Silencing heat shock factor 1 by small interfering RNA abrogates heat shock-induced cardioprotection against ischemia–reperfusion injury in mice

Journal of Molecular and Cellular Cardiology 39 (2005) 681–689 www.elsevier.com/locate/yjmcc

Original article

Silencing heat shock factor 1 by small interfering RNA abrogates heat shock-induced cardioprotection against ischemia–reperfusion injury in mice Chang Yin, Lei Xi 1, Xiaoyin Wang 1, Mareen Eapen, Rakesh C. Kukreja * Division of Cardiology, Department of Internal Medicine, Virginia Commonwealth University Medical Center, Richmond, VA 23298-0281, USA Received 17 May 2005; received in revised form 10 June 2005; accepted 13 June 2005 Available online 24 August 2005

Abstract Induction of heat shock factor 1 (HSF1) is known to associate with cellular response to divergent pathophysiological stresses including whole body hyperthermia (WBH) and ischemia-reperfusion. However, a direct cause-effect relationship between HSF1 activation and cytoprotection induced by myocardial preconditioning has not been conclusively established, mainly due to the limitations of available experiment tools. In the present studies, we used a novel approach to block HSF1 with small interfering RNA (siRNA) technique in vivo. Male adult ICR mice were treated intraperitoneally with amine (vehicle) or siRNA specific to HSF1 (siRNA-HSF1). Three days later, WBH preconditioning protocol (rectal temperature 42 °C for 15 min) was applied to these mice under light anesthesia. WBH preconditioning resulted in 2.7-fold and 3.4-fold increase in cardiac HSF1 mRNA and protein expression respectively 2 hours after WBH, which was inhibited in the siRNA-treated mice. The silencing effect of siRNA on HSF1 was associated with complete loss of the infarct- limiting protection by WBH preconditioning after 48 hours. Pretreatment with siRNA-HSF1 had no effect on infarct size in the sham control animals as compared with the amine-treated group. DNA micro-array analysis revealed that siRNA-HSF1 caused a general inhibition on multiple members of HSP family, except Hsp32, Hsp47 and Hsp60. In addition, the silencing effect of siRNA on HSF1 and HSPs gene expression was transient and its inhibitory effect disappeared by 10 days after treatment. siRNA-HSF1 also impaired the thermotolerance of the heat shocked mice as indicated by higher mortality following WBH. For the first time, we have applied siRNA technique in the field of myocardial preconditioning to demonstrate HSF1 activation as an essential step in WBH preconditioning against cardiac ischemia-reperfusion injury. © 2005 Elsevier Ltd. All rights reserved. Keywords: Ischemia–reperfusion; Infarct size; Heat stress; Heat shock proteins; siRNA; Gene micro-array

1. Introduction Heat shock proteins (HSPs) are a group of protective proteins that can be induced by many stressful stimuli, such as hyperthermia, exercise, ischemia, hypoxia and inflammation [1,2]. Adaptation to stress leads to the elevated expression of heat shock genes such that molecular chaperones are rapidly synthesized and deployed to prevent protein misfolding and to assist in their refolding to the native state [3]. The protec* Corresponding author. Rakesh C. Kukreja, PhD, Professor of Medicine, Physiology, Biochemistry and Emergency Medicine, Division of Cardiology, Box 980281, Virginia Commonwealth University Medical Center, Richmond, Virginia 23298, USA, Tel.: 804-828-0389 fax: 804-828-8700. E-mail address: [email protected] (R.C. Kukreja). 1 Authors contributed equally to this study. 0022-2828/$ - see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2005.06.005

tive nature of HSPs has been documented mainly by the observation that a mild heat shock (42 °C) confers resistance to the cell against a subsequent lethal heat shock (45 °C) [4]. This phenomenon usually referred to as thermotolerance is a transient resistance to the cytotoxic effects of a subsequent lethal hyperthermic treatment which is induced by a non-lethal heat treatment. The synthesis and degradation of heat-shock proteins directly correlates with the development and decay of thermotolerance [5]. This fact has been taken as evidence that these proteins are involved in the acquisition, maintenance, and decay of thermotolerance. The increased expression of HSPs, which occurs within minutes after exposure to noxious stimuli, is accomplished through mechanisms that involve both transcriptional activation and preferential translation of heat shock transcription factor 1 (HSF1) [6,7]. HSF1 is present in the cytoplasm in an

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inactive, monomeric form. However, under stressful conditions, trimerization as well as phosphorylation occur and HSF1 migrates to the nucleus, where it binds to triplet repeats of a nucleotide recognition motif (nGAAn) within promoter/enhancer regions of downstream target genes that encode the classical HSPs, and other proteins as well [8,9]. It has been observed that enhanced HSF1 DNA binding and associated increased HSPs are closely related with cardioprotection observed in the poly(ADP-ribose) polymerase1 knockout mice following ischemia/reperfusion injury [10]. HSF1 is efficiently activated with reduced oxygen species in ischemic-reperfused heart [11,12] and has been suggested to have theoretical and experimental basis for WBH preconditioning to protect the heart against ischemia-reperfusion injury [13–15]. Three other heat shock transcription factor isoforms (HSF2–4), products of distinct genes [1] are present in mammalian cells but are not sufficient to preserve the heat shock response in the absence of HSF1 [13]. Both HSF and HSP partners have been recognized as potential targets in therapeutic strategies designed to stimulate endogenous protective mechanisms against deleterious consequences of oxidative stress [14]. Disruption of the HSF1 gene in mice eliminates the heat shock response, abolished thermotolerance in vitro, and increased susceptibility of HSF1-deficient cells to heat-induced apoptosis [13]. While these studies suggest an important role of HSF1 in cytoprotection, a direct cause and effect relation between HSF1 induction and subsequent cardioprotection is not estab-

lished conclusively in part due to the methodological limitations. In the present study, we applied a novel approach with siRNA for blocking HSF1 in vivo in order to determine its effect on WBH-induced cardioprotection in mice [15,16].

2. Materials and Methods 2.1. Animals Adult male outbred ICR mice were purchased from Harlan (Indianapolis, IN). All animal experiments were conducted under the guidelines on humane use and care of laboratory animals for biomedical research published by NIH (No. 85-23, revised 1996). 2.2. Synthesis of siRNA-HSF1 Four pairs of small interfering RNA of HSF1 (siRNAHSF1) were synthesized and purified using SilencerTM siRNA Construction Kit (Ambion Inc., Austin, TX). As shown in Fig. 1, the siRNA-HSF1 were 21-bp long, double-stranded and corresponding to the positions at 353-373, 398-418, 554574 and 726-746 of HSF1 cDNA sequence (Accession Number: XM_128055). The specificity of siRNA-HSF1 was confirmed by BLAST program. siRNA-HSF1 was incubated in amine solution at 25oC for 30 minutes to form siRNAHSF1 + amine complexes for an effective intracellular entry.

Fig. 1. Preparation and validation of siRNA targeted on HSF1. Four pairs of small interfering RNA of HSF1 were synthesized and purified using SilencerTM siRNA Construction Kit (Ambion Inc., Austin, TX), which are shown in the circled positions (353-373, 398-418, 554-574 and 726-746 of HSF1 cDNA sequence according to accession# XM_128055).

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The siRNA-HSF1 + amine complexes (1.2 µg per g body weight) was then injected intraperitoneally into the mice, whereas volume-matched amine-saline mixture treated animals served as the Amine control group. 2.3. Whole body hyperthermia pretreatment Three days after receiving the siRNA or amine injection, the mice were subjected to whole body hyperthermia (WBH) under light anesthesia (sodium pentobarbital, 50 mg/kg, i.p.), which was accomplished by raising the rectal temperatures to 42 °C for 15 min [16]. At the end of WBH the mice were allowed to recover under room temperature for 1, 2, 24, and 48 hours respectively, before harvesting the heart for further biochemical or physiological experiments. Mice in the Sham control groups received the light anesthesia but their body temperature was not raised. In order to further examine the transient nature of the silencing effect of siRNA on HSF1, an additional subset of mice were subjected to WBH 10 days (in stead of 3 days) after the siRNA-HSF1 or amine injection. 2.4. Model of ischemia-reperfusion injury in Langendorff isolated mouse heart Forty eight hrs after WBH, the hearts were isolated and subjected to 20 min of normothermic global ischemia and 30 min of reperfusion in a Langendorff isolated, KrebsHenseleit buffer-perfused mouse heart model as described in our previous publications [16,17]. Ventricular contractile force was continuously measured with a force-displacement transducer (Grass, Model FT03) attached to the apex of heart and was recorded and analyzed with a computerized data acquisition/analysis system (AD Instruments, Model PowerLab 8SP). The heart temperature was precisely maintained at 37 °C throughout the experiment. Coronary flow rate was measured by timed collection of the outflow coronary perfusate. At the end of reperfusion, the heart was immediately removed from the perfusion apparatus, weighed, and stored under -20 °C overnight. The frozen heart was cut into 6 to 7 transverse sections (~1 mm thickness) across the long axis of heart and stained with 10% triphenyl tetrazolium chloride (TTC, Sigma-Aldrich) in phosphate buffer (pH 7.4) for 30 min under room temperature, and then fixed in 10% formaldehyde solution for at least 2 hours before the measurement with a computer morphometry system (Bioquant 98). The size of myocardial infarction (appeared in pale color) was quantified and calculated as % of risk area, which equals total area minus cavities.

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reverse 5’-GAGATCAGGAACTGAATGAGC-3’ [18]; Hsp72: forward 5’-AAGGTGGAGATCATCGCCAA-3’ and reverse 5’-GCGATCTCCTTCATCTTGGT-3’ [18]; GAPDH: forward 5’-GTCATTGAGAGCAATGC-CAG-3’ and reverse 5’-GTGTTCCTACCCCCAATGTG-3’. The RT-PCR products were electrophoresed on 1.5% Tris-Acetate-EDTA agarose gel. The target bands were identified based on their specific size using DNA standards. 2.6. Western immunoblotting Using previously described methods [19], heart samples were homogenized in ice-cold RIPA buffer (Upstate Biotechnology) and the homogenate was centrifuged at 10,000 g for 10 min under 4 °C, and supernatant was recovered as the total cellular protein. 70 µg total protein from each sample was separated by SDS/PAGE on 10% acrylamide gels and transferred to a nitrocellulose membrane, and then blocked with 5% non-fat dry milk in TBS. The membrane was subsequently incubated with the primary antibodies specific to HSF1 and Hsp70 (sc-9144 and sc-1060, Santa Cruz Biotechnology, Santa Cruz, CA) and subsequently the secondary antibody. The membranes were developed using enhanced chemiluminescence and exposed to x-ray film. For quantifying the mRNA or protein expression, the optical density for each RT-PCR or Western blot band was scanned and analyzed with a densitometric system (Molecular Dynamics 4.0). 2.7. DNA micro-array analysis Micro-array analysis of mRNA expression was performed at the Affymetrix facility in Virginia Commonwealth University. The RNA extracted from different mouse heart tissue samples was reverse transcribed, labeled with fluorescence and hybridized to Affymetrix murine genome array U74Av2 (Affymetrix, Santa Clara, CA). The hybridization signal was detected and analyzed at the Affymetrix facility. Changes between samples were determined by pairwise comparisons using Microsoft Excel software as recommended by Affymetrix. Each gene in the Affymetrix array was represented by perfectly matched (PM) and mismatched (MM) control oligonucleotides. Fluorescence intensity was read for each oligonucleotide to calculate the average signal intensity for each gene by subtracting the intensities of the PM from the intensity of the MM control, after discarding the maximum, the minimum and any outliers beyond three standard deviations. The calculation was performed using an Affymetrix algorithm.

2.5. RT-PCR 2.8. Data analysis and statistics RNA was extracted from the ventricular tissue using TRI Reagent (Molecular Research Center, Inc., Cincinnati, Ohio). RT-PCR was performed using OneStep RT-PCR Kit (Qiagen, Valencia, California). The primers used were as follows: HSF1: forward 5’-AAGTACTTCAAGCACAACAA-3’ and

The quantitative data (i.e. myocardial infarct size, ventricular contractile function, optical density of mRNA and protein expression) are presented as Mean ± SEM. The assessment of difference among the groups was performed using one-

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way ANOVA followed by Student-Newman-Keuls post-hoc test for pair-wise comparison. Normality Test and Equal Variance Test were used for comparing the gene array results between the siRNA-HSF1 treated group and amine-treated controls. P < 0.05 was considered as statistically significant.

3. Results 3.1. siRNA-HSF1 abolishes the cardioprotection afforded by WBH preconditioning Similar to our previous study [16], WBH preconditioning induced a delayed infarct-limiting cardioprotection 48 hours after WBH (Fig. 2 A). The averaged infarct size was reduced from 31.8 ± 3.2% in Amine + Sham group to 14.5 ± 4.0% in Amine + WBH group (Mean ± SEM, P < 0.05, n = 6 per group; Fig. 2 A). Pretreatment with siRNA-HSF1 completely blocked the WBH-induced cardioprotection (i.e. infarct size: 39.0 ± 2.5% in siRNA-HSF1 + WBH group; P < 0.05 versus Amine + WBH group, Fig. 2 A). The siRNA-HSF1 treatment did not significantly alter infarct size in the sham con-

trol animals (i.e. 29.9 ± 4.5% in siRNA-HSF1 + Sham group), as compared to Amine + Sham group (31.8 ± 3.2%, P > 0.05). In addition, ventricular contractile function, presented by rateforce product (i.e. the double product of heart rate and developed force) was not significantly different among the four experimental groups (Fig. 2B, P > 0.05). Also the preischemic basal values of coronary flow rate (ml/min) were not significantly different for the four groups (ranging from 1.65 ± 0.29 in siRNA-HSF1 + WBH group to 2.43 ± 0.45 in Amine + WBH group). To the contrary, the siRNAHSF1 treatment led to a significantly lower post-ischemic coronary flow in the heat-shocked mice (1.12 ± 0.18, P < 0.05 versus 2.63 ± 0.22 in Amine + WBH group or 2.32 ± 0.50 in siRNA-HSF1 + Sham group). 3.2. Effect of siRNA-HSF1 on HSF1 and Hsp70 mRNA expression WBH preconditioning resulted in a 2.7-fold increase in cardiac HSF1 mRNA in the amine-treated animals as compared with the sham control group (P < 0.05; Fig. 3 A and 3D). Similarly, a 3.4-fold increase in Hsp70 mRNA was observed (Fig. 3B and 3E). The WBH-induced enhancement of HSF1 and Hsp70 mRNA expression was effectively diminished by siRNA-HSF1 treatment (Fig. 3). 3.3. Effect of siRNA-HSF1 on HSF1 and Hsp70 protein expression Similar to the trend of mRNA, the mice responded to WBH with 3.4- and 3.9-fold increase in myocardial HSF1 and Hsp70 protein expression respectively, as compared to the corresponding amine-treated group (P < 0.05; Fig. 4). The upregulation of HSF1 and Hsp70 protein content was abolished by the treatment with siRNA-HSF1 (Fig. 4). 3.4. Effect of siRNA-HSF1 on gene expression of HSPs Our results of DNA micro-array analysis revealed that siRNA-HSF1 resulted in inhibition on the enhanced gene expression of HSP family members in response to WBH (Fig. 5 A). The quantitative analysis was achieved by calculating the percentage of inhibition of siRNA-HSF1 treated hearts on the fluorescence intensity of Affymetrix murine genome array against the values obtained from the aminetreated hearts (n = 3 per group). 3.5. Transient nature of the inhibitory effect of siRNA

Fig. 2. Effect of siRNA-HSF1 on infarct-limiting effect of hyperthermic preconditioning. Bar graphs show the mean values for myocardial infarct size (A) and post-ischemic ventricular contractile function (B) for all four experimental groups (mean ± SEM, n =6 per group). The ischemia-reperfusion studies were performed 48 hours after WBH.

When mice were subjected to WBH ten days after siRNAHSF1 treatment, there was no significant difference between siRNA- or amine-treated group in either HSPs gene expression (Fig. 5B) or mRNA/protein expression for both HSF1 and Hsp70 (Fig. 6), indicating a complete loss of the gene silencing effect of siRNA. The hearts regained the normal heat shock response that was similar to those in the Amine + WBH group.

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Fig. 3. Silencing effect of siRNA-HSF1 on WBH-induced gene transcription and expression of HSF1 and Hsp70 in heart. Representative pictures of RT-PCR experiments showing expression of mRNA for HSF1 (A), Hsp70 (B) and GAPDH (C). Lane 1: Amine + WBH; Lane 2: siRNA-HSF1 + WBH; Lane 3: Amine + Sham; Lane 4: siRNA-HSF1 + Sham. Densitometric quantification of RT-PCR signals is showed in Fig. 3D for HSF1 mRNA and in Fig. 3E for Hsp70 mRNA. All data were normalized with the values obtained from the Amine + Sham group (Mean ± SEM, n=3 per group). * indicates significant difference compared with all other groups (P < 0.05). The hearts were collected after 1 hour (for measurement of HSF1 mRNA) and 2 hours (for measurement of HSP70 mRNA) after WBH.

These results suggest a transient gene silencing effects afforded by siRNA-HSF1, which are distinctive from the permanent HSF1 gene knockout techniques [13,20,21]. 3.6. Impairment of thermotolerance caused by siRNA-HSF1 As shown in Fig. 7, injection of siRNA-HSF1 did not increase mortality as compared with the amine-treated mice suggesting that the siRNA preparation did not produce severe systemic toxicity in the sham control mice. On the other hand, 3 days after the pretreatment, a remarkably higher death rate was observed during or immediately after the WBH protocol

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Fig. 4. Silencing effect of siRNA-HSF1 on WBH-induced HSF1 and Hsp70 protein expression. Representative pictures of Western blots in detecting protein expression of HSF1 (A), Hsp70 (B) and GAPDH (C). Lane 1: Amine + WBH; Lane 2: siRNA-HSF1 + WBH; Lane 3: Amine + Sham; Lane 4: siRNA-HSF1 + Sham. Densitometric quantification of the Western blot bands is showed in Fig. 4D for HSF1 protein content and in Fig. 4 E for Hsp70 protein content. All data were normalized with the values obtained from the Amine + Sham group (Mean ± SEM, n = 3 per group). * indicates significant difference compared with all other groups (P < 0.05). The hearts were collected after 2 hours (for measuring HSF1 protein) and 24 hours (for measuring Hsp70 protein) after WBH.

in the siRNA-HSF1 treated mice as compared with the aminetreated group (Fig. 7). 4. Discussion The primary goal of the present study was to demonstrate the direct cause and effect relationship between activation of HSF1 and WBH-induced enhancement of myocardial ischemic tolerance with a novel approach of using interfering

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Fig. 5. Gene microarray analysis showing inhibition of HSPs by siRNA-HSF1. The bar graphs demonstrate the inhibition on gene expression of various HSPs 30 min after WBH preconditioning in the mice 3 days (Graph A) or 10 days (Graph B) after siRNA-HSF1 treatment. The averaged data (n = 3 per group) are presented as the percentage of decrease relative to the amine-treated control animals which are listed sequentially from the lowest inhibition of Hsp84 to the highest inhibition of Hsp70 (Graph A). On the other hand, Hsp60, Hsp47 and other members of HSP family such as Hsp32 (not shown) were neither inhibited nor activated by siRNA-HSF1. The inhibition of HSPs by siRNA-HSF1 was completely lost 10 days after the siRNA treatment (Graph B).

RNA technology. Our results show that silencing of HSF1 with siRNA completely abolished the WBH-induced protective effects against myocardial infarction following global ischemia-reperfusion (Fig. 2). The inhibitory effects of in vivo administration of siRNA-HSF1 on WBH-induced enhancement of cardiac mRNA and protein level of HSF1 and Hsp70 were also evident (Fig. 3 and Fig. 4). DNA microarray showed that the inhibitory effects of siRNA-HSF1 were wide-spread across the HSP family, from the smallest member Hsp10 to the largest Hsp105 (Fig. 5 A). Furthermore, we identified the transient nature of the silencing effect of siRNA on WBH-induced HSPs gene expression (Fig. 5B) as well as HSF1 and Hsp70 mRNA and protein expression (Fig. 6), which completely disappeared by 10 days after the siRNA injection. Additional results also indicated siRNAHSF1 caused impairment in thermotolerance (Fig. 7). To our knowledge, this is the first report showing systemic delivery of siRNA in vivo that has selectively targeted one of the key signaling components of the myocardial preconditioning pathways. It has been known for more than a decade that HSF1 is the principal transcription factor that regulates the heat shock response in all eukaryotes including mammalian cardiomyocytes [1,3,14,22]. It was reported that the homozygous HSF1 knockout (HSF1-KO) mice had characteristics that included a lack of the “classic” heat shock response to ther-

mal stress, and an exaggerated cytokine production and mortality following endotoxin challenge [13]. In addition, despite HSF1-KO mice could survive into adulthood, they also showed multiple developmental defects and female infertility [13,20,21], suggesting that HSF1 has a broader role in the developmental pathways beyond its well-known role in stress response. On the other hand, a recent study by Zou et al. [23] has provided strong evidence demonstrating that overexpression of HSF1 protected the transgenic mouse heart against apoptosis and necrosis following ischemia-reperfusion in vivo. Using a more physiological approach of temporary suppression of HSF1 activation with siRNA, the current study provides evidence supporting an essential role of HSF1 activation inWBH-induced cardioprotection. The identification of exact member(s) of HSP family that mediate the WBH-induced cardioprotection remains elusive [15]. Although Hsp70 was previously proposed by Hutter et al. [24] as the primary mediator of ischemic protection, a close link between the induction of Hsp70 and WBH-induced cardioprotection has not been confirmed by several other studies [16,25–27]. In the present study, the DNA micro-array results showed that silencing HSF1 with siRNA led to a broad inhibition of HSP genes, which included Hsp10, Hsp25, Hsp40, HSC70, Hsp70, Hsp84, Hsp86 and Hsp105 (Fig. 5 A). These data are compatible with the concept that HSF1 regulates multiple HSP genes [14]. Further studies are needed to

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Fig. 6. Transient inhibitory effect of siRNA-HSF1 on mRNA and protein expression of HSF1 and Hsp70. The inhibitory effect of siRNA-HSF1 on whole body hyperthermia-elicited induction of HSF1 mRNA (A) or protein (C) and Hsp70 mRNA (B) or protein (D) was completely lost when the heat shock was given 10 days after the intraperitoneal injection of siRNA-HSF1 (group siHSF1) in the mice.

examine the role of each of the HSP family members that are potentially responsible for the WBH-induced protective effects. We injected the siRNA-HSF1 intraperitoneally rather than direct injection into the left ventricle in order to eliminate the confounding effect of injuries involved with thoracic surgery, which would have obscured the interpretation of the silencing effect of siRNA. A few recent studies have also shown that intraperitoneal or intravenous injection can be used as an efficient means to deliver siRNA into target organs [28–30]. In the present study we did not observe any appreciable adverse effect associated with systemic administration of siRNA-HSF1 in the non-heat shocked control mice. How-

Fig. 7. Impairment of thermotolerance with siRNA-HSF1. The mortality data indicate that in vivo administration of siRNA-HSF1 did not cause any increase of mortality in the non-heat shocked sham control animals, whereas higher mortality rate was observed in the mice treated with siRNA-HSF1 (group siHSF1) as compared with amine-treated control mice.

ever the siRNA-HSF1 treatment resulted in intolerance to heat stress in the animals as indicated by approximately 50% mortality in mice treated with siRNA-HSF1 during or immediately after the WBH preconditioning procedure (Fig. 7). The surviving mice following WBH also appeared to be less resistant to ischemia-reperfusion injury when further subjected to ischemia-reperfusion 48 hours after WBH (Fig. 2C). These results are consistent with the notion that HSF1 is an important transcription factor controlling the cellular tolerance to various pathological stressors including hyperthermia [4,5] and ischemia-reperfusion [10,23]. There are several advantages of this new approach. For example, it is a normal endogenous RNA metabolic pathway that naturally exists in cells [31–33], unlike the “invading” pharmaceutical blockers or transgenic interventions. The siRNA technique uses a small interfering RNA (21-bp), which is short enough to not induce the host immune-response, but long enough to give a specific degradation of target mRNA [34,35]. Second, the silencing effect of siRNA appears transient and reversible (Mike Byrom, Technotes 11(3), Ambion, Inc.), which allowed these studies to be conducted under more physiological conditions than the HSF1 knockout mice where certain adaptive changes seem to be unavoidable. Similarly, the pharmacological inhibitors of HSF1 such as quercetin, lack specificity and known to have other effects such as scavenging of ROS [36–38] as well as block signaling pathways through inhibiting protein kinases and tyrosine kinases, independent of HSF1 inhibition. Furthermore, studies using antisense may involve the use of a long stretch of dsRNA to block

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a specific target, but the long dsRNA could elicit host immunoreactions that is cytotoxic [39–41]. Therefore, the use of siRNA overcomes the methodological limitations of the previous studies. In the present study, the effect of siRNA on gene expression of HSPs was performed in tissue extracts, which does not provide precise information on the cell-sepecific knockdown of HSPs in the heart. Future studies using immunohistochemical techniques and microscopic imaging would be necessary to demonstrate the gene silencing effect in various population of cells in the myocardium following systemic administration of siRNA. In conclusion, the current study using siRNA technique has provided evidence for an indispensable involvement of HSF1 in the WBH-induced protection against myocardial ischemia-reperfusion injury. This study has also validated a novel and relatively noninvasive approach to deliver siRNA into myocardium via intraperitoneal injection, which may be utilized as a new tool for future investigations on the function of a specific gene(s) or protein(s) in the heart in vivo.

Acknowledgements This study was supported by grants from National Institutes of Health (HL51045, HL59469, and HL79424 to R. C. K.). C. Y. was a postdoctoral trainee on the NIH T32 training grant (HL07537).

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