Free Radical Biology & Medicine 46 (2009) 51–61

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Free Radical Biology & Medicine

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Original Contribution

Taurine prevents cardiomyocyte death by inhibiting NADPH oxidase-mediatedcalpain activation

Ying Li a,b, J. Malcolm O. Arnold a,b,c, Macarena Pampillo d,e, Andy V. Babwah d,e, Tianqing Peng a,b,f,⁎a Critical Illness Research, Lawson Health Research Instituteb Department of Medicine, University of Western Ontario, London, ON, Canada N6A 4G5c Department of Physiology and Pharmacology, University of Western Ontario, London, ON, Canada N6A 4G5d Children's Health Research Institutee Department of Obstetrics and Gynaecology, University of Western Ontario, London, ON, Canada N6A 4G5f Department of Pathology, University of Western Ontario, London, ON, Canada N6A 4G5

Abbreviations: NE, norepinephrine; ROS, reactive oxventricular cardiomyocytes; DPI, diphenyleneiodoniumpropidium iodide; GAPDH, glyceraldehyde-3-phosphate⁎ Corresponding author. Critical Illness Research, Law

Fax: +1 519 685-8341.E-mail address: tpeng2@uwo.ca (T. Peng).

0891-5849/$ – see front matter © 2008 Elsevier Inc. Aldoi:10.1016/j.freeradbiomed.2008.09.025

a b s t r a c t

a r t i c l e i n f o

Article history:

Taurine has been shown to p Received 6 June 2008Revised 8 September 2008Accepted 17 September 2008Available online 9 October 2008

Keywords:TaurineApoptosisCardiomyocytesNADPH oxidaseCalpainFree radicals

revent cardiomyocyte apoptosis. This study investigated the effects of taurine onNADPH oxidase and calpain activation in mediating apoptosis in cardiomyocytes. Apoptosis was induced bynorepinephrine (NE) in cultured adult rat ventricular cardiomyocytes. NE (5 μM) increased NADPH oxidaseactivation and reactive oxygen species (ROS) production and induced apoptosis. These effects of NE oncardiomyocytes were diminished by taurine (0.5 mg/kg) but not β-alanine. Inhibition of gp91phox-NADPHoxidase or ROS production protected cardiomyocytes from apoptosis. NE also induced calpain-1 activation incardiomyocytes. This effect of NE on calpain was abrogated by gp91phox-NADPH oxidase inhibition or ROSscavengers and was mimicked by H2O2 (25 μM) in cardiomyocytes. Pharmacological inhibitors of calpain oroverexpression of calpastatin, a specific calpain inhibitor, blocked calpain activation and preventedcardiomyocyte apoptosis during NE stimulation. Furthermore, taurine treatment inhibited NE- or H2O2-induced calpain activation in cardiomyocytes. In conclusion, NADPH oxidase induces calpain activation,leading to apoptosis in NE-induced cardiomyocytes. Taurine inhibits NADPH oxidase and calpain activation.Thus, inhibition of NADPH oxidase-mediated calpain activation may be an important mechanism for taurine'santiapoptotic action in cardiomyocytes.

© 2008 Elsevier Inc. All rights reserved.

Taurine, or 2-aminoethanesulfonic acid, is a major intracellular

sulfur-containing β-amino acid. It is ubiquitously found in relativelyhigh concentrations in animal tissues including myocardium. Taurineis not metabolized or incorporated into cellular proteins, suggestingan important requirement for free cytosolic taurine. Taurine has beensuggested to be the organic osmolyte present in high concentration inthe heart [1]. The largest shift in osmolar equivalents within a cell inresponse to osmotic stress results from taurine. Taurine uptake ismediated by a ubiquitous Na+ and Cl−-dependent transporter, whichuses the Na+ gradient across the cell membrane to drive taurineaccumulation [2]. Studies have suggested that taurine plays importantprotective roles in the heart under various pathophysiologicalconditions [3]. Taurine supplementation protects the diseased heartin an animal model [4] and human patients [5]. A recent study hasshown that knockout of the taurine transporter gene induces cardiac

ygen species; ARVC, adult rat; NAC, N-acetylcysteine; PI,dehydrogenase.son Health Research Institute.

l rights reserved.

atrophy and consequent cardiomyopathy, suggesting taurine isimportant in maintaining cardiac structure and function [6]. Althoughmechanisms by which taurine exerts cardiac protection have not beenclarified, taurine suppresses cardiomyocyte apoptosis, which maybenefit the diseased heart. Taurine prevents myocardial apoptosisduring ischemia–reperfusion [7] or doxorubicin treatment in intactanimals [8], attenuates apoptosis induced by Ca2+ overload in isolatedhearts [9], and inhibits apoptosis in ischemic cardiomyocytes [10].Further evidence to support the antiapoptotic role of taurine is thatdepletion of taurine increases myocardial apoptosis in taurine trans-porter knockout mice [6]. The antiapoptotic action of taurine may berelated to its antioxidant properties and down-regulation of intracel-lular Ca2+ levels through inhibition of L-type Ca2+ channels and pro-motion of Ca2+ uptake by Ca2+ ATPase [2,4,11,12], leading to inhibitionof the Apaf-1/caspase-9 pathway [13]. However, the precise mechan-isms remain not fully elucidated in cardiomyocytes.

The NADPH oxidase is a multicomponent enzyme complex thatconsists of the membrane-bound cytochrome b558, which containsgp91phox and p22phox, the cytosolic regulatory subunits p47phox andp67phox, and the small GTP-binding protein Rac. Activation of NADPHoxidase leads to superoxide generation [14]. NADPH oxidase is a majorsource of reactive oxygen species (ROS)1 in the myocardium [15].

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Increased ROS induce apoptosis in cardiomyocytes [16,17]. As such,inhibition of NADPH oxidase blocks ROS production and inhibitscardiomyocyte apoptosis [18]. It remains unclear whether taurine hasany effects on NADPH oxidase activation in cardiomyocytes.

Increased ROS have been shown to induce calpain activation inretinal photoreceptor cells [19]. Calpains are a family of calcium-dependent thiol-proteases [20]. Fifteen gene products of the calpainfamily are reported in mammals. Among them, calpain-1 and calpain-2 are ubiquitously expressed, and other calpain family members havemore limited tissue distribution. Both calpain-1 and calpain-2 arespecifically countered by the endogenous calpain inhibitor, calpastatin[21]. Ca2+ is required for calpain activation. Calpains respond to Ca2+

signals by cleaving specific proteins, frequently components of sig-naling cascades, thereby irreversibly modifying their function [21].Studies have suggested that calpain activation contributes to apopto-sis in cardiomyocytes during TNF-α treatment and ischemia–reperfusion [22–24]. Because taurine modulates intracellular Ca2+

levels, it is possible that taurine may play a role in regulating calpainactivation.

In this study, we hypothesized that taurine inhibits calpainactivation in cardiomyocytes via prevention of NADPH oxidaseactivity, leading to attenuation of apoptosis in cardiomyocytes. Totest this hypothesis, we employed an in vitro model of adult ratcardiomyocyte death. Apoptosis was stimulated by norepinephrine(NE). The effects of taurine on NADPH oxidase and calpain activationand of blocking NADPH oxidase and calpain, leading to inhibition ofapoptosis, were studied.

Materials and methods

Animals and adult rat ventricular cardiomyocyte cultures

This investigation conformed with the Guide for the Care and Use ofLaboratory Animals published by the U.S. National Institutes of Health(NIH Publication No. 85-23). All experimental procedures wereapproved by the Animal Use Subcommittee at the University ofWestern Ontario, Canada. Adult male rats (Sprague–Dawley, 200 gbody weight) were purchased from Charles River Labs.

Adult rat ventricular cardiomyocytes (ARVC) were isolated asdescribed [25]. Cardiomyocytes were resuspended in a plating buffer(MEM supplementedwith 5% FBS,10mM2,3-butanedionemonoxime,100 u/ml penicillin–streptomycin, 2 mM l-glutamine) and seeded in12-well laminin-coated dishes. After 2 h incubation at 37 °C (95% air/2% CO2), the cells were maintained in a culture medium (MEM) andwere used for experiments after 2 h. These cardiomyocytes cansurvive for at least 48 h in our laboratory.

Reagents

NE, adrenochrome, β-alanine, Nω-nitro-l-arginine methyl ester (L-NAME), apocynin, diphenyleneiodonium (DPI), N-acetylcysteine(NAC), and lucigenin were purchased from Sigma. NADPH, propidiumiodide (PI), PD150606, and calpain inhibitor-III were from Calbiochem.Annexin V conjugated with FITC, Hoechst 33342, 7-dichlorodihydro-fluorescein diacetate (DCF-DA), Oregon Green 488 BAPTA-1 AM werefrom Invitrogen. Peptides gp91ds-tat and scramble-tat were synthe-sized by ProImmune Ltd. (UK).

Adenoviral infection of cultured ARVC

ARVC were infected with adenoviral vectors containing ratcalpastatin (Ad-CAST; University of Buffalo, Buffalo, NY, USA) or β-gal (Ad-gal; Vector Biolabs) as a control at a multiplicity of infection of10 pfu/cell. Adenovirus-mediated gene transfer was implemented aswe previously described [26]. All experiments were performed after24 h of adenoviral infection.

Calpain activity

Calpain activity was determined by using a fluorescence sub-strate, N-succinyl-LLVY-AMC (Cedarlane Laboratories), as describedpreviously [27]. This assay measures the fluorescence intensity ofAMC (7-amino-4-methylcoumarin) cleaved from the peptide sub-strate. Briefly, cardiomyocyte lysates were centrifuged and thesupernatant was used for calpain activity measurement. Thesamples were incubated in reaction buffer (63 mM imidazole–HCl, pH 7.3, 10 mM β-mercaptoethanol, 1 mM EDTA, and 10 mMEGTA) with or without calcium chloride (5 μM or 5 mM) at 37 °Cfor 2 h. In some reaction samples, a calpain-selective inhibitor,PD150606 (5 μM), was added. The fluorescence intensity of cleavedAMC was quantified with a multilabel reader (excitation, 360 nm;emission, 460 nm) and calpain activity was determined as thedifference between calcium-dependent and calcium-independentfluorescence.

NADPH oxidase activity

ARVC were homogenized and sonicated in reaction buffer. Theprotein concentrations were then measured using the Bio-Rad DCprotein assay. NADPH oxidase activity was assessed in cell lysates bylucigenin-enhanced chemiluminescence (20 μg of protein, 100 μMNADPH, 5 μM lucigenin) with a multilabel counter (Victor3; Wallac)[28]. Each samplewas set in four different wells. DPI was added to twoof them (5 μM). The light signal was monitored for 5 s, and counts persecond as presented as NADPH oxidase activity. The NADPH oxidaseactivity was determined as the difference between wells without DPIand with DPI. In some reaction samples, an inhibitor of nitric oxidesynthase, L-NAME (200 μM), was added.

ROS measurement

The formation of ROS was measured by using the ROS-sensitivedye DCF-DA as an indicator. ARVC were homogenized in reactionbuffer. The protein concentrations in cell lysates were measured usingthe Bio-Rad DC protein assay. Samples (50 μg total proteins) wereincubated with 10 μl of DCF-DA (20 μM) for 3 h at 37 °C. Thefluorescent product formed was quantified by spectrofluorometerwith excitation at 485 nm and emission at 525 nm and expressed asROS production (arbitrary units, A.U.).

Active caspase-3

As described in detail previously [29], caspase-3 activity in cardio-myocytes was measured using a caspase-3 fluorescence assay kit(Biomol Research Laboratories). This assay measures the fluorescenceintensity of the chromophore AMC cleaved from the C terminus of thepeptide substrate. The fluorescence intensity of cleaved AMC wasquantified with a multilabel reader (excitation, 355 nm; emission,460 nm; Victor 3; Wallac) and normalized with inhibitor-treatedsamples as background.

Cell death measurement

Cardiomyocyte death was measured by annexin V, PI, and Hoechst33342 staining. Annexin V conjugated with FITC (60 ng/ml), PI(0.5 mM), and Hoechst 33342 (1 μg/ml) were added directly to theculture medium. Ten minutes later, cardiomyocytes were brieflywashed with PBS and photographed under both phase-contrast andfluorescence conditions. Annexin V-positive cells were determined asgreen staining on the membrane and PI-positive cells as nuclearstaining in red. Hoechst 33342 staining in bluewas used to localize thenucleus. At least 200 cardiomyocytes were examined from eachsample, and each condition was measured in triplicate.

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Western blot analysis

ARVC were lysed in SDS (0.5%)-containing buffer. The proteinconcentrations were determined using the Bio-Rad DC protein assay.A total of 30 μg of protein was fractionated on an SDS–polyacrylamidegel (10%) and electrotransferred to polyvinylidene difluoride micro-porous membrane. The membrane was blocked with 5% nonfat milkand then incubated with specific antibodies (rabbit antibodies)against rat calpain-1, calpain-2, calpastatin, or GAPDH proteins (CellSignaling; 1/1000). After being washed, the blots were incubatedwith anti-rabbit IgG peroxidase-conjugated antibody (1/2000). Thesignals were visualized using an enhanced chemiluminescence detec-tion system.

Confocal microscopy

ARVC were seeded on collagen-coated 35-mm glass-bottomedculture dishes and the next day, the cells were prepared for confocal-based Ca2+ imaging. This was done by preloading cells for 30 minwith10 mM Oregon Green 488 BAPTA-1 AM according to the manufac-turer's specifications (Invitrogen). Immediately after preloading, thecells were subjected to drug treatments in 1× HBSS and then imaged.Each cell was imaged for 500 s; this was repeated three times with 30-min intervals between each recording. An untreated control was also

Fig. 1. Effects of taurine on apoptosis in cultured ARVC. ARVC were incubated with NE (5 μM)Apoptosis was assessed by (A) caspase-3 activation, (B and C) annexin V (A-V) staining, andHoechst 33342 (blue signal) is shown from NE- and NE+taurine-treated ARVC. (C) Quantificnuclear stain for PI (red signal) and Hoechst 33342 from NE- and NE+taurine-treated ARVC.means±SD from at least three different cell cultures. ⁎pb0.05 vs sham+vehicle and #pb0.0

imaged. Confocal microscopy was performed on the Olympus FV 1000laser-scanning microscope using a 63× 1.4 numerical aperture oilimmersion lens. Oregon Green 488 BAPTA fluorescencewas visualizedwith excitation at 488 nm and emission at 515–540 nm emission filterset. Fluorescent signals were collected continuously and pixel inten-sity within a region of interest was subsequently analyzed using theFV10-ASW 1.6 software to generate amplitude and oscillatory fre-quency information.

Statistical analysis

All data are given as means±SD. Differences between two groupswere compared by unpaired Student's t test. For multigroupcomparisons, ANOVA followed by Newman–Keuls or nonparametricKruskal–Wallis with Dunn's posttest was performed. A value ofpb0.05 was considered statistically significant.

Results

Taurine prevents apoptosis in cardiomyocytes

NE has been demonstrated to induce apoptosis in cardiomyocytes[30,31]. Consistent with these previous reports, NE (5 μM) increasedcaspase-3 activity and induced cell death as evidenced by the eleva-

in the presence of taurine (0.5 mg/ml or 4 mM) or β-alanine (4 mM) or vehicle for 18 h.(D and E) PI nuclear staining. (B) A representative stain for annexin V (green signal) andation of the percentage of annexin V-positive cells induced by NE. (D) A representative(E) Quantification of the percentage of PI nuclear-positive cells induced by NE. Data are5 vs NE+vehicle.

Fig. 3. Effects of taurine on NADPH oxidase activity and ROS production. ARVC wereincubated with NE in the presence of taurine or vehicle. Six or 18 h after NE treatment,NADPH oxidase activity and ROS productionweremeasured. (A) NADPH oxidase activity.

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tion of the percentage of annexin V-positive cardiomyocytes (Fig. 1).Concurrent treatment with a selective caspase-3 inhibitor, AC-DEVD-CHO (5 μM), significantly attenuated the percentage of anne-xin V-positive cells by 75%, suggesting that most annexin V-positivecells were ongoing apoptotic cells. Thus, annexin V staining wasused as an indicator of apoptosis in this study. To study the effectsof taurine on apoptosis, cardiomyocytes were incubated with NE inthe presence of taurine (0.5 mg/ml) or vehicle for 18 h. Treatmentwith taurine significantly reduced caspase-3 activity and decreasedthe percentage of annexin V-positive cells in NE-stimulated cardio-myocytes (Fig. 1). The effect of taurine on cardiomyocyte death wasalso assessed by PI nuclear staining. Similarly, NE increased thepercentage of PI nuclear-positive cells, which was significantlyattenuated by taurine (Fig. 1). Although PI is a viability exclusion dyeand does not enter cells with an intact membrane, in the late stageof apoptosis there is also a loss of membrane integrity as the celldies [32]. As such, the population of PI-positive cells is a mixture ofnecrotic and apoptotic cells. Thus, taurine prevents NE-inducedapoptotic death in cardiomyocytes. The antiapoptotic effect oftaurine could not be mimicked by using the same concentrationof another organic osmolyte, β-alanine (Fig. 1).

NE is subjected to oxidation and the product, adrenochrome, maycause cytotoxicity in cardiomyocytes [33]. To examine whethertaurine also prevents adrenochrome-induced apoptosis in ARVC, weincubated ARVC with adrenochrome (100 μM) in the presence orabsence of taurine (0.5 mg/ml) for 18 h. Adrenochrome inducedcaspase-3 activation and increased the percentages of annexin V- andPI-positive cells. Treatment with taurine significantly reduced cas-pase-3 activity and decreased the percentages of annexin V-positivecells in adrenochrome-stimulated cardiomyocytes (Fig. 2). Thus,adrenochrome directly induces apoptosis, which is also inhibited bytaurine in ARVC.

Fig. 2. Effects of taurine on adrenochrome (ANC)-induced apoptosis. ARVC wereincubated with ANC (100 μM) in the presence of taurine (0.5 mg/ml) or vehicle for18 h. Apoptosis was assessed by (A) caspase-3 activation, (B) annexin V staining, and(C) PI nuclear staining. (B and C) Quantifications of the percentages of annexin V- andPI-positive cells. Data are means±SD from three different cell cultures. ⁎pb0.05 vssham+vehicle and #pb0.05 vs ANC+vehicle.

(B) ROS production. (C) ARVC were incubated with H2O2 (25 μM) in the presence oftaurine or vehicle for 18 h. ROS production was measured. Data are means±SD from atleast three different cell cultures. ⁎pb0.05 vs control or sham + vehicle, #pb0.05 vs 6 or18 h or H2O2+vehicle.

Taurine blocks NADPH oxidase activation and ROS production

NE treatment significantly increased NADPH oxidase activity incardiomyocytes. This NADPH oxidase activity was not changed whenL-NAME (200 μM) was added into the reaction buffer, excluding theconfounding effect of nitric oxide synthase in this assay. The up-regulation of NADPH oxidase activity was associated with increasedROS production in cardiomyocytes at 6 and 18 h after NE stimulation(Fig. 3). These results demonstrate that NE induces NADPH oxidaseactivation and subsequent ROS production. To investigate the effectsof taurine on NADPH oxidase activation, cardiomyocytes wereincubated with NE in the presence of taurine or vehicle. Six and18 h later, NADPH oxidase activity and ROS production were mea-sured. Taurine treatment abrogated NE-induced NADPH oxidaseactivity and ROS production in cardiomyocytes at both 6 and 18 h(Figs. 3A and 3B). To examine whether inhibition of NADPH oxidaseactivity is associated with down-regulation of NADPH oxidaseexpression, we measured gp91phox protein levels because gp91phox isthe predominant NOX in cardiomyocytes [28]. The protein levels ofgp91phox were not altered in cardiomyocytes during taurine incuba-tion (mean±SD of gp91phox/GAPDH ratio from three different culturesin vehicle+NE versus taurine+NE: 0.26±0.01 versus 0.29±0.011).Thus, taurine inhibits NADPH oxidase activation in cardiomyocytes atboth early (6 h) and late stages (18 h). To study whether taurine has adirect action on ROS, ARVC were incubated with H2O2 (25 μM) in thepresence or absence of taurine for 18 h. H2O2 increased ROS pro-duction in ARVC, which was reduced by taurine by 63% (Fig. 3C). Thissuggests that taurine directly decreases ROS production in ARVC.

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Inhibition of NADPH oxidase prevents cardiomyocyte apoptosis

To investigate if NADPH oxidase plays a role in NE-inducedapoptosis, cardiomyocytes were incubated with NE (5 μM) alone orin combination with DPI (5 μM) or apocynin (200 μM) for 18 h.Treatment with DPI (5 μM) blocked NADPH oxidase activation and ROSproduction in NE-stimulated cardiomyocytes (Figs. 4A and 4B). Car-diomyocyte apoptosis was then assessed by caspase-3 activity,annexin V staining, and PI nuclear staining. Concurrent treatmentwith DPI or apocynin abrogated the increase in caspase-3 activity andreduced the percentages of annexin V- and PI nuclear-positive cells(Fig. 5). To demonstrate that NADPH oxidase-mediated ROS inducecardiomyocyte apoptosis, an antioxidant, NAC, was employed.Similarly, NE-induced caspase-3 activity and increases in the percen-tages of annexin V- and PI nuclear-positive cells were abrogated byNAC (2.5 mM) (Fig. 5). To substantiate the role of NADPH oxidase inapoptosis, we used gp91ds-tat to specifically inhibit NADPH oxidaseactivation [34]. Cardiomyocytes were incubated with NE in the pre-sence of gp91ds-tat or scramble-tat (2.5 μM) for 18 h. Consistently,gp91ds-tat blocked NADPH oxidase activation (Fig. 4C), inhibitedcaspase-3 activity, and decreased the percentages of annexin V- and PInuclear-positive cells during NE stimulation (Fig. 5). Thus, these datastrongly suggest that NADPH oxidase-mediated ROS induce apoptosisin NE-stimulated cardiomyocytes.

NADPH oxidase-produced ROS induce calpain activation

We first examined the effect of NE on calpain activity incardiomyocytes. Cardiomyocytes were incubated with NE (5 μM) forvarious times (0, 1, 6, and 18 h) and calpain activity was analyzed.Calpain activity started to increase at around 1 h and reached the

Fig. 4. Effects of DPI or gp91ds-tat on NADPH oxidase activity and ROS production.ARVC were incubated with NE in the presence of DPI (5 μM) or gp91ds-tat (2.5 μM)or vehicle or scramble-tat for 18 h. NADPH oxidase activity and ROS production weremeasured. (A and C) NADPH oxidase activity. (B) ROS production. Data are means±SDfrom three different cell cultures. ⁎pb0.05 vs sham+vehicle or scramble-tat, #pb0.05vs NE+vehicle or scramble-tat.

Fig. 5. Role of NADPH oxidase in apoptosis. CulturedARVCwere incubatedwithNE in thepresence of DPI, apocynin, NAC, vehicle, gp91ds-tat, or scramble-tat for 18 h. Caspase-3activity and cell deathwere determined. (A) Caspase-3 activity. (B andC) The percentagesof annexin V-positive cells and PI nuclear-positive cells. Data aremeans±SD from at leastthree different cell cultures. ⁎pb0.05 vs sham+vehicle, #pb0.05 vs NE+vehicle.

maximal levels at 6 h, which were sustained up to 18 h after NEtreatment, which is in parallel with the up-regulation of NADPHoxidase and caspase-3 activation (Figs. 6A–6C). The calpain activitywas blocked using a calpain-specific inhibitor, PD150606 (5 μM),which prevents Ca2+ binding to calpain and does not significantlyinhibit either cathepsins or caspases [35], confirming that the assayspecifically measures calpain activity (Fig. 6D). The calpain activitywas also inhibited by another calpain inhibitor, calpain inhibitor-III(Fig. 6D). Thus, NE treatment results in calpain activation in parallelwith NADPH oxidase activation in cardiomyocytes. The up-regulationof calpain activity was not due to alterations of calpain-1 and calpain-2 expression because treatment with NE did not change the proteinlevels of calpain-1 and calpain-2 (Fig. 6E).

To clarify the isoform-specific activation of calpain in NE-stimulated cardiomyocytes, we measured calpain-1 activation using5 μM calcium because calpain activity at 5 μM calcium results fromcalpain-1 activity, whereas total calpain activity is obtained at 5 mMcalcium [36]. As shown in Fig. 6F, the proportion of calpain activityattributable to calpain-1 was more than 67% that of total calcium-stimulated calpain activity. This result suggests that NE activatescalpain-1 in cardiomyocytes.

To examine if NADPH oxidase-mediated ROS induces calpainactivation, cardiomyocytes were incubated with NE alone or in com-bination with gp91ds-tat (2.5 μM), DPI (5 μM), apocynin (200 μM), orNAC (2.5 mM). Eighteen hours later, calpain activity was measured.Inhibition of NADPH oxidase with gp91ds-tat or DPI or apocynin

Fig. 6. Calpain activation and expression in NE-stimulated cardiomyocytes. (A–C) Time course of calpain, NADPH oxidase, and caspase-3 activation in cardiomyocytes in response toNE. (D) ARVC were incubated with NE in the presence of PD150606, calpain inhibitor-III (CI-III), or vehicle for 18 h. Calpain activity was measured. (E) ARVC were incubated with NEfor 18 h. The protein levels of calpain-1, calpain-2, and GAPDHwere determined byWestern blot analysis. (F) ARVCwere incubatedwith NE for 18 h and calpain activitywasmeasuredusing 5 mM or 5 μM Ca2+ concentration. Data are means±SD from at least three different cell cultures. ⁎pb0.05 vs time point 0 or control+vehicle, #pb0.05 vs NE+vehicle.

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diminished NE-induced calpain activity in cardiomyocytes (Fig. 7A).Similarly, scavenging ROS by NAC blocked calpain activation in NE-stimulated cardiomyocytes (Fig. 7A). Finally, treatment with H2O2

(25 μM) induced calpain activation in cardiomyocytes, which wasinhibited by taurine (Fig. 7B). These results demonstrate that NEactivates calpain by NADPH oxidase-mediated ROS-dependentmechanisms in cardiomyocytes.

Taurine inhibits calpain activation

Because our data have shown that NADPH oxidase induces calpainactivation and taurine inhibits NADPH oxidase, we hypothesized thattaurine inhibits calpain activation in NE-stimulated cardiomyocytes.To test this hypothesis, cardiomyocytes were incubated with NE aloneor in combination with taurine. Under basal conditions, taurine didnot have any effect on calpain activity. Calpain activity was signi-ficantly decreased in taurine-treated compared to vehicle-treatedcardiomyocytes at both early (6 h) and late stages (18 h) after NEstimulation (Fig. 7D). This result demonstrates that treatment withtaurine inhibits calpain activation in NE-stimulated cardiomyocytes.Taurine also blocked adrenochrome-induced calpain activity incardiomyocytes (Fig. 7C).

Effects of taurine on Ca2+ signals in cardiomyocytes

Ca2+ signals in cardiomyocytes were analyzed by confocal micro-scopy. NE (5 μM) significantly increased oscillatory frequency in car-diomyocytes (Fig. 8). Concurrent treatment with taurine (0.5 mg/ml)blocked oscillatory frequency induced by NE. Although NE did notalter Ca2+ amplitude, taurine significantly decreased Ca2+ amplitude incardiomyocytes (Fig. 8). These data suggest that taurine inhibits NE-induced Ca2+ changes in cardiomyocytes.

Pharmacological inhibition of calpain protects cardiomyocytesfrom apoptosis

To elucidate the role of calpain in apoptosis, cardiomyocytes weretreated with NE in the presence of the pharmacological calpain inhi-bitors PD150606 (5 μM) and calpain inhibitor-III (5 μM). EitherPD150606 or calpain inhibitor-III blocked calpain activity in cardio-myocytes in response to NE (Fig. 6D). Under basal conditions,PD150606 or calpain inhibitor-III had no effect on caspase-3 activityor the percentage of annexin V- or PI nuclear-positive cells (Fig. 9). Inresponse to NE, caspase-3 activity was reduced to basal levels and thepercentages of annexin V- and PI nuclear-positive cells were signi-

Fig. 7. Effects of NADPH oxidase inhibition and taurine on calpain activation. (A) ARVCwere incubatedwith NE in the presence of DPI, apocynin, NAC, vehicle, gp91ds-tat, or scramble-tat for 18 h. Calpain activity was then measured. (B) ARVC were treated by H2O2 in the presence of taurine or vehicle. Eighteen hours later, calpain activity was determined. (C) ARVCwere incubated with adrenochrome (ANC) in the presence of taurine or vehicle for 18 hours. Calpain activity was determined. (D) ARVCwere incubated with NE in combinationwithtaurine or vehicle. Six or 18 h later, calpain activity was measured. Data are means±SD from at least three different cell cultures. ⁎pb0.05 vs sham or control+vehicle, #pb0.05 vs NEor ANC or 6 or 18 h+vehicle.

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ficantly attenuated in PD150606- or calpain inhibitor-III-treatedcompared to vehicle-treated cardiomyocytes (Fig. 9). These resultssuggest that pharmacological inhibition of calpain prevents cardio-myocyte apoptosis induced by NE.

Overexpression of calpastatin inhibits calpain activation andprevents apoptosis

Calpastatin is a highly specific inhibitor of calpain 1 and calpain 2that does not inhibit the activity of any other protease tested [21]. Thus,calpastatin expression is an effective means of globally inhibitingcellular calpain 1 and calpain 2 activities. To further confirm the role ofcalpain in apoptosis, we overexpressed calpastatin in cardiomyocytesby using an adenoviral vector containing calpastatin (Ad-CAST). Car-diomyocyteswere infectedwithAd-CASTorAd-gal for24h, followedbyNE treatment. The overexpression of calpastatin was verified byWestern blot analysis in Ad-CAST-infected cardiomyocytes (Fig. 10A).Western blot analysis also revealed that NE treatment did notsignificantly alter the protein levels of endogenous calpastatin (mean±SD of calpastatin/GAPDH ratio from three different cultures in shamversus NE: 2.45±0.526 versus 2.17±0.0729). Infection with Ad-gal or

Ad-CASTslightly increased thebasal levelsof annexinV-andPI-positivecells in cardiomyocytes. This may result from the enhancement ofspontaneous apoptosis due to a longer time (24 h) in culture withoutFBS. In NE-stimulated cardiomyocytes, overexpression of calpastatininhibited calpain activity, blocked caspase-3 activity, and reduced thepercentages of annexin V- and PI nuclear-positive cells (Figs.10B–10E).Thesedatastronglysupport thenotion that inhibitionofcalpain leads topreventionof cardiomyocyteapoptosis inducedbyNEand thusconfirmthat calpain induces apoptosis in NE-stimulated cardiomyocytes.

Discussion

Excessive sympathetic activation is a characteristic feature of heartfailure. NE, the primary neurotransmitter of the sympathetic nervoussystem, is able to induce cardiomyocyte apoptosis [30,31]. This studyinvestigated the mechanisms of the inhibitory effects of taurine onapoptosis in NE-stimulated cardiomyocytes. The major findings arethat taurine inhibits NADPH oxidase activation and ROS production,leading to prevention of apoptosis. Inhibition of NADPH oxidase abro-gates calpain activation in NE-stimulated cardiomyocytes. Preventionof calpain activation protects cardiomyocytes from apoptosis induced

Fig. 8. Effects of taurine on Ca2+ changes in NE-stimulated cardiomyocytes. ARVC wereincubated with NE in the presence of taurine or vehicle. Ca2+ (A) oscillatory frequencyand (B) amplitudewere determined (see details underMaterials andmethods). Data aremeans±SD from three different cells in each group. ⁎pb0.05 vs sham+vehicle, #pb0.05vs NE+vehicle.

Fig. 9. Effects of calpain inhibitors on apoptosis. ARVC were incubated with NE in thepresence of PD150606 (5 μM) or calpain inhibitor-III (CI-III; 5 μM) or vehicle for 18 h.Apoptosis was assessed by caspase-3 activation and annexin V staining as well as PInuclear staining. (A) Caspase-3 activity in cardiomyocytes. (B and C) The percentages ofannexin V-positive cells and PI nuclear-positive cells. Data are means±SD from at leastthree different cell cultures. ⁎pb0.05 vs control+vehicle, #pb0.05 vs NE+vehicle.

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by NE. Thus, inhibition of NADPH oxidase and calpain activation mayrepresent a novelmechanism for the antiapoptotic effects of taurine incardiomyocytes. Furthermore, we demonstrate for the first time thatcalpain activation is mediated by NADPH oxidase-dependent mechan-isms in NE-stimulated cardiomyocytes.

Taurine protects cardiomyocytes from apoptosis through downregulationof NADPH oxidase activation

This study demonstrates that taurine prevents NE-inducedapoptosis in cardiomyocytes. NE has been shown to induce apoptosisthrough activation of adrenoreceptors [37]. Although even higherdoses of NE (N5 μM) have been widely used to induce apoptosis incardiomyocytes [30,31], the 5 μM concentration of NE is more thanthat for receptors. It is currently unknown whether this dose of NEregulates adrenoreceptors in our system. NE has been also shown tobe oxidized to produce adrenochrome, which leads to damage tocardiomyocytes [33]. Our study confirms that adrenochrome inducesapoptotic cell death in cardiomyocytes. Thus, NE may also inducecardiomyocyte apoptosis through its adrenochrome product. Moreimportantly, this effect of adrenochrome on apoptosis is also abro-gated by treatment with taurine. However, the mechanisms by whichtaurine exerts its antiapoptotic effect remain not fully understood incardiomyocytes.

Taurine is an endogenous antioxidant. Studies have demonstratedthat taurine reduces ROS production induced by iron overload [4] orhyperhomocysteinemia in the heart [12]. Our results confirm thattaurine decreases H2O2-induced ROS in cardiomyocytes. However, it iscurrently unclear whether taurine regulates ROS generation. Becauseinhibition of ROS prevents apoptosis and NADPH oxidase is animportant source of ROS in cardiomyocytes, our working hypothesesfor the present study are that NE induces NADPH oxidase activationand taurine inhibits NADPH oxidase in cardiomyocytes. Our resultsshow that inhibition of NADPH oxidase blocks apoptosis in NE-stimulated cardiomyocytes. This is consistent with a recent report

demonstrating that activation of adrenoreceptors by NE inducesNADPH oxidase activation, which contributes to apoptosis [38]. Moreimportantly, we demonstrate for the first time that taurine preventsNADPH oxidase activation in NE-stimulated cardiomyocytes. Thus, inaddition to scavenging ROS, inhibition of NADPH oxidase activation isanother important mechanism for taurine's antiapoptotic action. Theunderlying mechanisms by which taurine inhibits NADPH oxidase arecurrently not fully understood. In neutrophils, it has been suggestedthat taurine may play an inhibitory role in p47phox phosphorylationand translocation, an important step in the assembly and activation ofNADPH oxidase [39]. Whether this mechanism is also operative incardiomyocytes requires future investigation.

NADPH oxidase contributes to calpain activation

Calpain activity is mainly regulated by altering the Ca2+ concentra-tion required for its proteolytic activity, normally with approximatelymicromolar concentrations of Ca2+ for the maximal calpain-1 andapproximately millimolar concentrations for calpain-2 activity [21].The Ca2+ requirement for calpain activity is modulated by severalmechanisms. For example, certain phospholipids, with phosphatidy-linositol being the most effective, would lower the Ca2+ concentrationrequired for autolysis of calpain-1 and calpain-2 [40]. Calpain activitycan also be regulated through phosphorylation by MAPK, proteinkinase C, and protein kinase A [41,42]. Nevertheless, the regulation ofcalpain activation has not been fully understood. In cardiomyocytes, itremains unknownwhether NE can induce calpain activation. A recentstudy has suggested that activation of adrenoreceptors by NE inducesthe [Ca2+]i elevation [43]. Similarly, our results show that NEtreatment increases Ca2+ oscillatory frequency in cardiomyocytes. An

Fig.10. Role of calpastatin in calpain activity and apoptosis in cardiomyocytes. ARVCwere infectedwith Ad-CASTor Ad-gal for 24 h, followed by incubationwith NE or vehicle for 18 h.(A) Calpastatin protein, (B) calpain activity, (C) caspase-3 activity, and (D) annexin V- and (E) PI nuclear-positive cells were determined. (A) A representative Western blot forcalpastatin protein (110 kDa) in triplicate from three different cultures in each group. (D and E) The percentages of annexin V- and PI nuclear-positive cells in NE-stimulatedcardiomyocytes. Data are means±SD from at least three different cell cultures. ⁎pb0.05 vs Ad-gal+vehicle, #pb0.05 vs Ad-gal+NE.

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increase in the intracellular Ca2+ concentration is an importantmechanism for calpain activation. In this regard, this study demon-strates that NE increases calpain activity in cardiomyocytes. Wefurther show that the proportion of calpain activity attributable tocalpain-1 (calpain activity at 5 μMCa2+) is more than 67% of total Ca2+-stimulated calpain activity in response to NE. Thus, NE treatmentinduces calpain-1 activation in cardiomyocytes.

This study has shown that NE activates NADPH oxidase. Activationof NADPH oxidase results in ROS production. Studies have demon-strated that ROS induce calpain activation, likely by increasingintracellular calcium levels through modulating L-type calciumchannels [44]. In this study, we also show that H2O2 increases calpainactivity in cardiomyocytes. We further show that calpain is activatedin parallel with NADPH oxidase activation in response to NE and, moreimportantly, inhibition of NADPH oxidase or ROS production abro-gates calpain activation in cardiomyocytes. Although pharmacologicalinhibitors of NADPH oxidase may have other nonspecific effects,peptide gp91ds-tat has been demonstrated to specifically blockNADPH oxidase assembly and thus inhibit its activation [34]. Thus,NE induces calpain activation through NADPH oxidase-mediated ROS-dependent pathway in cardiomyocytes.

Taurine prevents calpain activation

The present study also demonstrates that acute taurine supple-ment prevents calpain activation in NE-stimulated cardiomyocytes.

This is in agreement with a recent study showing that taurine down-regulates calpain activity in a rat model of focal cerebral ischemia [45].We further demonstrate that the inhibitory effect of taurine on calpainactivity is mediated via down-regulation of NADPH oxidase activationand reduction of ROS. IncreasedROShavebeen shown to elevate [Ca2+]i[45], which may be responsible for calpain activation. As such, thesignaling mechanisms in inhibition of calpain activation by taurinemay involve [Ca2+]i. Indeed, our results show that taurine inhibits Ca2+

oscillatory frequency and amplitude, suggesting that taurine decreasesCa2+ signals. The effect of taurine on modulation of Ca2+ homeostasishas been suggested in previous studies. Taurine has been shown todecrease [Ca2+]i induced by angiotensin-II in cardiomyocytes [46].Taurine also exerts potent inhibitory actions on ionic currents underCa2+ overload conditions in rabbit sinoatrial nodal cells [11]. However,it is currently unknown whether taurine modulates [Ca2+]i merelythrough inhibition of ROS production or throughmultiplemechanismsin cardiomyocytes, which merits further investigation.

Calpain mediates cardiomyocyte apoptosis

Studies have suggested that calpain is an important player in celldeath signaling. Partial cleavage of pro- or antiapoptotic proteins bycalpain might activate or inactivate, respectively, putative substrates,including caspase-3, -7, -8, -9, and -12; Bcl-2; Bcl-xl; Bid; Bax; and NF-κB [21,47]. Calpain has been implicated in ischemia/reperfusion-induced apoptosis in the heart [48,49] and plays a role in TNF-α-

Fig. 11. Schematic mechanism of taurine's antiapoptotic action in cardiomyocytesduring NE stimulation. NE induces NADPH oxidase activation and ROS production.NADPH oxidase-produced ROS activate calpain through Ca2+, leading to caspase-3activation and apoptotic death in cardiomyocytes. Taurine protects cardiomyocytesfrom apoptotic death by inhibiting NADPH oxidase, ROS production, and calpainactivation. Overexpression of calpastatin blocks calpain activation, leading to inhibitionof caspase-3 activity and apoptosis.

60 Y. Li et al. / Free Radical Biology & Medicine 46 (2009) 51–61

mediated apoptosis in cardiomyocytes [22]. These studies suggest thatcalpain activation may contribute to the progression of heart failure.This was indeed supported by a recent study that demonstrated thatcardiac overexpression of calpain-1 is sufficient to cause heart failurein transgenic mice [36]. In the present study, we also show that phar-macological inhibition of calpain blocks apoptosis in NE-stimulatedcardiomyocytes. The antiapoptotic effect of calpain inhibition is furtherconfirmed by overexpression of calpastatin in cardiomyocytes, whichspecifically blocks calpain activation, abrogates caspase-3 activation,and subsequently prevents apoptosis in cardiomyocytes. Thus, calpaininduces apoptosis in NE-stimulated cardiomyocytes, and this proa-poptotic action may be mediated by the caspase-3-dependent path-way because inhibition of caspase-3 activity significantly decreasescardiomyocyte death.

In summary, this study demonstrates a pivotal role for NADPHoxidase in mediating apoptosis in cardiomyocytes during NE stimula-tion. NADPH oxidase induces calpain activation, presumably throughCa2+ changes, leading to apoptosis. Taurine protects cardiomyocytesfrom apoptosis, at least in part by inhibiting NADPH oxidase-mediatedcalpain activation (Fig. 11). The discovery that inhibition of NADPHoxidase-mediated calpain activation contributes to the antiapoptoticeffects of taurine offers insights into the signaling mechanismsrequired for taurine's cardioprotective effects, which may helpprovide biological end-points for the clinical evaluation of taurine'spotential as a cardiovascular therapeutic.

Acknowledgments

This study was supported by an operating grant and Rick GallopAward for research excellence awarded to Dr. Tianqing Peng from theHeart & Stroke Foundation of Ontario (NA 5940). Dr. Tianqing Peng isthe recipient of a New Investigator Award from the Heart & StrokeFoundation of Canada.

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