Mitochondria play an essential role in improved cardiac ischaemic tolerance conferred by adaptation to chronic hypoxia. In the present study, we analysed the effects of continuous normobaric hypoxia (CNH) on mitochondrial functions, including the sensitivity of the mitochondrial permeability transition pore (MPTP) to opening, and infarct size (IS) in hearts of spontaneously hypertensive rats (SHR) and the conplastic SHR-mtBN strain, characterized by the selective replacement of the mitochondrial genome of SHR with that of the more ischaemia-resistant brown Norway (BN) strain. Rats were adapted to CNH (10% O2, 3 weeks) or kept at room air as normoxic controls. In the left ventricular mitochondria, respiration and cytochrome c oxidase (COX) activity were measured using an Oxygraph-2k and the sensitivity of MPTP opening was assessed spectrophotometrically as Ca2+-induced swelling. Myocardial infarction was analysed in anaesthetized open-chest rats subjected to 20 min of coronary artery occlusion and 3 h of reperfusion. The IS reached 68±3.0% and 65±5% of the area at risk in normoxic SHR and SHR-mtBN strains, respectively. CNH significantly decreased myocardial infarction to 46±3% in SHR. In hypoxic SHR-mtBN strain, IS reached 33±2% and was significantly smaller compared with hypoxic SHR. Mitochondria isolated from hypoxic hearts of both strains had increased detergent-stimulated COX activity and were less sensitive to MPTP opening. The maximum swelling rate was significantly lower in hypoxic SHR-mtBN strain compared with hypoxic SHR, and positively correlated with myocardial infarction in all experimental groups. In conclusion, the mitochondrial genome of SHR modulates the IS-limiting effect of adaptation to CNH by affecting mitochondrial energetics and MPTP sensitivity to opening.

Clinical perspectives

  • Ischaemic heart disease and its acute form, myocardial infarction, is the leading cause of morbidity and mortality in the western world. Therefore, the development of new cardioprotective strategies that decrease I/R injury has important clinical implications. Cardiac mitochondria are the main targets and sources of damage during I/R injury. On the other hand, mitochondria play an essential role in increased cardiac ischaemic tolerance conferred by various protective stimuli, including chronic hypoxia.

  • The present study showed that chronic hypoxia decreased myocardial IS in SHR, the animal model for human essential hypertension of neurogenic origin. The IS-limiting effect was accompanied by decreased sensitivity of cardiac MPTP to opening and increased reserve COX capacity. This finding suggests that chronic hypoxia can improve cardiac ischaemic tolerance in high-risk hypertensive rats.

  • Moreover, in the chronically hypoxic SHR-mtBN conplastic strain harbouring a mitochondrial genome of the more ischaemia-resistant BN strain, a stronger IS-limiting effect, lower sensitivity of MPTP to opening and improved mitochondrial respiration were observed compared with SHR. These results support the hypothesis that mtDNA can modulate chronic hypoxia-induced cardioprotective response to I/R. Understanding the role of the mitochondrial genome in ischaemic heart disease can help to design new cardioprotective strategies and their translation into clinical practice.

Introduction

Ischaemic heart disease is the leading cause of morbidity and mortality in the western world. The development of new procedures decreasing ischaemia–reperfusion (I/R) injury therefore has important clinical implications. Chronic hypoxia is one of the cardioprotective strategies discovered almost 30 years before the era of preconditioning [1], but its mechanism has not so far been satisfactorily explained. It has been shown that adaptation to chronic hypoxia is not just another form of hypoxic preconditioning because its infarct size (IS)-limiting effect needs several days to develop [2,3]. Moreover, cardiac ischaemic tolerance afforded by chronic hypoxia persists for weeks after cessation of the stimulus [35], making this phenomenon superior to transient effects of any form of conditioning. Recently, it has been shown that chronic hypoxia has a therapeutic potential in preventing post-ischaemic myocardial remodelling and progression of heart failure in experimental settings [68]. The current population study of Ezzati et al. [9] confirmed that adaptation to chronic hypoxia has a beneficial association with ischaemic heart disease in humans as well. A large body of research suggests that both short-lived conditioning and long-lasting effects of chronic hypoxia can utilize an essentially similar endogenous pool of protective pathways, although with different efficiencies. However, chronic hypoxia not only activates these signalling pathways but also alters the expression of their components and other proteins maintaining energy and oxygen homoeostasis, including those associated with mitochondria [10].

Over the last two decades, the interest of experimental cardiologists in the role of mitochondria in the heart has markedly increased. Besides their traditional role as ‘powerhouses’ of the cell in generating ATP, mitochondria play an important role in other aspects of normal cell functioning. In particular, the emerging role of mitochondria as regulators of cell life and death has received the greatest attention [11]. Mitochondria are one of the main sources and targets of damage during myocardial I/R injury. The heart is a strictly aerobic organ that is extremely vulnerable to a deficit in oxygen supply, which causes profound and immediate mitochondrial derangements. On the other hand, the activation of mitochondrial self-defence mechanisms by various protocols of conditioning has been shown as a powerful mechanism of cardioprotection [12]. Therefore, well-timed activation of pro-survival mitochondrial signalling, as well as adequate mitochondrial gene and protein expression, is crucial for the limitation of heart injury caused by I/R.

Mitochondrial proteins are encoded by two separate genomes (nuclear and mitochondrial). In mammals, up to 1000 nuclear-encoded mitochondrial genes lie on chromosomes, whereas mtDNA contains only 37 genes encoding 13 mRNAs for essential peptides of respiratory chain complexes, 2 rRNAs and 22 tRNAs [13]. Although mtDNA-encoded proteins represent a minor part, they play an important role in the function of organs including the heart. It has been shown that the mutations of mtDNA are associated with the variety of pathological states such as cardiomyopathies [14]. Recently, Muravyeva et al. [15] reported that the replacement of mtDNA modulated cardiac ischaemic tolerance in diabetic rats. We showed that the inherited alterations in mitochondrial genome predispose spontaneously hypertensive rats (SHR) to systemic metabolic disturbances relevant to the pathogenesis of common diseases, including cardiovascular ones [1618].

In the present study, we used SHR, a pathophysiological animal model for human essential hypertension of neurogenic origin [19], and a unique conplastic SHR-mtBN strain characterized by the selective replacement of the mitochondrial genome of SHR with that of the more ischaemia-resistant brown Norway (BN) strain [20]. With respect to the role of the mitochondrial genome, the effects of adaptation to chronic continuous hypoxia on cardiac ischaemic tolerance, and the expression of mitochondrial complexes and mitochondrial functions, were assessed. The extent of myocardial injury caused by acute I/R (and initiation of the necrotic and apoptotic processes) depends on the sensitivity of the mitochondrial permeability transition pore (MPTP) to opening [21]. Therefore, MPTP sensitivity to Ca2+-induced opening, as well as activity and expression of selective MPTP-regulating proteins, was also analysed.

Materials and methods

Animals

The SHR-mtBN conplastic strain harbouring the mitochondrial genome of a highly inbred BN strain on the nuclear genetic background of an SHR was created by selective replacement of an SHR mitochondrial genome with the mitochondrial genome of a BN rat, as described earlier [16]. Adult male SHR and SHR-mtBN strains (aged 12 weeks) were exposed to continuous normobaric hypoxia (CNH; inspired O2 fraction of 0.1) in a normobaric chamber equipped with hypoxic generators (Everest Summit, Hypoxico Inc.) for 3 weeks. No reoxygenation occurred during this period. The control rats were kept for the same period of time at room air. All animals were housed in a controlled environment (22±2°C; 12-h:12-h light–dark cycle; light from 5:00 hours) with free access to water and standard chow diet. At the end of the 3-week period, the haematocrit was measured in the tail blood. The present study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals (published by the National Academy of Sciences, National Academy Press, Washington, DC). Experimental protocols were approved by the Animal Care and Use Committee of the Institute of Physiology, the Czech Academy of Sciences. All chemicals were of the highest commercially available purity and were purchased from Sigma Aldrich.

Blood pressure measurements

The animals assigned to the invasive measurement of blood pressure in the pulmonary circulation (using a special cannula introduced into the right ventricle via the jugular vein) and the systemic circulation (the carotid artery) were anaesthetized with sodium pentobarbital (60 mg/kg i.p.). After the haemodynamic investigation, the animals were killed by cervical dislocation; the heart was dissected on ice into the right ventricle (RV), left ventricle (LV) and septum (S), each part was separately weighed, and LV samples were immediately frozen in liquid nitrogen and stored at −80°C until use for analysis of mitochondrial gene and protein expression and enzyme activities.

Real-time RT-PCR

RNA isolation and real-time RT-PCR were performed as published previously [22]. Briefly, total cellular RNA was extracted from each LV sample using the RNAzol and 1 μg of total RNA was converted to cDNA using the RevertAid H Minus First Strand cDNA Synthesis Kit (Biogen) and oligo(dT) primers according to the manufacturer's instructions. RT-PCR was performed on a Light Cycler 480 (Roche Applied Sciences) using Syber Green Master Mix. Gene-specific primer pairs for cytochrome c oxidase (COX) subunits were designed using the Universal Probe Library Assay Design Center (UPL, Roche Applied Science; see https://www.roche-applied-science.com/ sis/ rtpcr/upl/index.jsp) and the sequences of forward and reverse primers were as follows: 5′-CACTGCGCTTGTGCTGAT-3′ and 5′-CGATCAAAGGTATGAGGGATG-3′ for Cox4.1, and 5′-TGAGCCTTACTGCACAGAGC-3′ and 5′-AACTGGAGCCGGTACAAGG-3′ for Cox4.2, respectively. The levels of analysed transcripts were quantified after normalization to the level of the 18S rRNA reference gene transcript as shown previously [23].

Tissue homogenization and determination of enzyme activities

LVs were cut into small pieces in buffer containing 250 mM sucrose, 20 mM Tris/HCl and 1 mM EDTA, pH 7.4, followed by homogenization with a Potter–Elvehjem homogenizer (glass–Teflon, 10 strokes at 780 rev./min). The homogenates were filtered through gauze and homogenized once more using a Dounce homogenizer (three strokes). The final concentration of the homogenate was 10% (v/v). The protein concentration was determined by the Bradford method [24]. The samples were constantly kept on ice. For immunodetection, the protease inhibitor cocktail was added according to the manufacturer's instructions (Roche).

Spectrophotometric measurements were performed at 30°C using a Shimadzu UV-1601PC spectrophotometer by recording changes in the absorbance during the oxidation or reduction of the substrate, or by measurement of substrate oxidation/reduction in a coupled reaction. Homogenates were thawed and frozen twice to permeabilize mitochondrial membranes before conducting the measurements.

Citrate synthase (CS)-specific activity (a marker of mitochondrial quantity) was measured at 412 nm using 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB), which reacts with the free sulfhydryl group of coenzyme A and produces mercaptide ions with an absorption maximum at 412 nm [25]. The reaction was performed in 1 ml of buffer containing 100 mM Tris, 0.1 mM DTNB and 0.12 mM acetyl-coenzyme A. The reaction was started by adding oxaloacetate (freshly prepared, final concentration 0.5 mM). The specific activity was calculated in nanomoles/minute per milligram of protein using an absorption coefficient of 13.6 mM/cm.

The activity of NADH cytochrome c reductase (complexes I and III) was measured in a medium containing 60 mM potassium phosphate, 0.3 mM NADH and 2 mM KCN (to prevent reoxidation of cytochrome c by COX) [26]. The reaction was started by adding oxidized cytochrome c (final concentration 80 μM) and its reduction was measured at 550 nm. The specific activity was calculated as a rotenone-sensitive activity (2 μM rotenone) in nanomoles/minute per milligram of protein using an absorption coefficient of 19.1 mM/cm.

Activity of succinate–cytochrome c reductase (complexes II and III) was measured in a medium containing 60 mM potassium phosphate, 10 mM succinate, 0.002 mM rotenone, 2 mM KCN and 0.1% BSA [27]. The reaction was started by addition of oxidized cytochrome c (final concentration 80 μM) and its reduction was recorded at 550 nm; the specific activity was calculated in nanomoles/minute per milligram of protein using an absorption coefficient of 19.1 mM/cm.

COX activity (complex IV) in the homogenate was measured in 1 ml of 60 mM potassium phosphate (anionic concentration with the highest COX activity) containing 1–4 μg of mitochondrial protein at pH 7.4 [28]. The specific activity of COX in picomoles of O2/minute per milligram of protein was calculated.

The specific hexokinase (HK) enzyme activity was determined using enzyme-coupled assays by measuring the increase in absorbance at 339 nm with a multireader system Synergy HT (Bio Tek). The HK activity was assessed according to a slightly modified Worthington protocol (see http://www.worthingtonbiochem.com/HK/; Worthington Biochemical) and expressed as units/gram of protein. The detailed procedure was described earlier [29].

Western blot analysis

Proteins were separated by SDS/PAGE electrophoresis (10% or 12% gels) and transferred to nitrocellulose (Amersham Biosciences) or PVDF membranes (Merck Millipore). After blocking with 5% dry low-fat milk in TBS with Tween 20 (TTBS) for 60 min at room temperature, the membranes were washed and probed with mouse antibody cocktail: total OXPHOS (MitoProfile, Abcam; 1:250), mouse anti-mitochondrially encoded COX subunit (MTCO1; Abcam; 1:1000), mouse anti-cyclophilin D (Cyp-D; Abcam; 1:1000) and rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Santa Cruz Biotechnology; 1:5000) antibodies at 4°C overnight. The membranes were washed and incubated with anti-mouse (Santa Cruz Biotechnology; 1:10000, v/v, in TTBS) and anti-rabbit (Bio-Rad; 1:3000) horseradish peroxidase-labelled secondary antibodies, respectively, for 60 min at room temperature. Bands were visualized using enhanced chemiluminescence on the LAS system or membranes were scanned on an Odyssey scanner (LI-COR Biosciences). ImageJ or AIDA software was used for quantification of the relative abundance of proteins. To ensure the specificity of immunoreactive proteins, prestained molecular-weight protein standards (Bio-Rad) were used. The samples from each experimental group were run on the same gel and quantified on the same membrane. CNH did not affect the expression of GAPDH, which was used as a loading control.

Mitochondria isolation

Mitochondria were freshly isolated from a separate set of animals. LV free walls were homogenized at 0°C by a Teflon–glass homogenizer as 10% homogenate in a medium containing 250 mM sucrose, 10 mM Tris/HCl, 2 mM EGTA and 0.5 mg/ml of fatty acid-free BSA, pH 7.2. The homogenate was centrifuged for 10 min at 600g, and the supernatant was centrifuged for 10 min at 10000g. The mitochondrial sediment was washed twice in a sucrose medium without EGTA and BSA by centrifugation for 10 min at 10000g. Pellets of washed mitochondria were re-suspended in 0.5 ml of 250 mM sucrose, 10 mM Tris/HCl, pH 7.2. The integrity of each mitochondrial preparation was tested by determination of the respiratory control index (RCI; state 3/state 4) using an OROBOROS Oxygraph-2k for high-resolution respirometry as previously described [30].

Reserve COX activity and mitochondrial respiration

COX activity of freshly isolated mitochondria was determined in the medium used for the maintenance of mitochondrial structural and functional integrity containing 80 mM KCl, 10 mM Tris/HCl, 3 mM MgCl, 1 mM EDTA, 0.5 mM ADP and 0.025 mM cytochrome c, pH 7.4. Lauryl maltoside (LM; 0.02%) was used for the measurement of reserved COX activity as described earlier [31]. Oxygen consumption due to autooxidation was determined in the presence of 5 mM sodium ascorbate and 0.2 mM N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) as electron donors. The reaction was terminated by the addition of 0.25 mM KCN and the respiratory rate was corrected for the KCN-insensitive portion of the oxygen uptake.

The respiration of isolated mitochondria was determined using an Oxygraph at 30°C [32]. The incubation medium contained 80 mM KCl, 10 mM Tris/HCl, 3 mM MgCl2, 4 mM potassium phosphate and 1 mM EDTA, pH 7.2. To the medium were added, stepwise, mitochondria (0.1 mg of protein/ml), 2.5 mM malate, 10 mM oxoglutarate, 1 mM ADP, 5 μM cytochrome c and 10 mM succinate. The respiratory rates and activation effects of added cytochrome c and RCI were calculated.

Measurement of mitochondrial swelling

The parameters of mitochondrial swelling were measured as described previously [24] with minor modifications. Mitochondrial swelling was detected, as a decrease of absorbance at 520 nm, in a Perkin Elmer Lambda spectrophotometer at 30°C in swelling medium containing 125 mM sucrose, 65 mM KCl, 10 mM Hepes, pH 7.2, 5 mM succinate and 1 mM potassium phosphate [33]. Mitochondria (about 0.4 mg of protein) were added to 1 ml of incubation medium to provide an absorbance of about 1. After 1 min of preincubation of the mitochondrial suspension, CaCl2 solution was added and absorbance changes were detected in 10-s intervals for a further 5 min. Two parameters of the swelling process were evaluated: (i) the extent of swelling expressed as absorbance change per 5 min (∆A520/5 min), and (ii) the maximum swelling rate after addition of CaCl2, obtained after derivation of the swelling curve and expressed as absorbance change over 10 s (∆A520/10 s) [24].

Myocardial ischaemia–reperfusion

Separate sets of rats were subjected to acute I/R as described previously [34]. Animals that had been anaesthetized (sodium pentobarbital, 60 mg/kg i.p.) were intubated with a cannula connected to a rodent ventilator (Ugo Basile SRL) and ventilated with air at room temperature and 68–70 strokes/min (tidal volume of 1.2 ml/100 g of body weight). A heparinized cannula was placed in the left carotid artery for blood pressure monitoring (pressure transducer Gould P23Gb). A single-lead electrocardiogram (ECG) and blood pressure were analysed using custom-designed software. The rectal temperature was maintained between 36.5 and 37.5°C by a heated table throughout the experiment. Hypoxic rats were anaesthetized in the hypoxic chamber and their exposure to normoxic air before coronary artery occlusion was less than 40 min. This short reoxygenation has no effect on cardiac ischaemic tolerance as shown earlier [2].

Left thoracotomy was performed and a silk braided suture 5/0 (Chirmax s.r.o.) was placed around the left anterior descending coronary artery about 1–2 mm distal to its origin. After a 15-min stabilization, regional myocardial ischaemia was induced by tightening of the suture threaded through a poly(ethylene) tube. After a 20-min occlusion period, the ligature was released and the chest closed, air was exhausted from the thorax and spontaneously breathing animals were maintained in deep anaesthesia for 3 h.

Infarct size determination

Hearts were excised and washed with saline via the aorta. The area at risk (AR) was delineated by perfusion with 5% potassium permanganate as described previously [34]. Frozen hearts were cut into slices 1-mm thick, stained with 1% 2,3,5-triphenyltetrazolium chloride (pH 7.4 and 37°C) for 30 min, and fixed in formaldehyde solution. After 4 days, both sides of the slices were photographed. The IS, and the size of the AR and the LV, were determined using a computerized planimetric method with the software Ellipse (ViDiTo). The size of the AR was normalized to the LV (AR/LV), and the IS was normalized to the LV (IS/LV) and the AR (IS/AR). The incidence and severity of ventricular arrhythmias during the 20-min ischaemic insult and during the first 3 min of reperfusion were assessed as previously described [35].

Statistics

The results are expressed as means±S.E.M.s from the indicated number of experiments. One-way ANOVA or ANOVA for repeated measurements, and the subsequent Student–Newman–Keuls test, was used for comparison of differences in normally distributed variables between groups. Data not normally distributed (arrhythmias) are expressed as median±interquartile range. Differences in the number of premature ventricular complexes between the groups were compared using the Kruskal–Wallis non-parametric test. The incidence of tachycardia and fibrillation was examined using Fisher's exact test. Differences were assumed to be statistically significant when P<0.05.

Results

Basic parameters

Normoxic SHR and SHR-mtBN strains did not differ in haematocrit, heart weight and blood pressure parameters except for the relative LV weight, which was slightly (by 5.3%) but significantly higher in the conplastic SHR-mtBN strain (Table 1). Adaptation of rats to CNH caused retardation of body growth, increased RV systolic pressure (RVSP) and pronounced RV hypertrophy, and an increase in haematocrit compared with normoxic controls. In hypoxic SHR-mtBN strain, the relative RV weight was lower by 12.1% compared with hypoxic SHR, whereas the RVSP did not differ between the hypoxic groups. Compared with normoxic controls, both hypoxic groups had significantly higher heart rates during the invasive measurement of systemic blood pressure under isoflurane anaesthesia. CNH did not affect systolic (SBP) but slightly increased diastolic blood pressure (DBP), significantly in SHR-mtBN strain (Table 1).

Table 1
Heart weight and blood pressure parameters, the relative size of the AR and IS, and haematocrit in SHR and SHR-mtBN strains adapted to CNH and normoxic controls
 Normoxia Hypoxia 
Parameter SHR SHR-mtBN SHR SHR-mtBN 
BW (g) 314 ± 5 300 ± 4 267 ± 6* 269 ± 8* 
RV (mg) 189 ± 5 180 ± 3 374 ± 11* 331 ± 17* 
LV (mg) 596 ± 7 607 ± 15 592 ± 15 621 ± 17 
S (mg) 236 ± 4 229 ± 6 215 ± 5 220 ± 7 
RV/BW (mg/g) 0.60 ± 0.01 0.60 ± 0.01 1.40 ± 0.01* 1.23 ± 0.01* 
(LV+S)/BW (mg/g) 2.65 ± 0.02 2.79 ± 0.06 3.02 ± 0.04* 3.13 ± 0.03* 
HW/BW (mg/g) 3.25 ± 0.03 3.39 ± 0.06 4.42 ± 0.04* 4.37 ± 0.06* 
HR (min−1309 ± 11.7 302 ± 11.5 378 ± 6.3* 389 ± 3.9* 
SBP (mmHg) 157 ± 6.6 154 ± 8.5 153 ± 3.3 157 ± 2.3 
DBP (mmHg) 109 ± 6.0 102 ± 6.2 120 ± 1.4 119 ± 1.2* 
RVSP (mmHg) 28.3 ± 0.9 27.7 ± 0.5 63.2 ± 2.2* 62.1 ± 2.9* 
AR/LV (%) 41.3 ± 2.6 40.3 ± 3.6 41.9 ± 2.8 46.8 ± 3.5 
IS/LV (%) 27.8 ± 2.3 26.4 ± 4.3 19.5 ± 2.3* 15.3 ± 1.6* 
Haematocrit (%) 47.4 ± 0.6 48.3 ± 0.6 69.9 ± 0.7* 71.8 ± 0.9* 
 Normoxia Hypoxia 
Parameter SHR SHR-mtBN SHR SHR-mtBN 
BW (g) 314 ± 5 300 ± 4 267 ± 6* 269 ± 8* 
RV (mg) 189 ± 5 180 ± 3 374 ± 11* 331 ± 17* 
LV (mg) 596 ± 7 607 ± 15 592 ± 15 621 ± 17 
S (mg) 236 ± 4 229 ± 6 215 ± 5 220 ± 7 
RV/BW (mg/g) 0.60 ± 0.01 0.60 ± 0.01 1.40 ± 0.01* 1.23 ± 0.01* 
(LV+S)/BW (mg/g) 2.65 ± 0.02 2.79 ± 0.06 3.02 ± 0.04* 3.13 ± 0.03* 
HW/BW (mg/g) 3.25 ± 0.03 3.39 ± 0.06 4.42 ± 0.04* 4.37 ± 0.06* 
HR (min−1309 ± 11.7 302 ± 11.5 378 ± 6.3* 389 ± 3.9* 
SBP (mmHg) 157 ± 6.6 154 ± 8.5 153 ± 3.3 157 ± 2.3 
DBP (mmHg) 109 ± 6.0 102 ± 6.2 120 ± 1.4 119 ± 1.2* 
RVSP (mmHg) 28.3 ± 0.9 27.7 ± 0.5 63.2 ± 2.2* 62.1 ± 2.9* 
AR/LV (%) 41.3 ± 2.6 40.3 ± 3.6 41.9 ± 2.8 46.8 ± 3.5 
IS/LV (%) 27.8 ± 2.3 26.4 ± 4.3 19.5 ± 2.3* 15.3 ± 1.6* 
Haematocrit (%) 47.4 ± 0.6 48.3 ± 0.6 69.9 ± 0.7* 71.8 ± 0.9* 

AR/LV, the relative size of the AR normalized to LV; BW, body weight; HR, heart rate; HW/BW, relative heart weight; IS/LV, the relative IS normalized to LV; (LV+S)/BW, relative LV weight; RV/BW, relative RV weight.

Values are means ± S.E.M.s; *P<0.05 vs corresponding normoxic group; P<0.05 vs corresponding SHR group.

Mitochondrial enzyme expression and activity

Normoxic SHR and SHR-mtBN strains did not differ in CS activity in LV homogenates (335 ± 7 and 308 ± 6 nmol/min per mg of protein, respectively) and CNH had no effect on CS activity in any strain (313 ± 13 and 320 ± 6 nmol/min per mg of protein, respectively). These findings suggested that normoxic and hypoxic groups did not differ in mitochondrial quantity.

Western blot analysis of mitochondrial complexes of oxidative phosphorylation showed a decrease in protein levels in both mtDNA-encoded and nuclear-encoded subunits of complex IV (MTCO1, COX-4) in LV homogenates of normoxic SHR-mtBN strain compared with the SHR progenitor strain (Figure 1A and B). In both strains, CNH significantly decreased protein expression of MTCO1 (by 12.2% and 13.1%, respectively) and COX-4 (by 11.2% and 19.4%, respectively). In agreement with protein levels, CNH reduced mRNA levels of COX subunits 4.1 and 4.2 (Figure 1C and D). COX activity also decreased by 11–12% in both strains under hypoxic conditions, but the difference did not reach statistical significance (Figure 1E). With respect to other mitochondrial complexes, there were no differences in protein expressions and activities between normoxic strains (Figure 1A, B, F and G). CNH tended to reduce protein expression of complexes I, II and III in SHR only. The observed decrease in complex I protein level in hypoxic SHR resulted in significantly higher levels of complex I in hypoxic SHR-mtBN strain (Figure 1A and B).

Mitochondrial protein expression and Western blots

Figure 1
Mitochondrial protein expression and Western blots

(A) Mitochondrial protein expression and (B) representative Western blots of mitochondrial protein expression using OXPHOS antibody cocktail against subunits of complex I (Ndufa9), complex II (Sdha), complex III (Uqcrc2), complex IV (Cox-4) and complex V (Atp5a1) and antibody against mtDNA-encoded subunits of complex IV (MTCO1); and mRNA expression of complex IV subunits (C) Cox4.1 and (D) Cox4.2, and (E–G) enzyme activities of mitochondrial complexes I–IV in hearts of SHR (white columns) and SHR-mtBN strains (hatched columns) adapted to CNH and normoxic controls. GAPDH was used as a loading control. Values are means ± S.E.M.s from 6–10 hearts in each group; *P<0.05 vs corresponding normoxic group; P<0.05 vs corresponding SHR group.

Figure 1
Mitochondrial protein expression and Western blots

(A) Mitochondrial protein expression and (B) representative Western blots of mitochondrial protein expression using OXPHOS antibody cocktail against subunits of complex I (Ndufa9), complex II (Sdha), complex III (Uqcrc2), complex IV (Cox-4) and complex V (Atp5a1) and antibody against mtDNA-encoded subunits of complex IV (MTCO1); and mRNA expression of complex IV subunits (C) Cox4.1 and (D) Cox4.2, and (E–G) enzyme activities of mitochondrial complexes I–IV in hearts of SHR (white columns) and SHR-mtBN strains (hatched columns) adapted to CNH and normoxic controls. GAPDH was used as a loading control. Values are means ± S.E.M.s from 6–10 hearts in each group; *P<0.05 vs corresponding normoxic group; P<0.05 vs corresponding SHR group.

As we observed decreased gene and protein expressions of COX subunits, but not its activity, in LV homogenates of rats adapted to CNH, the COX activity in the freshly isolated intact mitochondria, in the absence or presence of the detergent LM, was determined in an additional set of experiments. COX activity measured in the presence of detergent indicates its maximum (reserve) capacity, whereas activity determined in intact mitochondria in the absence of detergent reflects the situation in intact cardiomyocytes [23]. The typical respiratory trace of isolated mitochondria is shown in Figure 2(A). COX activity in intact mitochondria was markedly higher compared with activities in the homogenates (Figure 2B vs Figure 1G). Neither CNH nor genetic manipulation (conplastic strain) affected COX activity. LM increased the oxygen consumption approximately 4-fold compared with intact mitochondria in both normoxic groups. Adaptation to CNH elevated stimulated COX activity by 47% and 60%, respectively, in SHR and SHR-mtBN strains compared with the corresponding normoxic rats (Figure 2B); nevertheless, the maximum COX activity did not differ between CNH-exposed animals.

Representative record of COX activity measurement in the presence of detergent LM

Figure 2
Representative record of COX activity measurement in the presence of detergent LM

(A) COX activity was corrected to the oxygen consumption rate for autooxidation of cytochrome c (Cyt c) and ascorbate (Asc) and TMPD (see Materials and methods). (B) COX activity in intact (−LM) heart mitochondria isolated from SHR (white columns) and SHR-mtBN strains (hatched columns) adapted to CNH and normoxic controls and in the presence of detergent (+LM). Values are means ± S.E.M.s from 6–9 hearts in each group. *P<0.05 vs corresponding normoxic group.

Figure 2
Representative record of COX activity measurement in the presence of detergent LM

(A) COX activity was corrected to the oxygen consumption rate for autooxidation of cytochrome c (Cyt c) and ascorbate (Asc) and TMPD (see Materials and methods). (B) COX activity in intact (−LM) heart mitochondria isolated from SHR (white columns) and SHR-mtBN strains (hatched columns) adapted to CNH and normoxic controls and in the presence of detergent (+LM). Values are means ± S.E.M.s from 6–9 hearts in each group. *P<0.05 vs corresponding normoxic group.

Mitochondrial respiration

Figure 3A demonstrates the typical results of the oxygen consumption curve of isolated mitochondria. Using only glutamate and malate as substrates, we observed very low respiration in mitochondria isolated from LVs of normoxic and hypoxic rats. However, significantly higher oxygen uptake was determined in SHR-mtBN strain adapted to CNH (by 41%) compared with normoxic controls. When the respiratory chain was coupled to ATP synthesis by the addition of ADP, respiration increased markedly in all experimental groups and was still higher by 37% in hypoxic SHR-mtBN strain compared with normoxic SHR-mtBN ones. The RCI, the indicator of the quality and coupling of mitochondria, did not differ between groups, reaching 12.7 ± 1.4 and 15.1 ± 1.7 in normoxic and 12.7 ± 0.4 and 13.8 ± 0.6 in hypoxic SHR and SHR-mtBN strains, respectively. Activation of respiration by cytochrome c and succinate increased oxygen consumption in all groups, although the effect did not reach statistical significance in normoxic and hypoxic SHR-mtBN strain (Figure 3B).

Oxygraph record of respiration measurements and mean respiration rates of mitochondria

Figure 3
Oxygraph record of respiration measurements and mean respiration rates of mitochondria

(A) Representative Oxygraph record of isolated mitochondrial respiration measurement in vitro (see Materials and methods) and (B) mean respiration rates of mitochondria (using different substrates) from SHR (white column) and SHR-mtBN strains (hatched column) adapted to CNH and normoxic controls. Values are means ± S.E.M.s from 6–9 hearts in each group. *P<0.05 vs corresponding normoxic group.

Figure 3
Oxygraph record of respiration measurements and mean respiration rates of mitochondria

(A) Representative Oxygraph record of isolated mitochondrial respiration measurement in vitro (see Materials and methods) and (B) mean respiration rates of mitochondria (using different substrates) from SHR (white column) and SHR-mtBN strains (hatched column) adapted to CNH and normoxic controls. Values are means ± S.E.M.s from 6–9 hearts in each group. *P<0.05 vs corresponding normoxic group.

MPTP opening, HK activity and CyP-D expression

Opening of the MPTP was detected as a Ca2+-induced decrease in absorbance (at 520 nm). It was evident that the same mass of mitochondria from normoxic and hypoxic hearts gave the same value for initial absorbance, whereas its drop differed among the groups after addition of CaCl2 (Figure 4A). The maximum rate of swelling (Figure 4B) was calculated from curves obtained after derivatization of the curves shown in Figure 4(A). Neither CNH nor mitochondrial genome replacement affected the time when the maximum MPTP opening rate occurred, suggesting a similar delay of MPTP opening after addition of CaCl2 (20–30 s) in all experimental groups (Figure 4B).

Records and maximum rate of mitochondrial swelling

Figure 4
Records and maximum rate of mitochondrial swelling

(A) Representative record of Ca2+-induced mitochondrial swelling and (B) curves of maximum rate of swelling, (C) mean extent of swelling and (D) mean maximum rate of swelling in mitochondria isolated from SHR (white columns) and SHR-mtBN strains (hatched columns) adapted to CNH (H) and normoxic controls (N). Values are means ± S.E.M.s from 6–7 hearts in each group. *P<0.05 vs corresponding normoxic group; P<0.05 vs corresponding SHR group.

Figure 4
Records and maximum rate of mitochondrial swelling

(A) Representative record of Ca2+-induced mitochondrial swelling and (B) curves of maximum rate of swelling, (C) mean extent of swelling and (D) mean maximum rate of swelling in mitochondria isolated from SHR (white columns) and SHR-mtBN strains (hatched columns) adapted to CNH (H) and normoxic controls (N). Values are means ± S.E.M.s from 6–7 hearts in each group. *P<0.05 vs corresponding normoxic group; P<0.05 vs corresponding SHR group.

In normoxic SHR-mtBN strain the extent and the maximum rate of swelling reached 0.227 ± 0.013 ΔA520/5 min and 0.0283 ± 0.0024 ΔA520/10 s, respectively, and were lower compared with normoxic SHR (0.265 ± 0.013 ΔA520/5 min and 0.0397 ± 0.0023 ΔA520/10 s, respectively). Adaptation to CNH slowed down the rate of mitochondrial swelling in SHR to 76.2% of normoxic rats. Reduced sensitivity to Ca2+-induced swelling was more pronounced in chronically hypoxic SHR-mtBN strain (Figure 4C and D).

Next we analysed selected components regulating MPTP opening. The specific enzyme activity of HK was significantly higher in LV homogenates of normoxic SHR-mtBN strain compared with normoxic SHR (13.5 ± 0.7 units/g vs 11.6 ± 0.2 units/g). Adaptation to CNH increased HK activity by 54% and 63%, respectively, in SHR and SHR-mtBN strains, and thus significantly higher HK activity persisted in hypoxic SHR-mtBN strain compared with SHR (Figure 5A). Protein expression of CyP-D did not differ in LV homogenates of normoxic SHR and SHR-mtBN strains. The significant decrease in CyP-D protein expression (by 15%) was observed in hypoxic SHR but not in hypoxic SHR-mtBN strain (Figure 5B).

Specific HK activity and Western blots of CyP-D

Figure 5
Specific HK activity and Western blots of CyP-D

(A) The specific HK activity and (B) representative Western blot and expression of CyP-D in hearts of SHR (white columns) and SHR-mtBN strains (hatched columns) adapted to CNH and normoxic controls. GAPDH was used as a loading control. Values are means ± S.E.M.s from 6–10 hearts in each group. *P<0.05 vs corresponding normoxic group; P<0.05 vs corresponding SHR group.

Figure 5
Specific HK activity and Western blots of CyP-D

(A) The specific HK activity and (B) representative Western blot and expression of CyP-D in hearts of SHR (white columns) and SHR-mtBN strains (hatched columns) adapted to CNH and normoxic controls. GAPDH was used as a loading control. Values are means ± S.E.M.s from 6–10 hearts in each group. *P<0.05 vs corresponding normoxic group; P<0.05 vs corresponding SHR group.

Cardiac ischaemic tolerance

Normoxic groups exhibited similar incidence and severity of various types of ischaemic ventricular arrhythmias. CNH did not induce any antiarrhythmic effect. At the start of reperfusion, hypoxic SHR and SHR-mtBN strains exhibited less reperfusion arrhythmias (by 64% and 52%, respectively) compared with normoxic controls. However, the effect of CNH was not statistically significant due to high variability (Table 2).

Table 2
Premature ventricular complexes (PVCs) occurring as singles, salvos and VT, incidence of VT and VF, and total duration of VT and VF during 20 min of coronary artery occlusion and PVCs at the start of reperfusion in hearts of SHR and SHR-mtBN strains adapted to CNH and normoxic controls
  Ischaemia  
  Number of arrhythmias  Incidence (%) Duration (s) Reperfusion Number of arrhythmias 
Group n Singles Salvos VT PVCs VT VF VT VF PVCs 
Normoxia 
SHR 12 237(665) 126(215) 704(3875) 995(4237) 100 25.0 49(412) 0(17) 53(247) 
SHR-mtBN 10 219(411) 114(241) 647(2241) 1054(2466) 100 40.0 61(227) 0(41) 67(305) 
Hypoxia 
SHR 11 106(339) 48(213) 585(2038) 1027(1907) 100 9.1 59(201) 0(28) 16(163) 
SHR-mtBN 128(347) 52(166) 1206(3266) 1662(3318) 100 44.4 114(349) 0(38) 27(132) 
  Ischaemia  
  Number of arrhythmias  Incidence (%) Duration (s) Reperfusion Number of arrhythmias 
Group n Singles Salvos VT PVCs VT VF VT VF PVCs 
Normoxia 
SHR 12 237(665) 126(215) 704(3875) 995(4237) 100 25.0 49(412) 0(17) 53(247) 
SHR-mtBN 10 219(411) 114(241) 647(2241) 1054(2466) 100 40.0 61(227) 0(41) 67(305) 
Hypoxia 
SHR 11 106(339) 48(213) 585(2038) 1027(1907) 100 9.1 59(201) 0(28) 16(163) 
SHR-mtBN 128(347) 52(166) 1206(3266) 1662(3318) 100 44.4 114(349) 0(38) 27(132) 

Values are the median (range).

VF, ventricular fibrillation; VT, ventricular tachycardia.

The normalized area at risk (AR/LV) was 40–47% and did not differ among the groups (see Table 1). The IS reached 67.7 ± 3.0% and 65.1 ± 5.1% of the AR in normoxic SHR and SHR-mtBN strains, respectively (Figure 6A). CHN significantly decreased myocardial infarction to 46.0 ± 3.2% in SHR. In hypoxic SHR-mtBN strain, IS/AR reached 33.0 ± 2.3% and was significantly smaller compared with hypoxic SHR. Figure 6(B) shows a decreased slope for the linear dependence of IS on the AR in both hypoxic groups.

Myocardial IS and AR

Figure 6
Myocardial IS and AR

(A) Myocardial IS expressed as a percentage of the AR, (B) relationship between AR and IS, and (C) relationship between the maximum rate of mitochondrial swelling and IS in SHR (white column) and SHR-mtBN strains (hatched column) adapted to CNH (H) and normoxic controls (N). Values are means ± S.E.M.s from 9–12 hearts in each group. *P<0.05 vs corresponding normoxic group; P<0.05 vs corresponding SHR group.

Figure 6
Myocardial IS and AR

(A) Myocardial IS expressed as a percentage of the AR, (B) relationship between AR and IS, and (C) relationship between the maximum rate of mitochondrial swelling and IS in SHR (white column) and SHR-mtBN strains (hatched column) adapted to CNH (H) and normoxic controls (N). Values are means ± S.E.M.s from 9–12 hearts in each group. *P<0.05 vs corresponding normoxic group; P<0.05 vs corresponding SHR group.

The relationship between the mean values of the maximum rate of mitochondrial swelling and the mean values of IS/AR is shown in the Figure 6(C). Regression analysis demonstrated a positive linear relationship between these variables, with a correlation coefficient of 0.83.

Discussion

The main novel finding of the present study is that adaptation to CNH decreased myocardial IS in both progenitor and conplastic SHR strains. The improved cardiac ischaemic tolerance was accompanied by low sensitivity of MPTP to opening, increased specific enzyme activity of HK and reserve COX capacity, and decreased myocardial protein expression of COX subunits. Importantly, in hypoxic SHR-mtBN strain harbouring the mitochondrial genome of the more ischaemia-resistant BN strain, a stronger IS-limiting effect, lower sensitivity of MPTP to opening, and increased mitochondrial respiration and HK activity were observed compared with the chronically hypoxic progenitor SHR strain. Taken together, these findings suggest that mtDNA can modulate adaptive responses to chronic hypoxia, resulting in myocardial ischaemic tolerance.

SHR were developed as a pathophysiological animal model for human essential hypertension of neurogenic origin. Besides hypertension, many other manifestations of the metabolic syndrome (insulin resistance, dyslipidaemia and other risk factors for cardiovascular diseases) were identified in SHR [16]. SHR also exhibited an increased sensitivity to acute I/R injury [3640] that is in line with most studies analysing cardiac ischaemic tolerance in many models of myocardial hypertrophy. However, the ability of various interventions to stimulate intrinsic cardioprotection is present in SHR. It has been shown that preconditioning reduced deleterious manifestations of acute I/R insult in SHR [38,4043]. Likewise, chronic social stress before I/R led to better SHR heart function restoration on reperfusion [40]. Our observation of reduced myocardial infarction in both progenitor and conplastic SHR strains is in line with previous studies demonstrating the IS-limiting effect of CNH in normotensive rats [2,22,44,45]. There is a single study of Belaidi et al. [46] that reported impaired cardiac ischaemic tolerance in SHR exposed to chronic intermittent hypoxia (CIH), mimicking obstructive sleep apnoea (OSA) syndrome (the fast repeated cycles of severe hypoxia and normoxia in seconds). This finding is not surprising. OSA syndrome also has detrimental effects on cardiac ischaemic tolerance in normotensive animals [47,48] compared with the cardioprotective regimens of CNH or CIH with much longer (hours) normoxic periodicity [2,32,49,50].

In the present study, the SHR-mtBN conplastic rat strain harbouring the mitochondrial genome of BN was compared with progenitor SHR. Complete mitochondrial DNA sequence analysis in SHR-mtBN strain revealed polymorphisms of potential functional significance in 7 of 13 mRNA genes, which were predicted to cause amino acid substitution in mitochondrial respiratory complex subunits. As shown earlier, the selective replacement of the mitochondrial genome of normoxic SHR with the genome of the BN strain did not affect liver mitochondrial complex activities and protein levels, except for complex IV–COX [16]. COX represents a terminal part of the mitochondrial respiratory chain and its activity indicates the maximum capacity for the transfer of reducing equivalents from nutritional substrates to molecular oxygen [51]. It has been proposed that COX is an oxygen sensor regulating the switch from oxidative phosphorylation to anaerobic ATP production [52]. Therefore, the importance of COX for cellular energetic homoeostasis increases under conditions of reduced oxygen availability, i.e. in chronic hypoxia. In line with an earlier report of Pravenec et al. [16], we observed lower COX subunit protein levels in LV homogenates of normoxic SHR-mtBN strain than in SHR. Adaptation to CNH down-regulated gene and protein expression of COX in both SHR strains, but it did not have a statistically significant effect on COX activity in LV homogenates and intact, freshly isolated mitochondria. Similarly, the cardioprotective regimens of chronic hypoxia had no effect on myocardial COX activity in normotensive rats [5355].

COX enzyme activity is obviously affected (down-regulated) by its regulatory subunits, which can be released by membrane fragmentation or detergent solubilization [56]. Therefore, the assessment of detergent-stimulated COX activity reflects its maximum (reserve) enzymatic capacity. In the present study we show that CNH equally elevated the detergent-stimulated COX activity in both progenitor and conplastic SHR. It has been reported that the maximum stimulated COX activity, as well as the cardiac ischaemic tolerance, decreases during postnatal development [31,57]. Similarly, preconditioning failed to reduce anoxic cell death in cardiomyoblasts transfected with the specific siRNA targeted to the nuclear-encoded COX subunit IV [58]. These findings are in line with enhanced COX activity in mitochondria of preconditioned rat hearts isolated after the protective stimulus as well as in reperfusion [59]. The authors of this later study suggested that increased COX activity can improve myocardial recovery of ATP production and more rapid return of oxidative phosphorylation functions in reperfusion. Moreover, it has been shown that protein kinase C isoform ε (PKCε), which is also involved in the cardioprotective mechanism of CNH [45], interacts with COX and enhances its activity [59,60]. Finally, it appears that COX activity can also modulate the sensitivity of MPTP to opening, and vice versa. The acute knockdown of COX subunit IV decreased the resistance of MPTP to opening [58]. On the other hand, the inhibition of MPTP by cyclosporine A increased COX activity and ATP production in failing cardiomyocytes [61]. Altogether, the improved myocardial COX activity and ischaemic tolerance of chronically hypoxic SHR strains is in line with these reports. Adaptation to CNH increased maximum stimulated COX capacity in both progenitor and conplastic SHR strains, although COX activity was down-regulated in intact mitochondria. It is possible to speculate that membrane fragmentation associated with acute I/R can release the elevated reserve COX capacity in chronically hypoxic mitochondria, which can contribute to the cardioprotective phenotype.

In SHR-mtBN strain, but not in progenitor SHR, adaptation to CNH increased mitochondrial respiratory rates in the presence of substrates only (state 2), as well as after stimulation by ADP (state 3). Earlier studies reported decreased [62,63], unchanged [49] or increased [64,65] rates of respiration and ATP synthesis in mitochondria isolated from non-ischaemic hearts of chronically hypoxic normotensive rats, guinea-pigs and neonatal rabbits. Maslov et al. [66] showed higher mitochondrial rates of respiration after I/R in CNH rat hearts than in normoxic controls. Due to these controversial results, the role of mitochondrial respiration in cardioprotection conferred by chronic hypoxia remains unclear. Both species (rat vs rabbit vs guinea-pig) and ontogenetic (adult vs neonatal) differences may account for these discrepancies. Nevertheless, we still cannot exclude the observed increase in mitochondrial respiration possibly playing a role in the more pronounced cardioprotection afforded by CNH in SHR-mtBN strain.

It is generally accepted that MPTP plays a crucial role in the sensitivity of myocardium to acute I/R injury. Its opening allows water and solutes to enter the mitochondrial matrix, leading to matrix swelling, and uncoupling of the respiratory chain, calcium overload and release of small proteins such as cytochrome c. The inhibition of MPTP is associated with increased cardiac ischaemic tolerance (reviewed in the literature [21,67]). In the present study, CNH decreased mitochondrial sensitivity to Ca2+-induced swelling in hearts of both SHR strains. Similarly, the sensitivity of MPTP to opening was reduced in normotensive rats adapted to CNH [62,66], as well as to cardioprotective CIH regimens [49,68,69], and acute administration of atractyloside, an MPTP opener, before ischaemia or at the start of reperfusion inhibited the cardioprotection conferred by chronic hypoxia [49,69,70]. Altogether, these findings support a key role of MPTP in cardioprotection afforded by chronic hypoxia.

The results of the present study suggest that high blood pressure and metabolic abnormalities described in progenitor and conplastic SHR strains [16] do not interfere with CNH-induced desensitization of MPTP opening by Ca2+. Interestingly, conplastic SHR-mtBN strain adapted to CNH had smaller myocardial infarcts compared with corresponding progenitor SHR, and this stronger cardioprotective effect of CNH was associated with more pronounced resistance of MPTP to opening. The mean values of the maximum rate of mitochondrial swelling positively correlated with the size of the myocardial infarct among groups. It suggests that the mitochondrial genome can modulate myocardial MPTP sensitivity to opening and IS in chronically hypoxic SHR. Recently, Muravyeva et al. [15] analysed the efficacy of anaesthetic preconditioning in two conplastic rat strains sharing nuclear genome from animals with type 2 diabetes mellitus, and with a distinct mitochondrial genome of normotensive and hypertensive strains. They found that the selective replacement of the mitochondrial genome with that of hypertensive rats abolished the cardioprotective effect of anaesthetic preconditioning and sensitized MPTP to reactive oxygen species (ROS)-induced opening. These observations support the view that the mitochondrial genome can affect the efficacy of cardioprotective interventions.

The regulation of MPTP sensitivity to opening is a complex process and its modulators include ions, lipids and components of the mitochondrial membrane, as well as soluble and membrane proteins [21,67,71]. It is well known that Cyp-D is one of the key MPTP modulators and its inhibition or transgenic down-regulation attenuates myocardial I/R injury [72,73]. In the present study, CNH decreased the MPTP sensitivity to opening in both hypertensive strains, but it significantly reduced CyP-D protein levels in SHR only. Likewise, Magalhães et al. [68] have shown that the cardioprotective regimen of CIH desensitized MPTP to opening, but did not alter Cyp-D protein expression in normotensive rats. Therefore, the role of Cyp-D in cardioprotection conferred by chronic hypoxia seems unlikely, because its expression does not correspond with the sensitivity of MPTP to opening and cardiac ischaemic tolerance. It has also been shown that neonatal and adult rat hearts did not differ in Cyp-D protein expression in spite of the fact that neonatal myocardium is more ischaemic tolerant and has a decreased MPTP sensitivity to opening [32,57,74].

It is well known that chronic hypoxia increases the myocardial capacity for glucose utilization and shifts myocardial energetics from lipid oxidation to glycolysis [75,76]. Various regimens of chronic hypoxia increase specific enzyme activity of HK, a key glycolytic enzyme, and elevate the expression of mitochondria-binding isoforms HK1 and HK2 in normotensive rat hearts [29,76]. HK2 activity can play an important role in improved cardiac ischaemic tolerance and its binding to the outer mitochondrial membrane decreases the sensitivity of MPTP to opening [21]. The results of the present study showed that total HK activity was higher in LV homogenates from SHR-mtBN strain than from SHR, and that adaptation to CNH increased HK activity equally in both strains. These findings are in line with the view that up-regulation of HK activity by chronic hypoxia may lead to a higher stimulation of the respiratory chain via ADP recycling, which can reduce formation of ROS and maintain energy homoeostasis [29]. Increased HK activity corresponded to the IS-limiting effect afforded by CNH in SHR and SHR-mtBN strains, suggesting that it may be involved in the protective mechanism. However, its potential role (with respect to other chronic hypoxia-induced cardioprotective events preventing oxidative stress, improving mitochondrial energetics and desensitizing MPTP to opening) in this scenario remains to be examined.

In conclusion, the results of the present study showed that adaptation to CNH reduced the susceptibility to I/R injury in the hearts of SHR strains, the effect being more pronounced in SHR-mtBN strain. The mitochondrial genome modulates the cardioprotective effect of CNH in SHR by affecting mitochondrial energetics and MPTP sensitivity to opening.

Author contribution

J. Neckář primarily conceived of and designed the study, performed the experiments, analysed and interpreted the data, and wrote the manuscript. F. Kolář designed the study and contributed to the manuscript preparation. M. Pravenec generated the SHR-mtBN strain. All the authors were involved in performing the experiments, data collection, analysis and interpretation, contributed to the intellectual content and editing of the manuscript, and approved its final version.

This work was supported by the Czech Science Foundation [13-10267S to J. Neckář and 14-36804G to M. Pravenec], the Ministry of Education, Youth and Sports of the Czech Republic [LL1204 grant within the ERC CZ program to M. Pravenec], the Grant Agency of the Charles University in Prague [1214214 to I. Brabcová] and the institutional research projects 67985823 (Institute of Physiology, Czech Academy of Sciences) and 00023001 (Institute for Clinical and Experimental Medicine - IKEM). IKEM received financial support from the European Commission within the Operational Programme Prague–Competitiveness project ‘Rozvoj infrastruktury PEM’ [CZ.2.16/3.1.00/28025].

Competing interests

The Authors declare that there are no competing interests associated with the manuscript.

Abbreviations

     
  • AR

    area at risk

  •  
  • BN

    Brown Norway rat

  •  
  • CIH

    chronic intermittent hypoxia

  •  
  • CNH

    continuous normobaric hypoxia

  •  
  • COX

    cytochome c oxidase

  •  
  • CS

    citrate synthase

  •  
  • CyP-D

    cyclophilin D

  •  
  • DBP

    diastolic blood pressure

  •  
  • DTNB

    5,5´-dithio-bis(2-nitrobenzoic acid)

  •  
  • GAPDH

    glyceraldehyde-3-phosphate dehydrogenase

  •  
  • HK

    hexokinase

  •  
  • I/R

    ischaemia–reperfusion

  •  
  • IS

    infarct size

  •  
  • i.p.

    intraperitoneally

  •  
  • LM

    lauryl maltoside

  •  
  • LV

    left ventricle

  •  
  • MPTP

    mitochondrial permeability transition pore

  •  
  • MTCO1

    mitochondrially encoded COX subunit 1

  •  
  • OSA

    obstructive sleep apnoea

  •  
  • RCI

    respiratory control index

  •  
  • ROS

    reactive oxygen species

  •  
  • RT-PCR

    real-time PCR

  •  
  • RV

    right ventricle

  •  
  • RVSP

    RV systolic pressure

  •  
  • S

    septum

  •  
  • SBP

    systolic blood pressure

  •  
  • SHR

    spontaneously hypertensive rat

  •  
  • TMPD

    N,N,N´,N´-tetramethyl-p-phenylenediamine

  •  
  • TTBS

    Tween with TBS

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