection (11-13).

Above mentioned compensatory mechanisms induced by theexperimental DM facilitate the ability of myocardium to copewith pathological load, thus initiating a cardioprotective

JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2018, 69, 5, 685-697www.jpp.krakow.pl | DOI: 10.26402/jpp.2018.5.03

M. FERKO1, V. FARKASOVA1, M. JASOVA1, I. KANCIROVA1, T. RAVINGEROVA1, A. ADAMEOVA1, 2, N. ANDELOVA1, I. WACZULIKOVA3

HYPERCHOLESTEROLEMIA ANTAGONIZED HEART ADAPTATION AND FUNCTIONALREMODELING OF THE MITOCHONDRIA OBSERVED IN ACUTE DIABETES MELLITUS

SUBJECTED TO ISCHEMIA/REPERFUSION INJURY

1Center of Experimental Medicine, Slovak Academy of Sciences, Institute for Heart Research, Bratislava, Slovak Republic; 2Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University, Bratislava, Slovak Republic;

3Division of Biomedical Physics, Department of Nuclear Physics and Biophysics, Faculty of Mathematics, Physics and Informatics,Comenius University, Bratislava, Slovak Republic

Intrinsic cardioprotective mechanisms become activated in the early diabetes mellitus (DM) and this may protect theheart from ischemia/reperfusion (I/R) similarly as in case of ischemic preconditioning. However, this protection may byblunted in the presence of cardiovascular risk factors. Assuming that hypercholesterolemia (HCH) frequentlyaccompanies DM, this study extends findings from separate models of DM and HCH by investigation the impact ofHCH on DM-induced changes, including those of compensatory nature, in rat heart and its mitochondria. We used afactorial design with all combinations of treatment factors DM and HCH: control rats (C) and streptozotocin-treated rats(DM), both on standard chow (C and DM) and fed fat-cholesterol diet (HCH and DM-HCH). Isolated, Langendorffperfused hearts were subjected to 30 min global ischemia followed by reperfusion. Significantly increased levels ofcholesterol in DM-HCH after I/R injury abrogated compensatory fluidization characteristic of DM mitochondriamembranes. Concomitantly, the mitochondrial Mg2+-ATPase activity in DM-HCH was depressed. In comparison withDM, which showed significantly reduced size of myocardial infarction with simultaneously improved recovery ofcontractile function due to conditioning, DM-HCH hearts exhibited attenuated resistance to I/R injury. Taken together,cholesterol-enriched diet was associated with inflicting damage and has been implicated in the mechanisms leading tosuppression of cardiac protection presented in diabetic group. Apparently, DM and HCH are factors which are notadditive in their effects, therefore, caution should be exercised, when interpreting findings from studies consideringthese factors in isolation. Our findings suggest that this complex condition could accelerate the development of latediabetic complications.

K e y w o r d s : myocardial hypercholesterolemia, acute diabetes mellitus, heart mitochondria, ischemia/reperfusion injury,cardioprotection

adaptation (14). The degree of adaptation can be quantified, first,by physiological measures that assess cardiac function undervarious experimental conditions such as infarct size (IS),ventricular tachycardia, severity of arrhythmia or recovery ofleft ventricular developed pressure (LVDP) (15-17), and second,by measures that assess the structural and functional propertiesof heart mitochondria (6, 18, 19).

Recently, several research groups have begun to explore theactive role of cardiac mitochondria in the processes ofendogenous protection. Commonly used models ofpreconditioning have yielded results pointing to themitochondria that appear to be a key and effective end-effectorresponsible for myocardial energy maintenance under I/R injury(20). Although abnormal glucose metabolism defines DM andaccounts for many of its symptoms and associated adverse andcompensatory events, efforts to understand these processes areincreasingly focused on disordered lipid metabolism. Recentstudies have found that anti-contractile effects exertedbyperivascular adipose tissue on the vasculature are reduced inhypertension and obesity (21). Many animal and human studieshave been published that explore the mechanistic links betweendisorders of fatty acid-/lipid metabolism and insulin resistance(22), however, to our knowledge, none of the reviewed studiesaddressed the issue whether or not the cholesterol levels, whichare naturally increased in DM, can affect developingcompensatory responses. Here we present findings from a studydesigned to investigate individual and combined effects of theSTZ-induced DM and cholesterol-enriched diet. We chose amodel of the acute DM condition in order to better controlexperimental conditions, since disordered lipid metabolismwould reflect effect of the both factors, developing diabetes andthe cholesterol-enriched diet. The outcome - the cardiovascularadaptation - was evaluated in terms of parameters ofmitochondrial energy production, as well as in terms of cardiacphysiological parameters based on our previous findings ofincreased resistance to diabetic myocardium I/R injury (23).

The second reason why the diabetic-hypercholesterolemic(DM-HCH) model was chosen, is its relevance to clinical situationwhere diabetic patients suffer from both, altered lipid metabolismand simultaneously occurring ischemic heart disease (22).

Moreover, enhanced resistance to I/R in the early stage ofexperimental DM has been found to share several molecularmechanisms of protection conferred by ischemicpreconditioning (IPC) (24, 25). Endogenous cardioprotective

mechanisms become activated in early stage of DM and this mayprotect the heart from I/R injury through enhancement ofendothelial nitric oxide synthase (eNOS) expression, NOformation, activation of cell survival signals, and decreasedoxidative stress, which are the mechanisms responsible forcardioprotective action of IPC as well (24). However, it has beenwell documented that cardiovascular risk factors may interferewith cardioprotective interventions such as IPC (26). Similarly,under different experimental conditions it has been observed thatthe cardioprotective mechanisms present in experimental earlyDM are blunted or even abolished in both conditions, theadvanced stages of DM and the combination of early DM withhypercholesterolemia (HCH) (15, 27, 28).

Changes in mitochondrial membrane lipid composition andcontent modify all lipid-dependent processes that take place inthe membrane (29, 30), e.g. mitochondrial respiration (31, 32)oxidative phosphorylation, and other mitochondrial processessuch as generation of reactive oxygen species (ROS) (33) andCa2+-induced mitochondrial permeability transition (30, 33). Inthis respect especially the cholesterol content is of importance.Although plasma cholesterol correlates with membranecholesterol in a non-trivial manner, in vivo exposure tohypercholesterolemic environment increases incorporation ofcholesterol into the plasma membranes as well as in subcellularmembranes thus influencing their physicochemical properties(34-36). Cholesterol consists of four hydrocarbon rings, whichare strongly hydrophobic - however, the hydroxyl (OH) groupattached to one end of cholesterol is weakly hydrophilic,meaning that cholesterol is an amphiphatic molecule. Thus, theoptimal membrane location of cholesterol is with its backboneembedded in the hydrocarbon core, roughly along the membranenormal, and with the OH group protruding into the polarheadgroup region of the membrane (37). Cholesterol moleculesinteract with phospholipid molecules within one monolayer of alipid bilayer (Fig. 1) (38) and, unlike phospholipids whichspontaneous ‘flip-flop’ from one leaflet of a bilayer to the otheris extremely low, cholesterol can move easily from one leaflet ofthe bilayer to the other (39). The intercalation of cholesterolleads to straightening of the phospholipid acyl tails and preventsthe molecules from diffusing. All these events result inrigidization (stiffening) of the membranes (40) which is moreintense, when cholesterol is present in an oxidized form (38).

High cholesterol concentrations most often lead to adecrease in membrane fluidity and thickness thereby hindering

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Fig. 1. The cholesterolmolecule inserts itself in themembrane with the sameorientation as the phospholipidmolecules. Note that the polarhead of the cholesterol isaligned with the polar head ofthe phospholipids. The arrowdepicts spontaneous ‘flip-flop’of the cholesterol moleculefrom one leaflet of a bilayer tothe other. Processed accordingto Wolfe (38).

the mobility of the intermembrane components (41, 42).Although the amount of cholesterol found in the inner membraneis low in comparison with that in the outer membrane, theincorporated amount of cholesterol is sufficient to influenceboth, the activity of mitochondrial ATP synthase (43) and thefluidity of inner mitochondrial membrane (34). Lateraldisplacement of membrane proteins due to cholesterolincorporation results in the formation of aggregates in themembrane and the physical separation of the proteinsresponsible for electron transport (44). Higher than normalmitochondrial cholesterol levels are also connected with theinhibition of the pro-apoptotic-protein-Bax insertion in themitochondrial outer membrane owing to an increase in themembrane cholesterol (45). However, when it is present in highconcentrations, cholesterol can substantially disrupt the abilityof the phospholipids to interact among themselves, whichincreases fluidity and lowers the gel-sol transition temperature(39). The above mentioned makes the dependence of fluidity onthe amount of membrane cholesterol more elaborate, thusquantitative measurements of membrane fluidity should alwaysbe interpreted with respect to the technique and the referencevalues in the experimental setup. In the light of the above, usinga spatially and time-averaged (i.e. steady-state) fluorescenceanisotropy (r) may be a good choice in situations wherescientists are interested in distinguishing between variousexperimental conditions. Since animal cell plasma membranescan contain substantial quantities of cholesterol, this issue maybe significant in some pathologies, such as familiarhypercholesterolemia (46), atherosclerosis and coronary heartdisease (47), hypertension, diabetes and others cardiovascularand metabolic diseases (48).

Hyperlipidemia has been found to attenuate thecardioprotective effect induced by IPC as a form ofcardioprotection (49, 50). This finding is also supported by otherstudies confirming the interference between cardioprotective

effect of ischemic preconditioning and HCH (16, 51, 52). HCHaccelerates the evolution of myocardial ischemia, delays recoveryon reperfusion, and deteriorates the anti-ischemic effect ofpreconditioning in humans (53). All these observations raiseconcern that HCH might have a negative influence on adaptation,presented in the experimental model of acute STZ-induced DM,which is the reflection of the cardioprotection (16, 18). Wefocused on verifying whether the HCH induced by a cholesterol-enriched diet would influence the degree of cardioprotectioninduced by acute STZ-induced DM. Specifically, we aimed oninvestigating the impact of HCH on mitochondrial membranefluidity, mitochondrial energetics and related pathophysiologicalchanges in myocardial injury and function.

MATERIALS AND METHODS

All animal experiments were performed in strict accordancewith the rules issued by the State Veterinary Administration ofthe Slovak Republic, legislation No 289/2003 and with theregulations of the Animal Research and Care Committee ofInstitute for Heart Research.

Experimental animal models

Male Wistar rats 10 – 12 weeks aged (229 ± 20 g b.w.) werekept under conditions of standard light regimen (D:L, 12:12) at22 ± 2ºC in cages (max. 5 animals per cage) with free access towater. We used control healthy rats and STZ-induced earlydiabetic rats, both with normal chow and fed with fat-cholesteroldiet. Control animals were fed with standard chow (C rats; n = 8per group) and with fat-cholesterol diet (HCH rats; n = 8 pergroup). HCH was induced by cholesterol-enriched dietconsisting of 1% cholesterol, 1% coconut oil, 20 g/day (HCHrats; n = 8 per group) for five days. Acute experimental DM was

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Fig. 2. Experimental design of myocardial ischemic-reperfusion injury induced in control group, cholesterol-enriched diet group (HCHgroup), streptozotocin induced acute diabetic group (DM group), and combination of cholesterol-enriched diet and streptozotocin inducedacute diabetic group (DM-HCH group).

induced by a single dose of streptozotocin (STZ) (80 mg.kg–1;intraperitoneally) dissolved in 0.1 mol.L–1 citrate buffer, pH 4.0(DM rats; n = 8 per group). Animals with simultaneouslyinduced diabetes mellitus and hypercholesterolemia for fivedays were used for the induction of the diabetic-hypercholesterolemic state (DM-HCH rats; n = 8 per group).The DM and DM-HCH rats had been receiving 5% glucosesolution per os during the first 24 h after STZ application toprevent transient hypoglycaemia (15, 51). A modified double-disease model of the DM-HCH rats were developed by Jiao et al.and Kusunoki et al. (54, 55). Progress of disease was beingmonitored during five days by measuring of glycosuria usingGluko PHAN strips (Erba-Lachema, Brno, Czech Republic).Experimental protocol is depicted in Fig. 2.

Anesthesia

Prior to heart excision the experimental animals wereanesthetized with thiopental (50 – 60 mg.kg–1; intraperitoneally)administered together with heparin (500 IU; intraperitoneally).

Determination of metabolic status of animals

The samples of blood for estimation were collected from theabdominal aorta of the animals from all groups. Metabolic statewas evaluated by determination of glucose and triacylglycerolcontents, total cholesterol content, low-density lipoproteincholesterol (LDL), high-density lipoprotein cholesterol (HDL),very low-density lipoprotein (VLDL), glycohemoglobin byusing test strips of MultiCare and LUX multiparameter system,(Biochemical system internation, Florence, Italy) in the blood(blood sample volume was 10 – 15 µL) as well as insulin in theserum (RIA kit, Linco Researech USA).

Langendorff perfusion

Perfusion protocol was performed as previously described(15, 51). The hearts of anesthetized animals were rapidly excisedand perfused at 37ºC in the Langendorff mode at a constantperfusion pressure of 73 mmHg. The perfusion solution was amodified Krebs-Henseleit buffer infused with 95% O2 and 5%CO2 (pH 7.4) containing (mmol.L–1): 118 NaCl, 3.2 KCl, 1.2MgSO4, 25 NaHCO3, 1.18 KH2PO4, 2.5 CaCl2 and 5.5 glucose.An epicardial electrogram was recorded by two electrodesattached to the apex of the heart and the aorta. Left ventricular(LV) pressure was measured by means of a nonelastic ballooninserted into the LV cavity (water-filled to obtain end-diastolicpressure of 5 – 7 mmHg and connected to a pressure transducer(MLP844 (ADInstruments, Germany)). LV systolic pressure(LVSP); LV diastolic pressure (LVDiP); LV developed pressure(LVDP [systolic minus diastolic pressure]), maximal rates ofpressure development and fall ± [dP/dt]max, respectively) as theindexes of contraction and relaxation, heart rate (HR) andcoronary flow (CF) were measured during a preischemicstabilization period and were continuously recorded until the endof 40 min of reperfusion using PowerLab/8SP Chart 7 software(ADInstruments, Germany).

Protocols of global ischemia

The hearts of all rats were assigned to the following protocol(n = 8 per group). After a 20 min stabilization period inLangendorff mode, the hearts were subjected to 30 min of globalischemia followed by 40 min of reperfusion by clamping andunclamping of aortic inflow for the evaluation of post-ischemiccontractile dysfunction (myocardial stunning). Recovery of LVDPat the end of 40 min reperfusion served as the end point of injury.

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C HCH DM DM-HCH Glucose [mmol.L–1] Triacylglycerol [g.L–1] Cholesterol [mmol.L–1] LDL cholesterol [mmol.L–1] HDL cholesterol [mmol.L–1] VLDL cholesterol [mmol.L–1] Glycohemoglobin [%Hb] Insulin [ng.mL–1]

5.33 ± 0.16

1.25 ± 0.10

1.76 ± 0.12

0.24 ± 0.10

0.72 ± 0.11

0.28 ± 0.05

4.05 ± 0.13

1.04 ± 0.15

5.99 ± 0.23

1.33 ± 0.13

1.79 ± 0.12

0.32 ± 0.05

0.80 ± 0.12

0.37 ± 0.09

4.21 ± 0.33

1.02 ± 0.15

17.80 ± 0.89**

4.63 ± 0.32**

2.40 ± 0.15*

0.59 ± 0.08*

0.81 ± 0.11

0.31 ± 0.10

7.68 ± 1.02**

0.49 ± 0.09**

20.89 ± 1.57**

4.90 ± 0.42**

3.03 ± 0.56**

1.38 ± 0.39**

0.86 ± 0.15

0.63 ± 0.13*

7.81 ± 1.11**

0.48 ± 0.12**

Table 1. Determination of metabolic status of animals. Effect of the hypercholesterolemia induced by cholesterol-enriched diet andexperimental model of streptozotocin-induced diabetes mellitus on the metabolic parameters.C, control group; HCH, hypercholesterolemic group (hypercholesterolemia induced by cholesterol-enriched diet); DM, group withstreptozotocin-induced diabetes mellitus; DM-HCH, diabetic-hypercholesterolemic group (simultaneously streptozotocin-induceddiabetes mellitus with hypercholesterolemia induced by cholesterol-enriched diet); LDL, low density lipoprotein; HDL, high densitylipoprotein; VLDL, very low density lipoprotein; ND, not determined. Values are presented as means ± SEM; n = 8 per each group;**P < 0.01; *P < 0.05 versus control.

Isolation of mitochondria

Each excised heart was cut into small pieces by scissors in thepresence of small volume of ice-cold isolated solution (containingin mmol.L–1): 180 KCl, 4 EDTA, 1% bovine serum albumin, pH7.4. Minced heart tissue was suffused with 20 mL of ice-coldisolated solution with addition of protease 2.5 mg.g–1 of tissue(Sigma P-6141) and subsequently homogenized in teflon/glasshomogenizer on the ice during 2 – 3 min. Prepared homogenatewas processed by differential centrifugation. After 10 mincentrifugation at 1000 g, the supernatant containing the part ofmitochondria which were in direct contact with protease wasobtained and poured out afterwards. The pellet containingmitochondria was homogenized again in 20 mL of ice-coldisolated solution without protease and centrifuged at 1000 g for 10min. Resulting supernatant was centrifuged at 6200 g for 10 minand pellet containing mitochondria was then again re-suspendedin an albumin-free isolation solution (containing in mmol.L–1: 180KCl, 4 EDTA). It was centrifuged down at 6200 g for 10 min andsubsequently used for estimation of protein concentration as wellas for further biophysical and biochemical investigations.

Determination of Mg2+-ATPase activity

The Mg2+-ATPase activity represents the activity ofmitochondrial ATP synthase working in the presence of Mg2+

cations in a reverse direction, i.e. splitting ATP molecule to ADPand inorganic phosphate Pi. The activity was determined in 1 mLof incubation medium containing: 200 µL imidazole buffer (250mmol.L–1), 100 µL MgCl2 (40 mmol.L–1), 100 µL ATP-Tris (40mmol.L–1) and 50 µg of mitochondrial fraction withconcentration (1 µg.µL–1) in the presence and absence of 100 µLDNP (0.1 mmol.L–1). The addition of DNP results in thedisruption of all mitochondrial membranes, the influx of Mg2+

cations into the mitochondria, which allows measuring the totalactivity of mitochondrial Mg2+-ATPase. The reaction started bythe addition of ATP after 10 min pre-incubation with 50 µg ofmitochondrial fraction at 37ºC. After 20 min the reaction wasterminated by 1 mL of ice-cold 12% trichloroacetic acid (TCA).The activity of mitochondrial Mg2+-ATPase was determinedspectrophotometrically at 700 nm as the amount of inorganic

phosphate (Pi) liberated by ATP splitting per unit of the time(µmol.Pi.g–1.h–1).

Determination of mitochondrial membrane fluidity

Mitochondrial membrane fluidity was determined in terms offluorescence anisotropy of the lipophilic fluorescence probe 1,6-diphenyl-1,3,5-hexatriene (DPH). Fluorescence anisotropymeasurements are widely used as sensitive indicators of cellmembrane fluidity. The DPH probe incorporates into themembrane in parallel to the acyl chains of lipid bilayer and‘senses’ movement of lipids in the membrane. Increased rate ofits movement is associated with a decrease in the fluorescenceanisotropy. Isolated mitochondrial fraction was resuspended to a0.5 mg.mL–1 of protein in the solution containing: 180 mmol.L–1

NaCl, 4 mmol.L–1 EDTA (pH 7.4) and were marked by 0.25 µLof DPH solution resuspended in a mixture of acetone and water(1:250). Samples were incubated at 22 ± 1ºC. The fluorescencewas determined at an excitation wavelength of 360 nm and at anemission wavelength of 425 nm according to Shinitzky (56).

Determination of conjugated dienes content

The content of conjugated dienes in the lipids frommitochondrial membranes was assessed by the method (57)adapted to the estimation of conjugated dienes in the membranesof heart mitochondria. Isolated heart mitochondria weresuspended at a concentration of 1 mg.mL–1 in a solutioncontaining 180 mmol.L–1 KCl and 4 mmol.L–1 Na2EDTA adjustedto pH 7.4 by Tris-HCl. Lipids were extracted from 500 µl of themembrane suspension with 1000 µl of chloroform:methanol (1:2v/v) under vortexing for 30 s. Subsequently, 500 µl of chloroformwas added and vortexed again for 30 s. Extraction was terminatedby addition of 500 µl of 15 mmol.L–1 Na2EDTA containing 4%NaCl and spinning down for 10 min at 1900 g. Then 600 µl of thelipid-containing lower layer of chloroform: methanol wastransferred to a separate test tube and carefully evaporated atroom temperature under a continuous stream of nitrogen toprevent oxidation. Beginning from this point, all the procedurewas performed in a nitrogen atmosphere. The dry lipids were thendissolved in 3 mL of cyclohexane, vortexed for 30 s, and used

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Fig. 3. Effect of the hypercholesterolemiainduced by cholesterol-enriched diet andexperimental model of streptozotocin-induced diabetes mellitus on the size ofmyocardial infarction. C, control group;DM, group with streptozotocin-induceddiabetes mellitus; HCH, group withhypercholesterolemia induced bycholesterol-enriched diet; DM-HCH,diabetic-hypercholesterolaemic group(simultaneously streptozotocin-induceddiabetes mellitus with hypercholesterolemiainduced by cholesterol-enriched diet);IS/AR, infarct size/area at risk. Data aremeans ± SEM; n = 8 per each group; *P <0.05 versus DM, #P < 0.05 versus control.

directly for spectrophotometric determination of the conjugateddienes at λ = 233 nm, ε = 29,000 L.mol–1.cm–1.

Determination of myocardial infarction

Separate set of animals was used to determine the size ofmyocardial infarction. Langendorff perfused hearts of allexperimental groups were exhibited to 30 min global ischemiafollowed by 40 min (with baloon) plus 80 min (without baloon).After this protocol the size of the infarcted area and the area atrisk (AR) size were delineated by staining with 2,3,5-triphenyltetrazolium chloride (TTC) and determined by acomputerized planimetric method as previously described (58).Since the AR represents the entire area of the LV in the globalischemia protocol, IS was expressed as a percentage of LV size.

Statistical analysis

Descriptive and univariate analyses were performed on allselected animals' characteristics. Mean ± SEM (standard error ofmean) is given for the normally distributed variables or a medianand interquartile range if data showed substantial deviationsfrom normality. Categorical variables are presented as relativecounts and percentages.

We used a one-way analysis of variance (ANOVA) to analyzebetween-group differences in metabolic parameters, and a two-wayANOVA to analyze data from factorial experiments: treatmenteffect (cholesterol-enriched diet as the first main factor) in ratswhich were grouped by presence of diabetes condition (DM as thesecond main factor). Two-way ANOVA with repeated measureswas used in case of time-dependent recordings with a pre-plannedpost-hoc comparison at the end of reperfusion (40. min).

Level for statistical significance for testing between-groupdifferences was set at α = 0.05 and that for testing interaction atless conservative α’ = 0.15. All statistical tests were two-sided.

Statistical analyses were performed using StatsDirect3.0.191 software (Stats Direct Ltd., Cheshire, UK) andGraphPad Prism 7.0 (GraphPad Software, Inc., USA).

RESULTS

Development of diabetes and hypercholesterolemia

Effect of the HCH induced by cholesterol-enriched diet andexperimental model of streptozotocin-induced DM on themetabolic parameters are shown in Table 1. Rats in the DMgroups exhibited significant elevated levels of glucose (P <0.01), triacylglycerols (P < 0.01), total cholesterol (P < 0.05),LDL cholesterol (P < 0.01), glycohemoglobin (P < 0.01),decreased level of insulin (P < 0.01), non-significant increasedlevels of HDL cholesterol and VLDL cholesterol on the 5th dayafter the application, thus confirming the development ofdiabetes. The increase in the mentioned parameters was morepronounced in the DM-HCH group on comparison with controlgroup HCH group did not show any significant effect ofmetabolic parameters on comparison with control rats (Table 1).

Characteristic of functional parameters and lethal injury inisolated rat hearts

Preischemic stabilization parameters (LVSP, LVDiP, LVDP,±[dP/dt]max, HR and CF) exhibited no significant changes amongthe groups and these data are summarized in Table 2. Hearts fromDM rats on the standard diet showed a significantly reduced sizeof myocardial infarction in comparison with control hearts (Fig.3). Accordingly, recovery of contractile function (LVDP) afterI/R was improved in the diabetic animals. Similarly hearts fromDM rats on the standard diet exhibited a significantly betterrecovery of LVDiP, +dP/dtmax as well as –dP/dtmax compare to

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Parameters C DM HCH DM-HCH HR [beats/min] CF [ml/min] LVSP [mmHg] LVDiP [mmHg] LVDP [mmHg] +dP/dtmax [mmHg/s] –dP/dtmax [mmHg/s]

255 ± 6

10.8 ± 1.0

82.3 ± 5.2

6.0 ± 1.0

76.8 ± 2.3

2295 ± 123

1425 ± 118

249 ± 8

11.3 ± 1.2

88.4 ± 7

6.7 ± 1.5

82.6 ± 5.9

2214 ± 187

1368 ± 123

259 ± 9

12.5 ± 0.9

85.0 ± 5.6

5.9 ± 0.8

78.2 ± 8.7

2168 ± 203

1325 ± 99

248 ± 9

12.2 ± 09

87.0 ± 5.3

6.1 ± 0.9

80.9 ± 8.2

2183 ± 152

1359 ± 160

Table 2. Preischemic values of parameters of myocardial function in Langendorff-perfused rat hearts.C, control group; DM, group with streptozotocin-induced diabetes mellitus; HCH, hypercholesterolemic group (hypercholesterolemiainduced by cholesterol-enriched diet); DM-HCH, diabetic hypercholesterolemic group (simultaneously streptozotocin-induced diabetesmellitus with hypercholesterolemia induced by cholesterol-enriched diet); HR, heart rate; CF, coronary flow; LVSP, left ventricularsystolic pressure; LVDiP, left ventricular diastolic pressure; LVDP, left ventricular developed pressure; +(dP/dt) max and – dP/dt)max,maximum rates of pressure development and fall; respectively; n = 8 per each group.

control hearts (Table 3). A one-week HCH diet did not affectresponse to I/R in the control hearts. However, it reduced theability of diabetic hearts to resist I/R, since diabetic rats on thecholesterol-enriched diet exhibited significantly increased size ofinfarction (Fig. 3) and depressed functional recovery (LVDP,LVDiP, +dP/dtmax) when compared with the DM group (Fig. 4,Table 3). After 40 min reperfusion, HR tended to decreasesimilarly in all groups by 12% to 20%, and CF ranged between73% and 83% of baseline values. There were no significantchanges in values of HR and CF among the groups after 40 minof reperfusion (data not shown).

Alteration in mitochondrial membrane properties in diabetesmellitus condition and high cholesterol diet

In order to find out whether cholesterol-enriched diet afterI/R injury might have modulated cardioprotection due tomembrane remodeling induced by diabetic condition andevaluated in terms of the markers of mitochondrial membranestructure, function and damage such as fluidity, DNP-stimulated

Mg2+-ATPase activity, and conjugated dienes as a marker ofoxidative damage, we have chosen a factorial experiment, andhave investigated whether the strength of relationship differedbetween DM animals and the controls. To do so, we also testedthe interaction between both factors - DM condition and thecholesterol-enriched diet in order to determine what proportionof variation in the above parameters of mitochondrial structureand function could be ascribed to DM when adjusting foradverse effect of cholesterol-enriched diet. Two-way ANOVAshowed that both main factors, DM and HCH, had statisticallysignificant effect on mitochondrial membrane fluidity (Fig. 5),which means that diabetic condition increased fluidity (ignoringcholesterol-enriched diet) and cholesterol-enriched dietdecreased fluidity (again, irrespective of DM status). DNP-stimulated Mg2+-ATPase activity was significantly increased inDM; the factor cholesterol-enriched diet non-significantlydecreased the activity across DM status levels (Fig. 6).Cholesterol-enriched diet also significantly increased levels ofconjugated dienes with non-significant contribution of thediabetic condition (Fig. 7).

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Fig. 4. Effect of the hypercholesterolemiainduced by cholesterol-enriched diet andexperimental model of streptozotocin-induceddiabetes mellitus on the recovery ofcontractile function after 40 min ofreperfusion. C, control group; DM, group withstreptozotocin-induced diabetes mellitus;HCH, group with hypercholesterolemiainduced by cholesterol-enriched diet; DM-HCH, diabetic-hypercholesterolaemic group(simultaneously streptozotocin-induceddiabetes mellitus with hypercholesterolemiainduced by cholesterol-enriched diet); LVDP,left ventricular developed pressure. Data aremeans ± SEM; n = 8 per each group; *P < 0.05DM-HCH versus DM, #P < 0.05 versuscontrol.

D

Parameters C DM HCH DM-HCH LVSP [mmHg] LVDiP [mmHg] +dP/dtmax [mmHg/s] –dP/dtmax [mmHg/s]

52.7 ± 4.8

30.6 ± 5.9

860 ± 186

492 ± 118

65.8 ± 5.3

17.9 ± 3.6#

1392 ± 228#

784 ± 305#

53.0 ± 6.0

26.2 ± 6.5

911 ± 165

587 ± 109

59.8 ± 6.2

29.2 ± 6.8*

874 ± 147*

558 ± 207

Table 3. Parameters of myocardial function in Langendorff-perfused rat hearts after I/R injury.C, control group; DM, group with streptozotocin-induced diabetes mellitus; HCH, hypercholesterolemic group (hypercholesterolemiainduced by cholesterol-enriched diet); DM-HCH, diabetic hypercholesterolemic group (simultaneously streptozotocin-induced diabetesmellitus with hypercholesterolemia induced by cholesterol-enriched diet); HR, heart rate, CF, coronary flow, LVSP, left ventricularsystolic pressure; LVDiP, left ventricular diastolic pressure; LVDP, left ventricular developed pressure; +dP/dtmax and –dP/dtmax, maximumrates of pressure development and fall, respectively. Data are means ± SEM; n = 8 per each group; *P < 0.05 versus DM group, #P < 0.05versus control.

However, we observed significant interaction effects in allthe variables (all P < 0.15). The presence of these interactionscan be seen in Fig. 8, when comparing lines connecting therespective means. Apparently, the lines are non-parallel in all theabove mentioned characteristics, which means that the factorswere not additive in the resulting effect, but the effect size of one

factor depended on the level of the second factor. Thus, the mainfactors, DM and HCH, should not be considered in isolation.

Significant differences in the simple mean effects of DM andHCH are marked in Figs. 5-7. When compared with C groupmitochondrial membrane fluidity in DM group was increased asreflected by significantly reduced rigidity index (Fig. 5). This

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Fig. 5. Effect of the hypercholesterolemiainduced by cholesterol-enriched diet andexperimental model of streptozotocin-induceddiabetes mellitus on the mitochondrialfluorescence anisotropy of DPH in the ratheart. C, control group; DM, group withstreptozotocin-induced diabetes mellitus;HCH, group with hypercholesterolemiainduced by cholesterol-enriched diet; DM-HCH, diabetic-hypercholesterolaemic group(simultaneously streptozotocin-induceddiabetes mellitus with hypercholesterolemiainduced by cholesterol-enriched diet). Dataare means ± SEM; n = 8 per each group; *P <0.05 versus DM, #P < 0.05 versus control.

Fig. 6. Changes in mitochondrial DNP-stimulated Mg2+-ATPase activity induced byhypercholesterolemia induced by cholesterol-enriched diet and experimental model ofstreptozotocin-induced diabetes mellitus. C,control group; DM, group withstreptozotocin-induced diabetes mellitus;HCH, group with hypercholesterolemiainduced by cholesterol-enriched diet; DM-HCH, diabetic-hypercholesterolaemic group(simultaneously streptozotocin-induceddiabetes mellitus with hypercholesterolemiainduced by cholesterol-enriched diet). Dataare means ± SEM; n = 8 per each group; *P <0.05 versus control.

Fig. 7. Effect of the hypercholesterolemiainduced by cholesterol-enriched diet andexperimental model of streptozotocin-induceddiabetes mellitus on the concentration ofconjugated dienes in the heart mitochondria.C, control group; DM, group withstreptozotocin-induced diabetes mellitus;HCH, group with hypercholesterolemiainduced by cholesterol-enriched diet; DM-HCH, diabetic-hypercholesterolaemic group(simultaneously streptozotocin-induceddiabetes mellitus with hypercholesterolemiainduced by cholesterol-enriched diet). Dataare means ± SEM; n = 8 per each group.

finding was paralleled by a significant increase in mitochondrialDNP-stimulated Mg2+-ATPase activity (Fig. 6) in the DM group incomparison with the control group. The positive effect of increasedmitochondrial membrane fluidity and DNP-stimulated Mg2+-ATPase activity was not found in the group on the cholesterol-enriched diet in which the diet significantly reduced fluidity (Figs.5 and 6). In both DM groups on standard and cholesterol-enricheddiet the membrane fluidity was comparable to that observed in

control group (Fig. 5). The content of conjugated dienes was notsignificantly different among experimental groups (Fig. 7).

DISCUSSION

The enhanced tolerance to I/R injury observed in the STZ-DMhearts can be considered as a form of intrinsic cardioprotection

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Fig. 8. Graphs on the interaction effectbetween the diet as the first main factor anddiabetes as the second main factor. Bothfactors had two levels - treatment withcholesterol-enriched diet (HCH present) ortreatment with standard diet (HCH absent),and presence (coded DM present) or absence(coded DM absent) of diabetes. Combinationsof these 2 × 2 levels define all four treatmentgroups:HCH absent and DM absent - stands forcontrol group C;HCH absent and DM present - stands for DMgroup with streptozotocin-induced diabetesmellitus;HCH present and DM absent - stands for HCHgroup with hypercholesterolemia induced bycholesterol-enriched diet;HCH present and DM present - stands for DM-HCH diabetic-hypercholesterolaemic group.Estimated marginal means for index of rigidity(8A), mitochondrial Mg2+-ATPase activity(8B), and conjugated dienes (8C) are presentedtogether with SEM error bars.

A

B

C

analogous to that induced by a short-term adaptive phenomenon ofischemic preconditioning in the normal heart, where numerousmetabolic stimuli, in particular those related to both, oxidativedamage and increased intracellular calcium signaling, can triggerprotection against I/R (59). Early period after onset of DM isassociated with activation of adaptive mechanisms whichsuccessfully counteract metabolic disorders leading to irreversiblecell damage and arrhythmias. Similar efficacy against reperfusion-induced arrhytmias as presented in early phase of DM can byobserved with the use of Na+/H+ exchanger inhibitors as well asNa+/Ca2+ exchanger inhibitors (60). Several mechanisms have beenproposed to explain a lower sensitivity to I/R in the diabetic heart.The alterations in the intracellular pH, a decreased clearance ofprotons via Na+/H+ exchanger, and decreased rate of glycolysis inthe diabetic myocardium, may represent pivotal factors attenuatedresponse to I/R injury (23). Accordingly, our results showed adecreased susceptibility of DM hearts to I/R injury represented bysignificantly improved recovery of contractile function after I/R aswell as decreased size of myocardial injury. Duration of thediabetic state plays a crucial role in the myocardial response toischemia and in IPC-like effects, thus initially increased resistanceto I/R injury declines in the chronic phase of diabetes (61).Improved postischemic contractile recovery in the acute (1 or 2week) phase of STZ-induced DM as well as infarct size reduction,which was also observed in the present study, both are abrogated inthe DM rats in the chronic phase of the disease (7, 27).

However, one should bear in mind that DM-mediatedcardioprotective mechanisms representing metabolicpreconditioning may deviate from its dynamic equilibriumtowards the damage, e.g. under the conditions of the chronicdiabetic condition, or by the presence of additional adversestimulus that interacts with, or more specifically suppresses thecompensatory mechanisms (5, 12).

Particularly, changes in fluidity of mitochondrial membranesare considered to be an adaptive response with a positive impacton physiological/functional performance of the membranes. Inthis regard, it can be attributed to endogenous protectivemechanisms. Our finding of increased mitochondrial membranefluidity in the diabetic group accompanied with enhanced ATPaseactivity (62) is in agreement with previously observedcardioprotective effects induced by ischemic preconditioning (63).

Further results have proved that the condition of acute DMleads also to increased levels of lipids, lipoproteins andglycoproteins (64), to remodeling of mitochondrial membraneswith a direct impact on the membrane proteins (5). It has beenshown that activity of mitochondrial ATP synthase is essentiallydependent on the movement of the protein molecule in themembrane lipid environment. Adaptive changes in thephysicochemical properties underlying membrane fluidity allowthe membrane proteins, including ATP synthase, to keepworking under suboptimal conditions (65).

The physiological state of an organism is the main factoraffecting the composition of the mitochondrial membrane. In thephysiological situation heart mitochondria have lower cholesterolconcentration (41, 66, 67), while in the plasma membrane is theconcentration higher (68). Conversely, in some pathologicalstates, such as hyperlipidemia or also after myocardial ischemia,cholesterol accumulates in the mitochondria in the highest content(69). Additionally, high-fat and high-cholesterol diet are alsoassociated with increased lipopolysaccharide plasma levels (70). Arecent study has shown that the attenuation of cardioprotectiveeffect of IPC in hyperlipidemic condition may be involved in aninhibition of protective signaling pathways upstream of glycogensynthase kinase-3β (GSK-3β) and in the opening of mitochondrialpermeability transition pores (mPTP) (71). Elevated formation ofmPTP is one of possible explanations of mitochondrial membranefluidization. Enhanced formation and mPTP opening during acute

STZ-DM occur in mitochondrial membrane fluidization andfacilitation of ATP transport without lethal damage (72).Hypercholesterolemia-exacerbated myocardial I/R injury mayrelate to enhanced oxidation due to disturbed regulatory systemsand antioxidant network required to minimize ROS production,further, to attenuation of PI3K/Akt pathways, and induction ofmitochondrial permeability transition pore (mPTP) opening (73,74). In addition, other experimental and clinical studies haverevealed that hypercholesterolemia abrogates the protective effectof preconditioning and postconditioning (75-77).

In addition to the effects found in preconditioning HCH isconsidered to impair the cardioprotective effect of ischemicpostconditioning by altering of nitrosative stress signal and byincreasing the production of several oxidants such asperoxynitrite and lipid peroxidation compounds (75).

Delineating the impact of various factors leading to structuraland functional remodeling of heart helps identify signaling andregulatory pathways involved in adaptive and maladaptiveprocesses. All this might have significant implications for seekingways how to delay or prevent the transition to heart failure.

Although it is widely accepted fact that DM adverselyaffects heart structure and function independently ofhypercholesterolemia, caution should be used, when interpretingfindings from studies considering these factors in isolation. Thereason is that ignoring combination of these conditions (DM andHCH) could confound results by exacerbating or weakening theobtained relationship between the intended factor and theselected outcome variable. In this study significantly increasedlevel of cholesterol registered in the DM-HCH groupantagonized the endogenous protective mechanisms byincreasing the rigidity of mitochondria membranes.Consequently, the mitochondrial Mg2+-ATPase activity in DM-HCH group became depressed.

On the organ level evaluated functional heart recovery(LVDP) after I/R was also significantly decreased in DM-HCHwhen compared to DM group, which may be partially linked tothe functional changes observed on level of heart mitochondria.Depressed LVDP recovery together with increased size ofmyocardial infarction observed in DM-HCH group incomparison to DM group suggest that protection conferred byacute DM was blunted or even abolished if HCH diet wasapplied. Taken together, HCH seems to interfere withcardioprotective mechanisms, which was reflected in attenuationof adaptive responses that would otherwise be developed indiabetes. As a result, vulnerability of the heart to I/R wasincreased and mitochondrial function compromised when ratswere exposed to both factors, DM and HCH. Since HCH dietitself caused no alteration either in mitochondrial function or inboth, myocardial function and size of lethal injury after I/R, thepresence of a specific interaction between hypercholesterolemiaand acute diabetic condition should be taken into consideration.

Conclusion

Although the short-term administration of cholesterol-enriched diet did not cause any significant differences in themonitored parameters, in diabetic hearts it was associated withsignificant alterations in the functional and structural propertiesof mitochondria as well as in myocardial structure and function.Altered composition and structure of the mitochondrialmembranes both have been proved to contribute to increasedresistance of diabetic heart to experimentally applied ischemicinsult, which has been attributed to compensatory adaptation. Inour present study, we have shown that the applied cholesterol-enriched diet induces adverse remodeling which negatively affectmitochondrial function in diabetic animals. This remodeling maybe related to distortion of the mitochondrial membrane protein-

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lipid interactions due to incorporation of cholesterol molecules.Altogether, our results show that the application of cholesterol-enriched diet led to inhibition of compensatory, endogenouslyinitiated cardioprotective mechanisms, observed in diabeticgroup of animals.

M. Ferko and I. Waczulikova contributed equally to this work.

Acknowledgements: The work was supported by ScientificGrant Agency of the Ministry of Education, Science Researchand Sport of the Slovak Republic (VEGA) 2/0133/15, 2/0151/17,2/0121/18, 2/0141/18 and Slovak Research and DevelopmentAgency (APVV) 15-0119, 15-0607; ITMS 26230120009, EU-CARDIOPROTECTION COST CA-16225.

Conflict of interests: None declared.

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R e c e i v e d : September 17, 2018A c c e p t e d : October 30, 2018

Author’s address: Dr. Miroslav Ferko, Center of ExperimentalMedicine, Slovak Academy of Sciences, Institute for HeartResearch, Dubravska cesta 9, 841 04, Bratislava, Slovak Republic.E-mail: usrdmife@savba.sk

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