S-diclofenac Protects against Doxorubicin-Induced Cardiomyopathy in Mice via Ameliorating Cardiac Gap Junction Remodeling

Hydrogen sulfide (H2S), as a novel gaseous mediator, plays important roles in mammalian cardiovascular tissues. In the present study, we investigated the cardioprotective effect of S-diclofenac (2-[(2,6-dichlorophenyl)amino] benzeneacetic acid 4-(3H-1,2,dithiol-3-thione-5-yl)phenyl ester), a novel H2S-releasing derivative of diclofenac, in a murine model of doxorubicin-induced cardiomyopathy. After a single dose injection of doxorubicin (15 mg/kg, i.p.), male C57BL/6J mice were given daily treatment of S-diclofenac (25 and 50 µmol/kg, i.p.), diclofenac (25 and 50 µmol/kg, i.p.), NaHS (50 µmol/kg, i.p.), or same volume of vehicle. The cardioprotective effect of S-diclofenac was observed after 14 days. It showed that S-diclofenac, but not diclofenac, dose-dependently inhibited the doxorubicin-induced downregulation of cardiac gap junction proteins (connexin 43 and connexin 45) and thus reversed the remodeling of gap junctions in hearts. It also dose-dependently suppressed doxorubicin-induced activation of JNK in hearts. Furthermore, S-diclofenac produced a dose-dependent anti-inflammatory and anti-oxidative effect in this model. As a result, S-diclofenac significantly attenuated doxorubicin-related cardiac injury and cardiac dysfunction, and improved the survival rate of mice with doxorubicin-induced cardiomyopathy. These effects of S-diclofenac were mimicked in large part by NaHS. Therefore, we propose that H2S released from S-diclofenac in vivo contributes to the protective effect in doxorubicin-induced cardiomyopathy. These data also provide evidence for a critical role of H2S in the pathogenesis of doxorubicin-induced cardiomyopathy.


Introduction
Doxorubicin is an anthracycline antibiotic widely used in the treatment of a variety of human neoplastic diseases and solid cancers, but its clinical application is hampered by progressive and does-related cardiotoxicity including transient electrocardiographic abnormalities, cardiomyopathy and heart failure. The efficacy of doxorubicin as a cytotoxic agent for the treatment of human tumors has prompted a search to develop new agents to reduce its cardiotoxicity [1][2][3][4][5].
Hydrogen sulfide, as a novel gaseous mediator, is generated endogenously during cysteine metabolism in many types of mammalian cells in the reaction catalysed by cystathionine bsynthase (CBS) and cystathionine c-lyase (CSE) [6]. Although CBS and CSE are widely distributed in tissues, CBS is the main H 2 Sforming enzyme in central nervous system while CSE is the major H 2 S-producing enzyme in cardiovascular system. It has become clear that H 2 S fulfills a wide range of physiological and pathological functions in cardiovascular system [6]. Recent study has shown that deficiencies of H 2 S synthesis may contribute to the pathogenesis of doxorubicin-induced cardiomypathy and that administration of NaHS, an H 2 S donor ameliorated doxorubicin-related cardiac dysfunction via inhibiting oxidative stress injury [7]. Therefore, application of H 2 S donors may be a promising therapeutic strategy to prevent doxorubicin cardiotoxicity. However, NaHS is a short-lasting H 2 S donor, and it releases H 2 S quickly, which may cause an acute alteration in hemodynamics. As a result, further studies are needed to develop novel and potent compounds that are capable of producing H 2 S slowly and stably.
S-diclofenac (2-[(2,6-dichlorophenyl) amino] benzene acetic acid 4-(3H-1,2,dithiol-3-thione-5-yl) phenyl ester) is a novel molecule comprising a H 2 S-releasing dithiol-thione moiety attached by an ester linkage to diclofenac. Unlike other H 2 S donors, S-diclofenac releases H 2 S slowly both in vitro and in vivo over a period of several hours [8,9]. Furthermore, S-diclofenac has comparable or even improved anti-inflammatory property while its gastrointestinal-damaging effect is considerably reduced as compared with the parent drug [8,9]. These findings suggest that S-diclofenac may be an attractive alternative to the available H 2 S donors. On the other hand, one recent study has shown that Sdiclofenac could protect against ischemia-reperfusion injury in the isolated rabbit heart [10]. The experiment, which indicates the beneficial effect of S-diclofenac in ischemic heart diseases, prompted us to investigate the possible protective effect of S-diclofenac against non-ischemic origin. We hypothesized that Sdiclofenac might be able to block the progression of cardiomyopathy induced by doxorubicin, known for its late-onset acute and chronic cardiotoxicity. To test that idea, in the present study, we examined the cardioprotective effect of S-diclofenac in a murine model of doxorubicin-induced cardiomyopathy and investigated the specific mechanisms of its effect. Our data suggests that Sdiclofenac may attenuate the cardiotoxicity of doxorubicin by reversing cardiac gap junction remodeling along with inhibiting oxidative injury and inflammation in hearts.

Deficiencies of H 2 S formation in doxorubicin-induced cardiomyopathy
The model of dilated cardiomyopathy was induced in male 10week-old C57BL/6J mice by a single intraperitoneal injection of doxorubicin at a dose of 15 mg/kg. The success of the model was confirmed by decreased LV function and enlarged left ventricle of the mice (by echocardiography). At necropsy, those mice exhibited ascites and lobulated enlarged livers, indicative of liver congestion, or foamy lungs indicative of lung edema, suggesting that they undergo congestive heart failure.
Administration of doxorubicin resulted in a significant downregulation of CSE expression in heart. The mRNA and protein levels of CSE were significantly decreased in hearts 14 days after doxorubicin injection (n = 6-8 in each group, P,0.01, Fig. 1A and 1B). As a result, a remarkable reduction in cardiac H 2 S synthesizing activity and plasma level of H 2 S was observed in doxorubicin injection group (n = 6-8 in each group, P,0.01, Figure 1C and 1D). Not surprisingly, consecutive administration with S-diclofenac for 14 days dose-dependently diminished the deficiencies of H 2 S formation induced by doxorubicin. As shown in Figure 1C and 1D, S-diclofenac, but not diclofenac dosedependently abolished doxorubicin-induced reduction in cardiac H 2 S synthesizing activity and plasma H 2 S concentration.

Alterations of gap junction proteins in doxorubicininduced cardiomyopathy
Gap junction channels are formed by the association of two connexons composed of six protein subunits termed connexins (Cxs) [11,12]. In mammalian hearts, gap junctions are principally composed of three major connexin isoforms, Cx43, Cx40 and Cx45. Therefore, we examined the cardiac expression levels of Cx43, Cx45 and Cx40 in doxorubicin-induced cardiomyopathy. As shown in Figure 2, the mRNA and proteins levels of Cx43 and Cx45 in animal hearts were drastically downregulated 14 days after doxorubicin injection (n = 6-8 in each group, P,0.01). However, no significant change in Cx40 mRNA and protein expression was observed in animals injected with doxorubicin (data not shown). It may be due to the fact that of most mammalian species, Cx40 is abundant in gap junctions of atrial myocytes but not of working ventricular myocytes [11,12].
Effect of S-diclofenac on doxorubicin-induced downregulation of gap junction proteins S-diclofenac at doses of 25 or 50 mmol/kg significantly alleviated doxorubicin-induced downregulation of Cx43 and Cx 45, as compared to a mole for mole equivalent dose of diclofenac (n = 6-8 in each group, P,0.05, Figure 2A-D). S-diclofenac at a higher dose was more potent than that at a lower dose to reverse the suppression of Cxs expression in doxorubicin-induced cardiomypathy. Furthermore, the effect of S-diclofenac on gap junction proteins was in large part resembled by treatment with NaHS, a conventional H 2 S donor. As shown in Figure 2E and 2F, NaHS at a dose of 50 mmol/kg prominently provoked the mRNA and protein expressions of Cx43 and Cx45 in doxorubicin-injected mice (n = 6-8 in each group, P,0.05).

Effect of S-diclofenac on doxorubicin-induced gap junction remodeling
Doxorubicin-associated gap junction remodeling in mouse heart was examined by transmission electron microscopy. Ultrastructural morphometry showed a significant reduction in gap junction density (number of gap junctions per unit intercalated disk length) 14 days after doxorubicin injection ( Figure 3A and Table 1). Aggregate gap junction length per unit intercalated disk length was also declined in doxorubicin group. Treatment with S-diclofenac but not diclofenac led to a remarkable increase in both the number and length of cardiomyocyte gap junctions ( Figure 3A and Table 1). A higher dose of S-diclofenac resulted in a more impressive rise in gap junction number and length as compared to a lower dose ( Figure 3A and Table 1). Similarly, NaHS significantly reversed the doxorubicin-induced gap junction remodeling in heart ( Figure 3B and Table 2).

Effect of S-diclofenac on doxorubicin-induced JNK activation in hearts
To get more insights into the intracellular mechanism by which S-diclofenac upregulated the cardiac expression of connexins in doxorubicin-induced cardiomyopathy, further experiments were performed to investigate the effect of S-diclofenac on JNK activation, known to play a part in modulating the connexin expression [13][14][15][16]. It was found that administration of doxorubicin significantly activated the phosphorylation of JNK in hearts ( Figure 4, n = 6 in each group). Treatment with S-diclofenac dosedependently inhibited doxorubicin-induced activation of JNK (n = 6 in each group, Figure 4A). NaHS at a dose of 50 mmol/kg also markedly blocked the phosphorylation of JNK in doxorubicin-injected mice (n = 6 in each group, P,0.05, Figure 4B). In contrast, diclofenac had no effect on JNK activation ( Figure 4A). In addition, we also examined the effect of H 2 S on ERK1/2 and p38 MAPK pathways in this animal model. Both S-diclofenac and NaHS failed to affect doxorubicin-evoked activation of ERK1/2 and p38 in hearts (data not shown).

Effect of S-diclofenac on doxorubicin-induced inflammation and oxidative stress
Overproduction and/or ineffective scavenging of reactive oxygen species (ROS) as well as inflammation play a crucial role in the pathogenesis of doxorubicin-induced cardiomyopathy [2,3]. Therefore, we examined the effect of S-diclofenac on lipid antioxidative mechanism (SOD and GSH activity), lipid peroxidation activity (MDA content), and neutrophil infiltration (MPO activtity) in hearts. As compared to a mole for mole equivalent dose of diclofenac, S-diclofenac more potently alleviated doxorubicin-induced inflammation in hearts as evidenced by a distinct reduction in cardiac MPO activity (Table 3). Consistently, the severity of oxidative stress caused by doxorubicin was also significantly reduced by treatment with S-diclofenac. As shown in Table 3, S-diclofenac, but not its parent drug markedly elevated the cardiac activity of SOD and GSH-px and decreased the level of MDA in hearts. The anti-inflammatory and anti-oxidative effect of S-diclofenac was more potent at a higher dose. In addition, treatment with NaHS also significantly inhibited doxorubicinassociated inflammation and oxidative damage (Table 4).  Effect of S-diclofenac on doxorubicin-induced cardiac injury and cardiac dysfunction Evidence of cardiac injury induced by intraperitoneal administration of doxorubicin was indicated by an obvious increase in plasma levels of cardiac markers (LDH and CK) ( Figure 5A), an evident depression in left ventricular systolic function (LVEF and LVFS) ( Figure 5B) and an apparent dilation of left ventricles ( Figure 5C). Treatment with S-diclofenac but not diclofenac protected mice against doxorubicin-induced cardiac injury. As shown in figure 5A, plasma activities of LDH and CK were significantly declined in mice treated with doxorubicin and Sdiclofenac as compared to those treated with doxorubicin and diclofenac. The impairment of cardiac function caused by doxorubicin was also attenuated by treatment with S-diclofenac, as characterized by a significant elevation in LVEF and LVFS (n = 6-8 in each group, P,0.05, Figure 5B, 5D). S-diclofenac at a dose of 50 mmol/kg, but not at a lower dose notably repressed the enlargement of left ventricle induced by doxorubicin (n = 6-8 in each group, P,0.05, Figure 5C, 5D). Similarly, NaHS significantly decreased the plasma levels of LDH and CK, improved the left ventricular dysfunction and attenuated left ventricle dilation in doxorubicin-injected mice (n = 6-8 in each group, Figure 5E, 5F, 5G).
In addition, administration with doxorubicin in mice resulted in a high 14-day mortality rate. Treatment with S-diclofenac but not diclofenac significantly improved the survival rate of the mice with doxorubicin-induced cardiomyopathy. The 14-day survival rate of the mice with doxorubicin-induced cardiomyopathy was significantly elevated when treated with S-diclofenac at a dose of 50 mmol/kg (n = 20 in each group, P,0.05, Figure 6).

Discussion
In the present study, we found that doxorubicin injection downregulated the cardiac expression of CSE along with reduced plasma H 2 S level and cardiac H 2 S synthesizing activity. Our findings are in accord with earlier observations and reinforce the significance of H 2 S insufficiency in the development of doxorubicin-induced cardiotoxicity [7]. These results provide a solid basis for the use of H 2 S donor drugs in the treatment of doxorubicininduced cardiotoxicity. We showed here that S-diclofenac, an H 2 S-releasing diclofenac derivative, provided marked cardioprotection in myocardial injury and cardiac dysfunction resulting from doxorubicin injection. The cardioprotective effect of Sdiclofenac could be mimicked in large part by a mole for mole equivalent dose of NaHS, a conventional H 2 S donor. Our findings suggest that H 2 S release (from S-diclofenac) protects animals against doxorubicin-associated cardiotoxicity and improves the outcome of animals with doxorubicin-induced cardiomyopathy.
Although the pathogenesis of anthracycline cardiotoxicity has been under intensive investigation for many years, the underlying mechanisms are still not fully understood. Multiple explanations have been proposed for the doxorubicin cardiotoxicity, e.g. oxidative stress, inflammation, DNA damage, attenuation in protein synthesis, alterations in cellular energetics and cardiomyocyte apoptosis [1][2][3][4][5]. However, the present study is focused on three aspects of the action of doxorubicin with respect to gap junction remodeling, oxidative injury and inflammation.
Gap junction channels are responsible for direct cell-to-cell communication and remodeling of gap junction organization is a conspicuous feature of heart diseases. In the present study, we have demonstrated for the first time that doxorubicin suppressed cardiac expression of Cx43 and Cx45, resulting in loss of Cx43/ Cx45 junction channels and consequent spatial remodeling of gap junctions, and thereby leading to progressive cardiac dysfunction and left ventricular remodeling. Treatment with S-diclofenac, but not diclofenac, not only remarkably upregulated the cardiac expression of Cxs (Cx43 and Cx45) mRNA and proteins, but also raised the density and length of gap junctions in doxorubicininduced cardiomyopathy. As a result, it considerably attenuated heart injury and left ventricular enlargement, and improved left ventricular ejection function and the survival rate of animals with doxorubicin-induced cardiomyopathy. Moreover, the protective effect of S-diclofenac could be mimicked by NaHS. Our data straightforwardly demonstrate that H 2 S may exert its cardioprotective effect in doxorubicin-induced cardiomyopathy via regulating connexin expression.
JNK is a crucial regulator of AP-1 activation which has been shown to be necessary for activating Cx43 and Cx45 proximal promoter [13,14]. Activation of JNK induced significant loss of Cx43 expression and decreased the numbers of gap junctions in cultured cardiomyocytes and transgenic mouse ventricular myocardium [15,16]. Therefore, we further examined the relevance of JNK pathway to the protective effect of S-diclofenac. We found that doxorubicin induced an obvious activation of JNK in hearts, suggesting that doxorubicin-induced downregulation of Cx43 and Cx45 is possibly mediated by JNK pathway. Treatment with Sdiclofenac or NaHS significantly inhibited the phosphorylation of JNK in hearts from doxorubicin-injected mice. Hence, it seems reasonable to assume that S-diclofenac derived H 2 S may abolish doxorubicin-associated downregulation of Cxs by inhibiting the cardiac activity of JNK. However, the precise points of interaction  between S-diclofenac and JNK pathway can not be determined in the present in vivo experiments and further work is needed most likely in an isolated cardiomyocyte system to probe the exact mechanisms involved. In addition to transcription, post-translational modifications such as phosphorylation are also important for connexin trafficking and assembly at gap junctions [17,18]. It has been shown that phosphorylation of connexins regulates many aspects of the gap junction life cycle including assembly, gating, internalization, and degradation [17]. Whether H 2 S could alter the biological functions of gap junctions via modulating the phosphorylation of Cxs in doxorubicin-induced cardiotoxicity will be the subject of our future study. Moreover, latest studies have proposed that doxorubicin-induced cardiomyopathy is primarily a stem cell disease [19,20]. Cardiotoxicity of the anthracycline is not restricted to cardiomyocytes but affects resident cardiac progenitor cells (CPCs) even more dramatically. Anthracycline not only inhibited the growth and survival of CPCs but also impaired global gene expression of CPCs. Reduction in the number of CPCs and defects in progenitor cell function finally lead to the development of cardiac myopathy or result in an adult heart that is more susceptible to stress. These findings offer an original possibility that H 2 S or H 2 S donor drugs may attenuate the loss of CPCs or the impairment of CPC capacity induced by doxorubicin, thus enhancing the myocardial regeneration and restoring the structural integrity of cardiomyopathic heart. Interestingly, in the present study, we did find that Sdiclofenac or NaHS significantly elevated the expression levels of junctional proteins (Cx43 and Cx45) and partially reversed the remodeling of gap junctions in hearts. Therefore, it is meaningful to further investigate the type of cardiac cells affected by H 2 S and the underlying molecular mechanisms in the future study.
On the other hand, we found that doxorubicin suppressed the activities of antioxidant enzymes and caused overproduction and/ or ineffective scavenging of ROS in hearts while S-diclofenac prevented these pathological processes. Similar antioxidative properties of H 2 S or H 2 S donor drugs were observed previously in neurons, vascular smooth muscle cells and neutrophils. H 2 S may suppress ROS generation and ameliorate oxidative insult by different mechanisms: 1) H 2 S may increase the activity of diverse antioxidant enzymes; 2) H 2 S may increase the levels of glutathione participating in regulating oxidative stress; 3) H 2 S, as a highly reactive molecule may easily react with ROS and/or reactive nitrogen species (NOS); 4) H 2 S may open ATP-dependent K + and Cl 2 channels [21][22][23]. Additionally, S-diclofenac has been shown to exhibit greater anti-inflammatory activity (compared to diclofenac) in the present study. A superior anti-inflammatory effect of S-diclofenac (compared to diclofenac) was also observed in animal models of hindpaw edema, acute panreatitis as well as endotoxemia [8,24,25]. The additional anti-inflammatory activity of S-diclofenac may be at least partially due to release of H 2 S. Sdiclofenac derived H 2 S inhibited inflammatory response by downregulating the transcription of inflammation related genes via NF-kB pathway [8]. However, it is to be noted that H 2 S has been shown to play complex but poorly understood roles in inflammation with evidence for proinflammatory as well as antiinflammatory properties. It was reported that H 2 S provoked the inflammatory response in sepsis, acute pancreatitis and endotoxemia [26,27,28]. In contrast, H 2 S inhibited leukocyte migration in animal models of paw edema and nonsteroidal anti-inflammatory drugs (NSAIDs)-induced gastric injury [29,30]. Although the role of H 2 S in inflammation remains controversial, it raises the possibility that H 2 S may act as a double-edged sword in Table 3. Effect of S-diclofenac on inflammatory response and oxidative stress in heart.  inflammatory conditions. It may act as an anti-inflammatory mediator to at physiological concentration, but may contribute to inflammation in an already inflamed area when overproduced. In addition, S-diclofenac has distinct advantages over NaHS in the treatment of doxorubicin-induced cardiomyopathy, although both of them protected animals against doxorubicin-induced myocardial injury. Firstly, H 2 S release from S-diclofenac is a slow process probably mediated by plasma/tissue esterase, which split the ester linkage of the parent compound to form ADT-OH. This molecule thereafter releases H 2 S either by spontaneous chemical degradation or by an as yet unidentified metabolic process. Hence, S-diclofenac has been considered as a slow-releasing H 2 S donor. Administration of S-diclofenac had minimal effect on blood pressure [8]. In stark contrast, NaHS breaks down rapidly in aqueous solution to yield Na + and HS 2 , the latter then reacting with H + to form H 2 S. Administration of NaHS caused a pronounced but transient decrease in blood pressure [28,31]. Secondly, recent studies have demonstrated that S-diclofenac may have a potential usefulness in cancer treatment [32]. S-diclofenac has been found to inhibit the activity and expression of carcinogen activating enzymes and may serve as effective chemoprevention agents by balance the equation of carcinogen activation and detoxification mechanisms [32]. Therefore, we propose that in comparison to NaHS, S-diclofenac may be a better option to prevent cardiac complications from anthracyclines in cancer patients undergoing chemotherapy with doxorubicin.
In summary, S-diclofenac attenuated doxorubicin-induced cardiac gap junction remodeling, oxidative stress and inflammatory response. The pharmacological profiles of S-diclofenanc, i.e. cardioprotective activity, suggest that this compound might constitute a potentially new therapeutic option to prevent doxorubicin-induced cardiotoxicity. However, further clinical studies will have to verify if this concept is valid in patients as well, and whether or not S-diclofenac treatment counteracts with the oncological effect of doxorubicin.

Chemicals
Doxorubicin was obtained from Wanle Pharmaceutical Industry (Shenzhen, China) as a 10 mg/bottle lyophilized powder. The malondialdehyde (MDA, a marker of lipid peroxidation and free radical activity), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) assay kits were purchased from Nanjing Jiancheng Bio-engineering Institute (Nanjing, China). S-diclofenac was obtained from CTG Pharma, Milan, Italy.

Animals and experimental protocols
The study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Shanghai JiaoTong University School of Medicine. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Shanghai JiaoTong University School of Medicine (Permit Number: [2011]-22). Male 10-week-old C57BL/6J mice were randomly assigned to receive doxorubicin (15 mg/kg, i.p.) or same volume of saline (i.p.). Two hours after doxorubicin injection, diclofenac (25 and 50 mmol/kg, i.p.), S-diclofenac (25 and 50 mmol/kg, i.p.), NaHS (50 mmol/kg, i.p.) or same volume of vehicle (0.5% w/v carboxymethylcellulose or saline, i.p.) were administered daily for 14 consecutive days. The dosage of S-diclofenac was determined based on previous study [8]. Fourteen days after doxorubicin injection, animals were sacrificed by an i.p. injection of a lethal dose of pentobarbitone (90 mg/kg). Samples of hearts and plasma were harvested and stored at 280uC for subsequent measurement. Measurement of plasma H 2 S Aliquots (120 ml) of plasma were mixed with distilled water (100 ml), trichloroacetic acid (10% w/v, 120 ml), zinc acetate (1% w/v, 60 ml), N, N-dimethyl-p-phenylenediamine sulfate (20 mM; 40 ml) in 7.2 M HCl and FeCl 3 (30 mM; 40 ml) in 1.2 M HCl. The absorbance of the resulting solution (670 nm) was measured 10 min thereafter by spectrophotometry (Tecan Systems Inc.). H 2 S was calculated against a calibration curve of sodium hydrosulfide (NaHS; 3.125-100 mM). Results were showed plasma H 2 S concentration in mM.
Assay of H 2 S synthesizing activity H 2 S synthesizing activity in heart homogenates was measured essentially as described elsewhere [26]. Briefly, the assay mixture contained 100 mM potassium phosphate buffer (pH 7.4), Lcysteine (20 ml, 20 mM), pyridoxyal 59-phosphate (20 ml, 2 mM), saline (30 ml) and 4.5% w/v tissue homogenate (430 ml). The reaction was performed in tightly sealed micro centrifuge tubes and initiated by transferring the tubes from ice to a water bath at 37uC. After incubation for 30 min, 1% w/v zinc acetate (250 ml) was added to trap evolved H 2 S followed by 10% v/v trichloroacetic acid (250 ml) to denature the protein and stop the reaction. Subsequently, N, N-dimethyl-p-phenylenediamine sulfate (20 mM; 133 ml) in 7.2 M HCl was added, immediately followed by FeCl3 (30 mM; 133 ml) in 1.2 M HCl. The absorbance of the resulting solution at 670 nm was measured by spectrophotometry (Tecan Systems Inc). The H 2 S concentration was calculated against a calibration curve of NaHS. Results were then corrected for the DNA content of the tissue sample [33] and were expressed as nmoles H 2 S formed/mg DNA.

RT-PCR analysis
Total RNA from heart was extracted with Trizol H reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. The concentration of isolated nuclei acids was determined spectrophotometrically by measuring the absorbance at 260 nm and the integrity was verified by ethidium bromide staining of 18S and 28S rRNA bands on a denaturing agarose gel. All samples were thereafter stored at 280uC until required. 1 mg RNA was reversely transcribed using iScript TM cDNA Synthesis Kit (Biorad, Hercules, CA, USA) at 25uC for 5 minutes, 42uC for 30 minutes, followed by 85uC for 5 minutes. The cDNA was used as a template for PCR amplification by iQ TM Supermix (Biorad, Hercules, CA, USA). The primer sequences for detection of CSE, Cx43, Cx45, Cx40 and 18S, optimal annealing temperature, optimal cycles and product sizes were as shown in table 5. PCR amplification was carried out in MyCycler TM (Biorad, Hercules, CA, USA). The reaction mixture was first subjected to 95uC for 3 minutes, followed by an optimal cycle of amplifications, consisting of 95uC for 50 s, optimal annealing temperature for 45 s and 72uC for 1 minute. PCR products were analyzed on 1.5% w/v agarose gels containing 0.5 mg/ml ethidium bromide.

Western immunoblot
Left ventricles were homogenized at 4uC in radioimmunoprecipitation assay lysis buffer supplemented with protease inhibitor cocktail (Roche, Switzerland). The tissue homogenates were centrifuged at 14,000 g for 10 min at 4uC. Protein concentration in the soluble fraction was determined by Bradford method. Protein samples (100 mg) were separated by 12% SDS-polyacrylamide gel and then transferred onto polyvinylidene difluoride membranes. Membranes were then washed, blocked, and probed overnight at 4uC with rabbit anti-Cx43

Myeloperoxidase estimation (MPO)
Heart samples were thawed, homogenized in 20 mM phosphate buffer (pH 7.4), centrifuged (13,0006 g, 10 min, 4uC) and the resulting pellets were resuspended in 50 mM phosphate buffer (pH 6.0) containing 0.5% w/v hexadecyltrimethylammonium bromide (Sigma). The suspension was subjected to four cycles of freezing and thawing and further disrupted by sonication (40 seconds). The samples were then centrifuged (13,0006 g, 5 min, 4uC) and the supernatants were used for the MPO assay. The reaction mixture consisted of the supernatant (50 ml), 1.6 mM tetramethylbenzidine (Sigma), 80 mM sodium phosphate buffer (pH 5.4), and 0.3 mM hydrogen peroxide (reagent volume: 50 ml). This mixture was incubated at 37uC for 110 sec. The reaction was terminated with 50 ml of 0.18 M H2SO4 and the absorbance was measured at 405 nm. This absorbance was then corrected for the DNA content of the tissue sample and the results were expressed as enzyme activity.

Assay of MDA content, activity of SOD and GSH-Px in heart
Cardiac tissue samples (25 mg) were homogenized in 0.5 ml of PBS at 4uC with a homogenizer. Homogenates were then centrifuged at 1500 rpm for 30 min and the supernatants were collected. MDA content as well as SOD and GSH-px activity in supernatants were evaluated with MDA, SOD and GSH-Px assay kits according to the manufacturer's protocol.
Assay of creatine kinase (CK) and lactate dehydrogenase (LDH) enzyme activities Plasma CK and LDH enzyme activities were measured using an autoanalyzer (Model 7020, Hitachi Medico, Japan).

Transmission electron microscopy
In order to analyze gap junction remodeling, left ventricular tissues were immersion-fixed in phosphate-buffered 2.5% glutaraldeyde (pH 7.4), post-fixed with 1% osmium tetroxide, dehydrated through a graded ethanol series and embedded in Epon medium. Ultrathin sections were observed by electron microscopy (JEM1230; JEOL Transmission Electron Microscope, Tokyo, Japan). Five hearts from each group were examined. Ten pictures were taken from each heart and analyzed morphometrically using methods described previously [34,35]. At a magnification of 30,000, every intercalated disk profile identified within each test area was rephotographed and the total number and length of gap junctions and the total length of intercalated disks were measured.
Gap junction number and length was calculated per unit intercalated disk length as previously described [34,35].

Echocardiography
Mice were lightly anesthetized with isoflurane gas. AUBM system (Vevo 770, Visualsonics, Toronto, Canada) equipped with a 7.5-MHz imaging transducer was used for all the examinations. The transducer was placed in gentle contact with the midprecordial area through a transmission medium. Left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular internal dimensions at the end of diastole (LVEDD) and at the end of systole (LVESD) were measured digitally on the M-mode tracing.

Statistics
The data were expressed as mean 6 SEM. The significance of differences among groups was evaluated by analysis of variance (ANOVA) with post-hoc Tukey's test when comparing three or more groups. The significance of differences between two groups was evaluated by T test. The overall survival analysis for lethal injury was described by Kaplan-Meier plots. The difference of mortality among groups was tested by log rank test. A P,0.05 was regarded as statistically significant.