Vol. 56 No. 4/2009, 687–694 Regular paper on-line at: www.actabp.pl

It is well known that 5-lipoxygenase derivates of arachidonic acid play an important pathogenic role during myocardial infarction. Therefore, the gene encoding arachidonate 5-lipoxygenase (ALOX5) appears to be an attractive target for RNA interference (RNAi) application. In experiments on cultivated cardiomyocytes with anoxia-reoxygenation (AR) and in vivo using rat model of heart ischemia-reperfusion (IR) we determined influence of ALOX5 silencing on myocardial cell death. ALOX5 silencing was quantified using real-time PCR, semi-quantitative PCR, and evaluation of LTC(4) concentration in cardiac tissue. A 4.7-fold decrease of ALOX5 expression (P < 0.05) was observed in isolated cardiomyocytes together with a reduced number of necrotic cardiomyocytes (P < 0.05), increased number live (P < 0.05) and unchanged number of apoptotic cells during AR of cardiomyocytes. Downregulation of ALOX5 expression in myocardial tissue by 19% (P < 0.05) resulted in a 3.8-fold reduction of infarct size in an open chest rat model of heart IR (P < 0.05). Thus, RNAi targeting of ALOX5 protects heart cells against IR injury both in culture and in vivo.

Despite fairly detailed investigation of 5-LO activity during IR and MI, changes in ALOX5 gene expression in heart tissue and cardiomyocytes under such conditions have not been studied.However, it is beyond any doubt that understanding of the genetic changes occurring during myocardial ischemia and ventricular remodeling is critical for developing therapies enhancing beneficial changes in gene expression and inhibiting potentially harmful ones.Gene therapy holds great potential for the ischemic heart disease treatment on many levels, including protection against IR injury, influence on cell death mechanisms, stimulation of angiogenesis, etc.Therefore, RNA interference (RNAi) technology, which is one of the most modern and specific methods of targeted gene silencing, holds much promise (Fire et 2009 O.O.Lisovyy and others al., 1998;Sledz & Williams, 2005;Sugano et al., 2005;Nordlie et al., 2006;Song et al., 2008).As far as we know, RNAi utilization for protection of heart cells against ischemic injury was described only in two published articles (Sugano et al., 2005;Song et al., 2008).Sugano et al. (2005) demonstrated that suppression of Src homology domain 2 (SH2)-containing tyrosine phosphatase-1 (SHP-1) with SHP-1-specific small interference RNAs (siRNA) markedly reduced infarct size in a rat in vivo model of heart IR (Sugano et al., 2005).In a second paper Song et al. (2008) have shown that siRNA's against protein tyrosine phosphatase 1B (PTP-1B) effectively protect cardiomyocytes against hypoxia-reoxygenation injury and inhibit apoptosis of cultivated cardiomyocytes (Song et al., 2008).Despite the effectiveness of cardioprotection achieved in the papers mentioned above it seems reasonable to underline that the choice of genes SHP-1 and РТР-1В was quite unexpected, the pathogenic role of the protein products encoded by those genes in heart IR being practically unstudied.
We have chosen in our study the ALOX5 gene encoding the enzyme 5-LO as the target for RNAi silencing considering the well-known role of 5-LO in pathogenesis of MI.We found that ALOX5 gene suppression in culture of isolated rat cardiomyocytes and in heart tissues in vivo provides a significant cyto-and cardioprotective effect reducing the ischemic injury range.
For gene silencing in vivo these siRNAs were injected in the tail vein of female Wistar rats (weight 300-340 g) in a dose of 80 µg twice with a 24-hour interval.
Anoxia-reoxygenation. After 24 h of cultivation cells transfected with scrambled or ALOX5specific siRNA were subjected to 30 min of anoxia followed by 60 min of reoxygenation.Anoxia was attained with an airtight jar from which O 2 was flushed with a gas mixture of 5% CO 2 and 95% Ar.Reoxygenation was realized by exchanging fresh medium and by its aeration with the standard gas mixture (Dosenko et al., 2005;2006).
Cell viability.The number of live, necrotic and apoptotic cells was determined by staining with 8.75 µM bisbenzimide (Hoechst 33342), propidium iodide and examination by fluorescence microscopy.Five-hundred cells were counted in each experiment (Tumanovs'ka et al., 2004).
Caspase-3 assay.After sonication, the remaining unlysed cells, membranes, and nuclei were removed by centrifugation at 5000 × g for 10 min.The supernatant was incubated in a buffer consisting of 100 mM Hepes (pH 7.4) and 5 mM dithiothreitol.The fluoropeptide substrates N-acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin (DEVD-AMC) and N-acetyl-Asp-Glu-Val-Asp-7-amido-4-trifluoro-methylcoumarin (DEVD-AFC) were used to measure the caspase-3 activity.After a 1-h incubation with 50 µM of these fluorogenic peptides, the fluorescence of the reaction products was monitored on a Hitachi 4000 spectrofluorimeter at 380 nm excitation and 460 nm emission (for DEVD-AMC) and 380/460 (for DEVD-AFC) using free 7-amino-4-methylcoumarin (AMC) as a standard.The reaction was carried out in the absence or presence of pan-caspases inhibitor -N-acetyl-Asp-Glu-Val-Asp-al (100 µM).Part of hydrolysis inhibition of the respective substrates by the inhibitor was taken as the caspase-3 activity and was expressed as nМ AMC per 10 6 cells/1 hour.
Ischemia-reperfusion in vivo.Four days after last injection of siRNA the rats underwent left anterior descending artery ligation for 1 h and following reperfusion for 3 h.Briefly after anesthesia with urethane (1 500 mg/kg) and intubation, animals were ventilated with a volume-cycled rodent respirator.Ligation of the left coronary artery was performed with a 7-0 monofilament polypropylene suture (SURGIPRO II, VPF-713-X, UK), 3-4 mm from the tip of the left auricle.Reperfusion was started ALOX5 gene silencing and myocardial infarction after 60 min of ischemia by cutting the knot of the suture.Myocardial ischemia confirmation was obtained by registration of ECG change -ST segment elevation relatively to base line that was 41.8 ± 5.1% in sham-operated animals and 181.8 ± 11.2% in ischemia-reperfusion group.Sham-operated animals underwent all surgical procedures besides ligation of coronary artery.The Standing Committee on Animal Research from our institution has approved the animal study protocol.
RNA isolation and reverse transcription.RNA isolation from cardiomyocytes and heart tissue was performed using Trizol RNA-prep kit (Isogen, Russia).RNA concentration was determined using a NanoDrop spectrophotometer ND1000 (NanoDrop Technologies Inc., USA).Reverse transcription (RT) was performed using RevertAid ™ H Minus First Strand cDNA Synthesis Kit (Fermentas), 1.2-1.5 µg of total RNA and random-hexamer primer.Singlestrand cDNA obtained was used for polymerase chain reaction (PCR) and real-time PCR.
Real-time PCR.We performed amplification in 10 µl of SYBR Green PCR Master Mix containing 30 pM of each primer.For amplification of ALOX5 and 18S rRNA gene (housekeeping gene) fragments we used the following primers: ALOX5-S-5'-GCC TAA GAA GCT CCC AGT GAC-3' and ALOX5-A-5'-ACT GGT GTG TAC AGG GGT CAG-3', 18S-S-5'-CTT AGA GGG ACA AGT GGC G-3' and 18S-A-5'-GGA CAT CTA AGG GCA TCA CA-3'.Sample volume was made to 20 µl with deionized water.Amplification was performed on a 7500 Fast Real-Time PCR System thermocycler.Amplification program consisted of initial AmpliTaq Gold® DNA polymerase activation step at 95°C for 10 min and following 50 cycles: denaturation (95°C for 15 s), annealing and elongation (64°C for 60 s).For control of specificity we analyzed the melting temperature of PCR product by sequential increase of temperature from 64°C to 99°C with registration of the drop in fluorescence strength of double-stranded DNA-SYBR Green complexes.We performed calculations using the 7500 Fast Real-time System SDS software provided.The Ct (cycle threshold) is defined as the number of cycles required for the fluorescence signal to exceed the detection threshold.We calculated the expression of the target gene relative to the housekeeping gene as the difference between the threshold values of the two genes.
5-LO activity measurement in heart tissues.In spite of the fact that the primary product of AA transformation by 5-LO is LTA 4 we evaluated 5-LO activity based on LTC 4 concentration as it is formed stoichiometrically from LTA 4 by LTC 4 -synthase.The amount of LTC 4 was determined in ethanolic extracts of heart tissue.The extracts were prepared by pulverizing pieces of heart tissue in liquid nitrogen.Collected ethanolic extracts were evaporated at 40°С in air bath and kept in a freezing chamber.LTC 4 determination was performed using radioimmunoassay technique and a standard assay kit (Amersham, USA).Sample radioactivity determination was performed on a liquid scintillation counter (Beckman Coulter Inc., USA).
Infarct size determination.Infarct size was determined as described (Ito et al., 1997;Wolff et al., 2000;Buehler et al., 2002;Martire et al., 2003) with two different methods -triphenyltetrazolium chloride (TTC) and propidium iodide staining.In our opinion propidium iodide staining is a more objective, accurate and up-to-date method for infarct size determination.The same conclusion was made by other researchers (Ito et al., 1997;Wolff et al., 2000).That is why we present only results of propidium iodide staining.Briefly, at the end of the 3-h reperfusion period, rats underwent propidium iodide intravenous injection (420 µl of 8.75 µM solution per one animal).After 20 min heart was removed and sliced perpendicularly to its long axis into four equal-thickness transverse slices.The slices were then photographed under a Nikon Eclipse E200 microscope.Tissue that showed bright red fluorescence of propidium iodide was presumed to be necrotic.Total necrotic area was digitized using computer-assisted planimetry software ImageJ 1.37 (National Institutes of Health, Bethesda, MD, USA).
Statistics.Statistical analysis was performed with Excel 2003, Оrigin 7.0 and 7500 Fast System SDS software.All data were expressed as the mean (S.E.) of replicate experiments performed in each assay.Statistical differences were evaluated using Stu-O.O.Lisovyy and others dent's t-test.P < 0.05 was taken to indicate a statistically significant difference.

ALOX5 gene expression in isolated cardiomyocytes at anoxia-reoxygenation and ALOX5 gene silencing
The level of ALOX5 mRNA in isolated rat cardiomyocytes deviates 7.5×10 -5 ± 7.5×10 -6 relative units in normal conditions and practically does not change after anoxia-reoxygenation (Fig. 1A).This shows that cardiomyocytes express ALOX5 and thus are likely to be a source of eicosanoids derived from AA via the 5-LO pathway.Introduction of ALOX5specific siRNA into a culture of cardiomyocytes caused a 4.7-fold downregulation of ALOX5 mRNA level (Р < 0.05) in comparison with scrambled siRNAtreated cells (Fig. 1B).

ALOX5 gene silencing and myocardial infarction
scrambled siRNA was reduced to 63 ± 1.9% (P < 0.05 compared to scrambled without AR), necrotic cells increased to 30 ± 2.6% (P < 0.05 compared to scrambled without AR) whereas the percentage of apoptotic cells was not changed significantly.The fractions of live and necrotic cells after AR of cultures with silencing of the ALOX5 gene were 76 ± 1.3% (P < 0.05 compared to scrambled group with AR) and 15 ± 1.3% (P < 0.05 compared to scrambled group with AR), while that of apoptotic cells was not changed (Fig. 2).The caspase activity was not changed in the culture with siRNA-induced ALOX5 silencing af-ter AR compared to the AR group without siRNA treatment (506.4 ± 10.26 nМ AMC per 10 6 cells/1 h vs 510.6 ± 12.06 nМ AMC per 10 6 cells/1 h, respectively, using DEVD-AMC, P > 0.05, 781.8 ± 13.2 nМ AMC per 10 6 cells/1 h vs 726.6 ± 28.2 nМ AMC per 10 6 cells/1 h, respectively, using DEVD-AFC, P > 0.05).Thus, RNA interference of the ALOX5 gene diminishes necrotic and does not affect apoptotic cell death at AR in cardiomyocyte culture.

Influence of ALOX5-specific siRNA on gene expression and 5-LO activity in heart tissues at ischemiareperfusion in vivo
Similarly as in cultured cardiomyocytes, expression of the ALOX5 gene in heart tissue samples obtained from control (sham-operated) animals and animals subjected to heart ischemia-reperfusion in vivo was not significantly different (Fig. 3).In scrambled siRNA-treated animals LTC 4 level was increased 1.75-fold after ischemia-reperfusion compared to sham-operated animals (Fig. 3).ALOX5-specific siR-NA application caused a 1.2-fold downregulation of the ALOX5 gene after ischemia-reperfusion comparing to scrambled siRNA-treated animals (P < 0.05) (Fig. 3).At the same time the LTC 4 level was decreased 3.3-fold (P < 0.05) (Fig. 3).

Myocardial infarct size after heart ischemia-reperfusion and ALOX5 gene silencing
Figure 4 presents pictures obtained by fluorescence microscopy of heart slices from one representative experiment.These pictures demonstrate a significant decrease of the necrotic area with red fluorescence in slices from myocardium of the rats with ALOX5 gene silencing after ischemia-reperfusion. Statistical analysis of six independent experiments revealed that after IR the total necrotic area of myocardium was 5.8-fold increased comparing to sham-operated rats (Р < 0.05).At the same time, the infarct area size in animals with suppressed ALOX5 gene was decreased 3.8-fold comparing to scrambled siRNA-treated animals (Р < 0.05) (Fig. 3).

DISCUSSION
Our data shows that ALOX5 gene silencing decreases the frequency of necrotic cell death after anoxia-reoxygenation of cultured cardiomyocytes as well as reduces heart infarct area size at ischemia-reperfusion in vivo.There is evidence that lipid hydroperoxidation mediated by 5-LO may lead to key features of necrosis development -disruption of organelle and plasma membranes (Kuhn et al., 1990;van Leyen et al., 1998;Spiteller, 2003;  O.O.Lisovyy and others Festjens et al., 2006).It is also well known that leukotrienes play an important role in coronary vessel constriction and PMN leucocyte recruitment, and exert an arrythmogenic effect (Feuerstein, 1986).Therefore, down-regulation of ALOX5 with the ensuring decrease of 5-LO protein formation reduces the possibility of realization of the above scenario.An involvement of lipoxygenases in TNFα-induced apoptosis and other apoptotic pathways has been reported several times (Maccarrone et al., 2001;Spiteller, 2003).However, according to our data, ALOX5 gene silencing does not affect the apoptotic death of cardiomyocytes.This indirectly points to an insignificant role of 5-LO and 5-LO-derived eicosanoids in the triggering of the apoptosis pathway at anoxia-reoxygenation of cardiomyocytes.Another possible mechanism which can explain the involvement of lipoxygenases in cardiomyocyte functioning and their resistance to ischemia involves activation of leukotriene receptors (CysLt1 and CysLt2) (Liu et al., 2003).CysLT mRNA was found recently in heart tissues using various methods, including in situ hybridization, Northern blotting and RT-PCR (Lynch et al., 1999;Heise et al., 2000;Kamohara et al., 2001;Ogasawara et al., 2002).Accordingly, leukotrienes can exert autocrine action on cardiomyocytes by interaction with specific receptors.Liu et al. (2003) demonstrated that cysteinyl leukotrienes (CysLT) mediate an angiotensin II-evoked increase in cytosolic free Ca 2+ concentration in neonatal rat cardiomyocytes which can be prevented by the 5-LO inhibitor AA-861 and the CysLT1-selective antagonist MK-571 (Liu et al., 2003).This data allows one to assume that the protective effect of ALOX5 silencing is related to reduced Ca 2+ overloading of cardiomyocytes occurring at anoxia-reoxygenation. Ca 2+ -dependent injury of cardiomyocytes, in particular with induction of necrosis, has already been described by several authors (Zimmermann et al., 2001;Nakayama et al., 2003).
In our opinion, RNA interference holds great potential in therapy of cardiovascular disorders, in particular MI.The advantages of this method involve the reversible nature of gene suppression and possibility of silencing multiple genes simultaneously.According to the latest data expression of genes encoding micro RNAs (mainly miR21, miR214, miR379 and miR146b) that regulate gene expression at the post-transcriptional level by either degradation or translational repression of a target mRNA, changes during experimental MI (Roy et al., 2009).In this aspect determination of the most effective strategy for MI gene therapy is not straight forward.It is essential to clarify which approach will be the most effective -suppression of key regulatory genes such as the genes encoding the micro RNAs or silencing of effector genes, for instance ALOX5.It is conceivable that RNA interference targeting genes encoding

Figure 3 .
Figure 3. ALOX5 mRNA, LTC 4 levels and infarct size in the heart after various treatments.Treatments: a, sham-operated; b, IR; c, IR after scrRNA administration; d, IR after ALOX5-specific siRNA administration.Results are expressed as means (±S.E.M.; bars), n = 10.* P < 0.05 compared to sham-operated, # P < 0.05 compared to scrRNA administration.Animals were subjected to different treatments, heart was removed and ALOX5 mRNA level was determined by reverse transcription PCR, that of LTC 4 by radioimmunoassay and infarct area by fluorescence microscopy of heart slices.

Figure 4 .
Figure 4. Myocardial necrosis after ischemia-reperfusion. Animals were subjected to IR after scrRNA (A) or ALOX5-specific siRNA (B) treatment.After propidium iodide injection hearts were removed, transverse slices were viewed under a fluorescence microscope."because It represents what were done in our experiments.