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Abstract

Background and aims:

The aim of this study was to investigate the early changes of cardiac uncoupling protein-2 (UCP2) expression following myocardial ischemia reperfusion in rats chronically treated with ramiprilat and losartan.

Methods:

Male Wistar rats were assigned into seven groups (six in each): intact (control); sham-operated; nontreated rats subjected to ischemia and reperfusion (IR); ramiprilat-treated rats with (Ram+IR) and without ischemia (Ram); losartan treated with (Los+IR) and without ischemia (Los). Quantitative evaluation of UCP2 mRNA was carried out using real-time reverse transcription-polymerase chain reaction (RT-PCR). Mitochondria were isolated, and protein expression was quantified by Western blotting.

Results:

In IR group: UCP2 protein but not mRNA level was increased in the ischemic area of the left ventricle (LV) (172% ± 26.7, p < 0.001 vs. LV of control). Following acute myocardial IR, UCP2 protein levels was increased in the ischemic area of the LV but not in RV, suggesting the local effect of ischemia on UCP2 expression. IR-induced overexpression of UCP2 was suppressed by ramiprilat and losartan.

Conclusion:

These findings suggest that losartan and ramiprilat can suppress UCP2 expression following myocardial IR, and by this mechanism may protect the myocardium against IR injury.

Keywords

Cardiac expression ischemia reperfusion UCP2 losartan ramiprilat

Introduction

Uncoupling proteins (UCPs) are mitochondrial inner membrane proteins that dissipate the proton electrochemical gradient across the inner mitochondrial membrane, and thereby reduce generation of mitochondrial reactive oxygen species (ROS). Decreased ROS levels may protect cardiomyocytes, but decreased intracellular adenosine triphosphate (ATP) generation due to uncoupling of the mitochondrial oxidative phosphorylation may cause less efficient metabolism.^1,2 UCPs are also thought to have a role in calcium homeostasis.³ UCP2 is the predominant isoform that is expressed in the mammalian heart,⁴ but the cardiac regulation of its expression in myocardium remains unclear, especially in relation to pathological conditions and drug treatments. The renin-angiotensin system (RAS) is an important factor in the pathogenesis of myocardial ischemia reperfusion (IR) injury. As a result, pharmacologic blockade of this system has turned out to be an attractive strategy for cardioprotection against IR injury.^5,6 Most of the experiments including our recent data published in this journal have shown that the angiotensin-converting enzyme (ACE) inhibitor ramiprilat and the angiotensin type 1 receptor blocker losartan are able to protect the myocardium against IR injury by reducing infarct size and ventricular arrhythmias even at non-hypotensive doses.^7,8 However, the key protective pathways and end effectors remain to be understood. Molecular studies can provide new insights into the early cellular mechanisms involved in myocardial responses to IR injury, and therefore may have implications for the specificity of diagnosis and treatment with these drugs. The aim of the present study was to investigate the cardiac UCP2 mRNA and protein levels following acute regional myocardial IR in rats chronically treated with losartan and ramiprilat.

Materials and methods

Experimental groups

Male Wistar rats (250–300 g) were purchased from the Pasteur Institute, Iran. The animals were kept on a 12-hour (h) light-12-h dark cycle in a temperature-controlled room. All experimental procedures were performed in accordance with the approved guidelines of the Ethics Committee on Animal Experiments of the Tarbiat Modares University, Tehran, Iran.

The animals were randomly assigned to seven groups (n=6 in each group). Two groups of rats received 10 mg/kg/day of losartan (Daru Pakhsh, Iran) and another two groups were given 50 µg/kg/day of ramiprilat (Sanofi-Aventis, France) during four weeks using a feeding tube. Our previous data had shown that four weeks of pretreatment with ramiprilat and losartan at the mentioned doses significantly decreases myocardial infarct size and ischemia-induced ventricular premature beats. One of the losartan- and one of the ramiprilat-treated groups underwent acute regional IR protocol (Los+IR and Ram+IR, respectively). Another group of untreated animals also underwent IR (IR group). In all of the IR groups the animals were subjected to 30 minutes (min) of left anterior descending artery (LAD) occlusion and subsequent reperfusion for 180 min. In the sham-operated group the animals underwent the similar surgical procedure without occlusion of LAD. The seventh group included intact animals that served as the controls.

IR protocols and sample collection

A rat model of myocardial IR was used as previously described.⁸ Anesthesia was induced by sodium pentobarbital (50 mg/kg, intraperitoneally (i.p.)). Body temperature was monitored with a rectal probe and maintained at 37^°C using a heating pad. The trachea was intubated and ventilated artificially with room air at a tidal volume of 1 ml/100 g and a frequency of 80 inflations/min. Lead II of electrocardiogram (ECG) was recorded using skin needle electrodes. Arterial blood pressure was monitored from the left carotid artery by a blood pressure transducer. The chest was opened by a left thoracotomy incision in the fourth intercostal space. The pericardium was incised and the heart was exposed. A 5/0 silk thread was passed below the LAD branch of the coronary artery, and occluded for 30 min by lifting the thread through a piece of polyethylene tube. Development of a pale color in the distal myocardium, ST elevation on ECG, and reduction of the blood pressure confirmed successful coronary occlusion. After 30 min of ischemia, loosening of the silk thread allowed reperfusion of ischemic myocardium for 180 min. At the end of the reperfusion the heart was then excised rapidly and cleared of blood by rinsing in cold isotonic saline. In the IR group, the ischemic area from the left ventricle (LV) was cut out, while in the control and sham-operated animals the corresponding normal left ventricular region was taken. In all of the groups a sample from the right ventricle (RV) was collected as well. The tissue samples were snap frozen in liquid nitrogen and stored at −80°C for further studies.

Cardiac mitochondrial isolation

Rat heart mitochondria were isolated using differential centrifugation according to the modified method of Butz et al.⁹ Briefly, tissue was placed in ice-cold cardiac homogenization and isolation buffer A (210 mM sucrose, 2 mM ethylene glycol tetraacetic acid (EGTA), 40 mM NaCl, and 30 mM 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES), pH 7.4) supplemented with protease inhibitors. Tissue was disrupted using a Teflon-glass homogenizer. The homogenate was centrifuged at 600 × g for 10 min at 4°C to eliminate nuclei and cell debris. One milliliter of supernatant was centrifuged again at 10,000 g for 20 min at 4°C. The supernatant fluid was discarded, and the pellet from this spin was washed in 1 ml of isolation medium B containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 10 mM Tris, pH 7.4. Final mitochondria pellet was resuspended in 200 μl of buffer B and 66 μl of 16% sodium dodecyl sulphate (SDS) and centrifuged at 1100 g at room temperature for 20 min to remove insoluble materials. Supernatants containing mitochondria were stored at −80°C.

Western blot analysis

Protein concentrations of homogenates were determined using the Bradford method.¹⁰ The protein extract was incubated for 7 min at 100°C in sample buffer (10% (v/v) glycerol, 5% (w/v) SDS, 0.25% (w/v) bromophenol blue, 5% (v/v) 2-mercaptoethanol, and 0.0625 M Tris-HCl, pH 6.8) before being loaded onto the wells. Equal amounts of homogenate protein were loaded on a 12.5% SDS-polyacrylamide gel electrophoresis mini-gel in each lane (Bio-Rad laboratories, Hercules, CA, USA). Electrophoresis was conducted for ~1.5 h at 110 V in running buffer (0.025 M Tris-HCl, 0.2 M glycine, 1 mM EDTA, and 3.5 mM SDS). Proteins were transferred from the gels to polyvinylidene fluoride membrane (Amersham Bioscience Co., UK) using a blot apparatus (Bio-Rad). The membranes were blocked with 2% ECL Advance blocking milk (Amersham Bioscience Co., UK) and 1% bovine serum albumin in Tris-buffered saline with Tween (TBST) (0.01 M Tris-HCl, pH 7.6, 1.5 mM NaCl, 0.1% (v/v) Tween-20) for 1 h. Western blotting was performed with polyclonal goat anti-UCP2 primary antibodies (Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA) and with donkey anti-goat horseradish peroxidase-conjugated secondary antibody (Santa Cruz) at a dilution of 1:500 and 1:10000, respectively. Then a chemiluminescent substrate (ECL Advance reagents, Amersham Bioscience Co., UK) was applied, and blots were exposed on film. Bands were scanned into a computer, and band density determined using ImageJ software (1.43). Data were normalized to control bands.

RNA extraction and real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis

Genes’ expression levels were determined by real-time RT-PCR. Total RNAs were extracted using RNeasy fibrous tissue mini kit (QIAGEN, USA) according to the manufacturer’s instructions. RNA concentrations were estimated by measuring the absorbance at 260 nm, and purity was assessed by 260/280 nm absorbance ratio (Eppendorf, Hamburg, Germany). First-strand cDNA synthesis was performed on 1 µg of total RNAs using random hexamers, deoxynucleoside triphosphate (dNTP) and Moloney murine leukemia virus reverse transcriptase (Fermentas, USA), in a total volume of 20 μL. The RT-PCR was performed using SYBR Green I and the Rotor Gene system (Corbett Research 2004, Australia). The relative quantification of gene expression was analyzed according to the Pfaffl method¹¹ (see supplementary information, Table).

Statistical analysis

One-way analysis of variance (ANOVA) was used to compare the differences between the control, sham and IR groups. Post hoc analysis was performed using the Tukey multiple comparison test. To detect whether losartan or ramiprilat treatment was interfering with IR, a two-way ANOVA was used. Statistical significance was designated at p < 0.05. Values are expressed as mean ± standard error of mean (SEM). Statistical analysis was performed using Prism software.

Results

Hemodynamic

The Table shows the effect of pretreatment with ramiprilat and losartan on blood pressure and heart rate before LAD occlusion, at the end of ischemia and at the sampling time. In all groups, shortly after LAD occlusion blood pressure was decreased significantly and returned to pre-occlusion values after reperfusion. In all groups, heart rate was stable throughout the experiments. There were no significant differences in heart rate or blood pressure between the control and IR groups at sampling time (p >0.05) (Table).

Table 1.

Mean blood pressure (MBP) (mmHg) and heart rate (HR) (beats min–1) obtained at baseline, at the end of ischemia (30 minutes after LAD occlusion) and at the sampling time (three hours after reperfusion) in rats treated with losartan (Los) and ramiprilat (Ram) with or without IR.

Groups	Baseline		Ischemia		Sampling time
	MBP	HR	MBP	HR	MBP	HR
Control	—	—	—	—	113 ± 5	379 ± 21
Sham	119 ± 8	373 ± 25	—	—	116 ± 7	361 ± 19
IR	115 ± 7	358 ± 19	84 ± 6^b	403 ± 18	107 ± 8	346 ± 16
Los	104 ± 11	382 ± 24	—	—	99 ± 8	397 ± 18
Los+IR	111 ± 6	361 ± 15	76 ± 8^a	392 ± 19	95 ± 9	375 ± 21
Ram	121 ± 8	394 ± 17	—	—	114 ± 7	372 ± 15
Ram+IR	116 ± 9	383 ± 21	81 ± 6^a	411 ± 13	119 ± 10	390 ± 23

Data are expressed as mean ± S.E.M. ^ap < 0.05 and ^bp < 0.01 compared with baseline values within the same group.

Effect of myocardial IR on cardiac UCP2 expression

We aimed firstly to assess whether the acute IR altered the cardiac UCP2 expression at protein and mRNA levels. Representative Western blot analysis on the mitochondrial fraction exhibited increased expression of UCP2 in the ischemic area of LV by 172% ± 26.7 (p < 0.001, vs. LV of control). In contrast to the LV, UCP2 protein level was not altered in the non-ischemic area from RV. At transcription level, UCP2 mRNA level in the IR group was decreased by 23% ± 7.2 in the ischemic area of LV as compared to the LV of control, which was not statistically significant. No difference in UCP2 protein and mRNA levels was observed between control and sham-operated rats (Figure 1).

Figure 1.

Uncoupling proteins-2 (UCP2) expression in the left (LV) and right (RV) ventricles following ischemia reperfusion (IR).The transcripts were quantified by real-time reverse transcription-polymerase chain reaction (RT-PCR) in total RNA isolated from rat hearts. Representative Western blot analyses of mitochondrial UCP2 are shown at the top of the panel. The statistical analysis reveals an increased protein level in the ischemic area of the LV of IR group. Values are mean ± S.E.M, n=6; ***p < 0.001: IR vs. control.

UCP2 transcription and expression in losartan-treated rats

To evaluate the effect of losartan on myocardial UCP2 expression, the mRNA and protein levels of UCP2 were assessed in cardiomyocytes of both ventricles after four weeks of pretreatment with losartan. As Figure 2 shows, in group of animals treated with losartan the mitochondrial UCP2 protein contents in the LV and RV were increased by 68% ± 11 (p < 0.05) and 61% ± 6.8 (p < 0.01), respectively. To further determine the profile of UCP2 expression in losartan-treated animals, one of the losartan-treated groups was subjected to acute IR (Los+IR), and protein expression was evaluated in response to it. In the Los+IR group exposure to myocardial IR increased mitochondrial UCP2 protein levels in LV and RV (83% ± 5.1 and 58% ± 8.7, respectively, compared to control hearts). Usage of losartan caused a significant difference in UCP2 protein expression between the Los+IR group and IR group (p < 0.001). In other words, in the Los+IR group, UCP2 protein expression in LV was decreased compared to the LV of untreated IR rats.

Figure 2.

The effects of losartan on uncoupling proteins-2 (UCP2) mRNA (A) and protein expression (B).Treatment of rats with losartan (Los) significantly increased UCP2 mRNA and protein levels compared to controls (C) (**p < 0.01). Ischemia reperfusion (IR)-induced upregulation of UCP2 protein was diminished by chronic administration of losartan (Los+IR). Values are mean ± S.E.M., n=6.

At transcription level, in the Los group the UCP2 mRNA level was increased in LV and RV by 66% ± 10.2 and 82% ± 14.7, respectively (p < 0.01 vs. control).

In the Los+ IR group the UCP2 mRNA level in LV was increased by 17% ± 4.9, which was not significant statistically. However, the UCP2 mRNA level in RV was increased by 63% ± 5.4, which was significant compared to the RV of the untreated IR group (p < 0.01).

UCP2 transcription and expression in ramiprilat-treated rats

To examine whether treatment with ramiprilat alters the UCP2 expression in the heart, UCP2 expression was assessed at protein and mRNA levels in the animals that had received ramiprilat. As is seen in Figure 3, there was no significant difference in UCP2 mRNA and protein expression between the Ram and control groups. In further experiments, the effect of IR on UCP2 expression was evaluated in a separate group of animals that had received ramiprilat (Ram+IR). Compared with controls, exposure to IR in the Ram+IR group increased the UCP2 protein level by 61% ± 5.3. Ram+IR significantly decreased UCP2 protein expression in LV compared to the untreated IR group as indicated in Figure 3 (p < 0.001).

Figure 3.

The effects of ramiprilat (Ram) on uncoupling protein 2 (UCP2) mRNA (A) and protein expression (B). Myocardial ischemia reperfusion (IR)-induced UCP2 upregulation was suppressed significantly in ramiprilat-treated rats subjected to IR (Ram+IR). C: control, values are mean ± S.E.M., n=6.

Discussion

This study was designed to investigate the expressional profile of cardiac UCP2 following myocardial IR injury in losartan- and ramiprilat-treated rats. Our main finding is that there are dynamic changes in cardiac UCP2 expression in response to myocardial IR. Our data indicate that following regional myocardial ischemia, UCP2 protein level was increased just in the ischemic area of the LV but not in the RV, suggesting the local effect of ischemia on the UCP2 protein levels. Regarding the fact that the UCP2 mRNA and protein levels didn’t change in the sham group, it should be accepted that the UCP2 changes observed following IR are not induced by surgical procedure. Furthermore, at the sampling time the blood pressure and heart rate were not different significantly between the experimental groups (Table), suggesting that the observed alteration in proteins and mRNA levels had not been affected by hemodynamic factors. The changes in the expression of UCP2 observed in our study are in agreement with the previous report showing that expression of the UCP2 is enhanced in mitochondria isolated from the ischemic area of swine hearts; however, the ischemia model was induced chronically and the UCP2 expression was measured 10 weeks after LAD stenosis.¹² Amplification of UCP2 expression in ischemic lesions of the human brain has also been shown.¹³ In another study the UCP2 mRNA level was increased in response to ischemia in the tissue surrounding the infarct zone; however, the mRNA transcription was assessed four weeks after LAD ligation, and the protein level had not been measured.¹⁴ To our knowledge, this is the first study to investigate the early UCP2 alteration at mRNA and protein levels in response to acute IR. What is the mechanism responsible for UCP2 over-expressions in response to IR? There is strong evidence that shows exposure to oxidative stress can upregulate and activate UCPs as a potential defensive mechanism by which cardiomyocytes can become protected against oxidative injury.^15,16 If UCP2 acts as a defensive mechanism against oxidative stress by decreasing the production of superoxide, the increased expression of UCP2 may provide a potential mechanism for mitochondrial adaptation in response to IR to deal with greater oxidative injury. Therefore, free radicals production may be the main factor responsible for increased UCP2 protein levels.

It should be noted that the partial dissipation of the electrochemical gradient lowers the rate of ATP production to oxygen consumption. Enhanced UCP expression would thus lower metabolic efficiency and hence ATP production. A relationship between cardiac UCP2 expression and cardiac dysfunction has been reported at the chronic stage of heart failure, hypertrophy and diabetes mellitus.^17–19 Almsherqi et al.²⁰ demonstrated that mitochondrial UCP3 expression was increased in the posterior non-ischemic wall following anterior myocardial infarction. The authors have suggested that an increase in UCP3 expression would explain the impaired energy metabolism of the non-ischemic area. However, there is also evidence supporting the idea that UCPs’ upregulation improves the mitochondrial efficiency of oxidative phosphorylation through increased removal of ROS and enhanced mitochondrial biogenesis.^21,22 Therefore, it seems more likely that the UCP2 upregulation observed in response to acute IR is a physiologically beneficial response that is triggered by ROS produced during ischemia and especially reperfusion, and prevents further oxidative damage in the heart.

Another finding of the current study was the discordance between UCP2 mRNA and protein expression following myocardial IR. The disparity between UCPs mRNA and protein has been reported previously,^23–25 suggesting that post-translational regulation may be involved in UCP2 protein induction. For example, the presence of an open reading frame in the UCP2 gene that encodes a peptide capable of mediating downregulation of the gene translation has been shown.²⁶ Therefore, any study investigating the physiological functions of UCP2 must confirm the presence of these proteins at both the mRNA and protein levels.

Another explanation for the observed disparity in our study may be related to the time of sampling. The mRNA expression was measured 240 min after LAD occlusion, and the mRNA expression has not been measured before this time; therefore, early increase and then degradation of mRNA before the sampling time is also supposed. In the rat heart perfused by a Langendorff system, 20 min of ischemia and 40 min of reperfusion increased the UCP2 and UCP3 mRNA levels. The difference between the results may stem from the sampling time.²⁷ Fert-Bober et al.²⁸ have found that the hearts subjected to ischemia with or without reperfusion show an increase in protein but a decrease in total mRNA levels. In our study four weeks of pretreatment with losartan increased UCP2 mRNA level in the ventricles, but following myocardial IR the mRNA expression was decreased in the ischemic area of the LV but not the RV. So, it can be proposed that in response to 30 min of ischemia and 180 min of reperfusion UCP2 mRNA content was decreased in the ischemic area at the sampling time. Measurement of mRNA and protein levels following different durations of ischemia and reperfusion can provide more complete data in this regard. UCPs are synthesized in the cytosol and imported into mitochondria with the aid of translocators, but the mechanism for the delivery of the protein is not known. Manipulation of the trafficking of these proteins into the mitochondria by IR may be an interesting mechanism to be investigated.

Here we demonstrate that an anti-ischemic dose (the dose that has prophylactic effects against ischemia) of losartan and ramiprilat suppressed UCP2 upregulation in the ischemic heart. It is clear that angiotensin II contributes to myocardial IR injury.^29,30 At the functional level, our recent study displayed the fact that losartan and ramiprilat protect the heart against IR injury independent of their hemodynamic effects. In that study four weeks of pretreatment with losartan and ramiprilat decreased myocardial infarct size and ischemia-induced ventricular premature beats.⁸ Therefore, in the current study both drugs are administered for four weeks so that we can be sure about their anti-ischemic effects. Other studies showed that angiotensin receptor blockers (ARBs) can modulate UCP in the mouse brown adipose tissue,³¹ rat liver³² and rat kidney.^33,34 In another study an ACE inhibitor (ACEi), perindopril, improved cardiac performance and also prevented decrease in the high-energy phosphates at the chronic stage of heart failure via suppression of UCP2 mRNA expression,¹⁸ which is in agreement with the relatively high mitochondrial content of UCP2 in rat kidney after administration of losartan.^35,36

The precise mechanism(s) for the observed effects is unclear. It may be due to the antioxidant effect of these drugs. Angiotensin II stimulates superoxide production by activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX); therefore, both ARBs and ACEi can decrease ROS generation.^37,38 Losartan can decrease renal mitochondrial H₂O₂ generation in hypertensive and diabetic rats.^35,36 Recent data from our lab have shown that administration of losartan suppresses IR and induces NOX2 upregulation in rats.³⁹ Thus a potential explanation may be that IR is associated with the burst of ROS generation and thus UCP2 expression, and losartan and ramiprilat suppress this upregulation via decreasing ischemia-induced oxidative stress, as suggested in another study that antioxidants could facilitate downregulation of UCP2 by limiting oxidative stress. Losartan and ramiprilat can suppress UCP2 protein expression under IR conditions compared to untreated IR.

In our study, four weeks of treatment with losartan but not ramiprilat increased cardiac UCP2 expression. Some evidence has shown that the nuclear transcription factor peroxisome proliferator-activated receptors (PPARs) are the key modulators of the expression of genes that encode UCPs.⁴⁰ Some of the ARBs, including telmisartan, irbesartan and losartan metabolite EXP3179, were shown to have PPARγ binding activity,^41–43 and therefore induce PPARγ target gene expression, but the possible effect of losartan on PPARγ transcription has not been shown. To examine whether PPARγ is responsible for UCP2 upregulation observed in the losartan-treated group, the level of PPARγ mRNA was measured. Our data show that administration of losartan but not ramiprilat increases PPARγ transcription in the heart, and this increase may be responsible for UCP2 upregulation observed following losartan treatment (see supplementary information, Figure 1). Therefore, the role of the PPAR family is considered as another mechanism for UCPs regulation.

Taken together, this is the first study that shows that UCP2 protein level is increased in the ischemic area following acute IR, and administration of a non-hypotensive anti-ischemic dose of ramiprilat and losartan can suppress this upregulation. Identification of the early changes in gene transcription and protein expression that take place following IR, especially in relation to drugs, may stimulate the search for new tools to diagnose and treat myocardial IR injury at the early stages.

Footnotes

Acknowledgements

The authors thank Dr. Naghdi for providing losartan and Dr. Richard Head for providing ramiprilat as a gift.

Conflict of interest

None declared.

Funding

This work was supported by research grants from Tarbiat Modares University, Iran.

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Supplementary Material

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Expressional profile of cardiac uncoupling protein-2 following myocardial ischemia reperfusion in losartan- and ramiprilat-treated rats

Abstract

Background and aims:

Methods:

Results:

Conclusion:

Keywords

Introduction

Materials and methods

Experimental groups

IR protocols and sample collection

Cardiac mitochondrial isolation

Western blot analysis

RNA extraction and real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis

Statistical analysis

Results

Hemodynamic

Effect of myocardial IR on cardiac UCP2 expression

UCP2 transcription and expression in losartan-treated rats

UCP2 transcription and expression in ramiprilat-treated rats

Discussion

Footnotes

Acknowledgements

Conflict of interest

Funding

References

Supplementary Material