antiarrhythmic effect of tamoxifen on the vulnerability induced by hyperthyroidism to heart...

1

2

3 Q1

Q24

5

6

7

8

9

10

11

12

13

14

15

16

17

Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx

G Model

SBMB 4215 1–8

Antiarrhythmic effect of tamoxifen on the vulnerability induced byhyperthyroidism to heart ischemia/reperfusion damage

Natalia Pavón b, Luz Hernández-Esquivel b, Mabel Buelna-Chontal a, Edmundo Chávez b,*aDepartamento de Bioquímica, Departamento de Biomedicina Cardiovascular, México, D.F., Mexicob Instituto Nacional de Cardiología, Ignacio Chávez, México, D.F., Mexico

A R T I C L E I N F O

Article history:Received 5 March 2014Received in revised form 6 May 2014Accepted 5 June 2014Available online xxx

Keywords:TamoxifenHyperthyroidismHeart damageIschemia/reperfusionMitochondriaPermeability transition

A B S T R A C T

Hyperthyroidism, known to have deleterious effects on heart function, and is associated with anenhanced metabolic state, implying an increased production of reactive oxygen species. Tamoxifen is aselective antagonist of estrogen receptors. These receptors make the hyperthyroid heart more susceptibleto ischemia/reperfusion. Tamoxifen is also well-known as an antioxidant. The aim of the present studywas to explore the possible protective effect of tamoxifen on heart function in hyperthyroid rats. Ratswere injected daily with 3,5,30-triiodothyronine at 2 mg/kg body weight during 5 days to inducehyperthyroidism. One group was treated with 10 mg/kg tamoxifen and another was not. The protectiveeffect of the drug on heart rhythm was analyzed after 5 min of coronary occlusion followed by 5 minreperfusion. In hyperthyroid rats not treated with tamoxifen, ECG tracings showed post-reperfusionarrhythmias, and heart mitochondria isolated from the ventricular free wall lost the ability to accumulateand retain matrix Ca2+ and to form a high electric gradient. Both of these adverse effects were avoidedwith tamoxifen treatment. Hyperthyroidism-induced oxidative stress caused inhibition of cis-aconitaseand disruption of mitochondrial DNA, effects which were also avoided by tamoxifen treatment. Thecurrent results support the idea that tamoxifen inhibits the hypersensitivity of hyperthyroid ratmyocardium to reperfusion damage, probably because its antioxidant activity inhibits the mitochondrialpermeability transition.

ã 2014 Published by Elsevier Ltd.

Contents lists available at ScienceDirect

Journal of Steroid Biochemistry & Molecular Biology

journal homepage: www.else vie r .com/locate / j sbmb

18

19

20

21

22

23

24

25

26

27

28

29

30

31

1. Introduction

Hyperthyroidism is commonly associated with an increase inmorbidity and mortality in the face of cardiovascular disease. Someof the cardiovascular characteristics of hyperthyroidism are hearthypertrophy, hyperdynamic circulation with improved cardiacoutput, ventricular arrhythmias, a faster heartbeat and a decreasein vascular peripheral resistance [1–3]. In addition, hyperthyroid-ism is associated with an enhanced metabolic state, implyingincreased oxygen consumption due to greater induction of geneexpression for proteins of the mitochondrial respiratory chain by T3 [4]. The latter hormone is linked to over-production of reactiveoxygen derived species (ROS), whose major generation sites aremitochondrial Complex I, II and III [5,6]. Therefore, an important

32

33

34

35

36

* Corresponding author at: Instituto Nacional de Cardiologia, Mexico, D.F.014080, Mexico. Tel.: +52 55 5573 2911; fax: +52 55 5573 0926.

E-mail address: [email protected] (E. Chávez).

http://dx.doi.org/10.1016/j.jsbmb.2014.06.0060960-0760/ã 2014 Published by Elsevier Ltd.

Please cite this article in press as: N. Pavón, et al., Antiarrhythmic effect ofischemia/reperfusion damage, J. Steroid Biochem. Mol. Biol. (2014), http

target of oxidative stress-induced injury due to hyperthyroidism isthe mitochondrion [7].

In animal models of hyperthyroidism, the antioxidant capacityof mitochondria is considerably reduced [8–10], leaving theseorganelles very vulnerable to oxidative stress. This process bringsabout a transition from specific to non-specific membranepermeability. The opening of a transmembrane pore to a diameterof 2–3 nm allows for the release of matrix molecules withmolecular weight up to 1500 Da, resulting in membrane leakage[11]. Furthermore, the increase in inner membrane permeabilityleads to detachment of cytochrome c from the cytosol side of theinner membrane [12,13], thus increasing susceptibility to apopto-sis [14]. It should be noted that non-specific mitochondrialpermeability is caused not only by greater ROS generation, butalso by massive Ca2+ accumulation [15].

Some reports point out that membrane permeability transitionunderlies heart ischemia/reperfusion injury induced by hyperthy-roidism [7,8,16]. Several drugs and chemicals have been used toprotect the heart from the deleterious effects of hyperthyroidism,

tamoxifen on the vulnerability induced by hyperthyroidism to heart://dx.doi.org/10.1016/j.jsbmb.2014.06.006

mailto:[email protected]

http://dx.doi.org/10.1016/j.jsbmb.2014.06.006



http://www.sciencedirect.com/science/journal/09600760

www.elsevier.com/locate/jsbmb

37 in38 [239 tr40

41 an42 Cu43 op44 an45 m46 ra47

48 to49 Al50 to51 to52 re53

54 th55 dr56 is57 m58 m59 le60 th61 le

62 2.

63 2.1

64

65 w66 3,67 pr68 ca69 an70 Bi71 ch72 m

73 2.

74

75 w76 no77 ra

78 2.

79

80 an81 m82 M83 el84 tr85 th86 w87 pe88 fir89 pr90 th

91 2.

92

93 le

94

95

96

97

98

99

100

101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142

143

144

2 N. Pavón et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx

G Model

SBMB 4215 1–8

cluding octylguanidine [16], b-blockers [17,18] and vitamin E1]. Octylguanidine is an inhibitor of mitochondrial permeabilityansition [20].Another drug that may be useful in this sense is the agonist/tagonist of estrogen receptors, tamoxifen, which according tostodio et al. [22] and Hernández-Esquivel et al. [23] inhibits theening of non-specific pores. Along the same line, Ek et al. [24]d Diez et al. [25] have reported that tamoxifen protectsyocardium from reperfusion-induced damage in ovariectomizedts.Regarding hyperthyroidism, tamoxifen alleviates the symp-

ms of this disorder that appear in the syndrome of Mc Guire-len [26]. Additionally, the fact that women are more susceptible

develop an autoimmune thyroid disease [27] adds plausibility the use of tamoxifen, which is an antagonist of estrogenceptors.The aim of the present study was to explore the possibilityat tamoxifen, through its antioxidant activity, could be a usefulug for circumventing the deleterious effects of hyperthyroid-m on heart function. For this purpose determinations wereade of the effect of tamoxifen on post-reperfusion arrhyth-ias, the concentration of inflammation markers (e.g., inter-ukins), the mitochondrial permeability transition, the level ofe cis-aconitase enzyme, mitochondrial DNA, and matrix Ca2+

vels.

Materials and methods

. Rat hyperthyroidism

Hyperthyroidism was established in female Wistar rats,eighing between 300 and 350 g, by a daily i.p. injection of5,30-triiodothyronine at 2 mg/kg body weight for 5 days, aseviously reported [16]. The protocol of the present study wasrried out according to the procedures published by NIH for cared handling of laboratory animals, and was approved by theoethics Commission of our institution. T3 was determined by aemiluminescence assay in rat blood serum and expressed as theean � SD of 9 different samples.

2. Tamoxifen administration

Tamoxifen was injected i.p. daily, at a dose of 10 mg/kg bodyeight during 5 days, following each injection of T3. It should beted that the LD50 for tamoxifen has been found to be 5 g/kg ints when administered i.p. [28].

3. Heart reperfusion

To evaluate heart reperfusion damage/protection, rats wereesthetized with sodium pentobarbital (55 mg/kg, i.p.) andaintained under assisted respiration through a tracheotomy.eanwhile, heart rate was monitored with a led-II surfaceectrocardiograph, and blood pressure measured with a pressureansducer attached to a femoral cannula. The chest was opened byoracotomy and the left coronary artery ligated near its originith an intramural 6.0 silk loop. Occlusion of the artery wasrformed by passing a short tube over the vessel and clamping itmly. The ischemic period lasted 5 min, in agreement withevious reports [16,29,30]. Reperfusion was started by removinge clamp, and also lasted 5 min.

4. Mitochondria preparation and function analysis

Mitochondria were prepared by homogenizing tissues from theft ventricle in 250 mM sucrose-1 mM EDTA adjusted to pH 7.3,

Please cite this article in press as: N. Pavón, et al., Antiarrhythmic effect oischemia/reperfusion damage, J. Steroid Biochem. Mol. Biol. (2014), ht

and then following the standard centrifugation procedure. Proteinlevels were determined according to the Lowry method [31]. Ca2+

uptake was tracked spectrophotometrically at 675–685 nm usingthe arsenazo III indicator. The transmembrane electric gradientwas assayed spectrophotometrically at 525–575 nm using Safra-nine dye. Mitochondrial inflammation was analyzed at 540 nm.Oxygen consumption was assayed polarographically using a Clarktype electrode. The incubation media are described in therespective figure legends.

2.5. Analysis of mitochondrial DNA disruption

Mitochondrial DNA was isolated as described by García et al.[32]. The genetic material was analyzed in 0.8% agarose gel andvisualized by adding ethidium bromide.

2.6. Superoxide dismutase activity

Superoxide dismutase activity was determined in mitochondriaby non-denaturating 8% acrylamide gel electrophoresis and nitroblue tetrazolium staining, as described by Pérez-Torres et al. [33].

2.7. Aconitase activity

Aconitase activity was analyzed according to the procedurereported by Hausladen and Fridovich [34]. Briefly, mitochondrialprotein was solubilized by adding 0.05% Triton X-100 containing25 mM phosphate, pH 7.2, followed by 0.6 mM manganesechloride, 1 mM citrate and 0.1 mM NADP. The cis-aconitase formedwas measured spectrophotometrically at 240 nm.

2.8. TBARS determination

Mitochondrial membrane lipid peroxidation was determinedspectrophotometrically as the concentration of thiobarbituric acidreactive substances (TBARS). A tetraethoxypropane curve was usedas the standard.

2.9. Analysis of cytochrome c

Mitochondrial cytochrome c content was analyzed by Westernblot. First, 15 mg of mitochondrial protein was loaded onto 15%acrylamide SDS-PAGE gel and then transferred to a PVDFmembrane for immunodetection. A primary monoclonal antibodyagainst cytochrome c (1:1000 dilution) and a secondary alkaline-phosphate conjugated antibody were used to evaluate themitochondrial content of this enzyme.

2.10. Determination of cytokines

At the end of the experiment, samples of the left ventricularwall were obtained to estimate the amount of IL1, IL 6 and TNFareleased, according to the method described by Pavón et al. [16].The values of cytokines were analyzed through a sandwich ELISAmethod [35].

2.11. Statistical analysis

The Student’s t test for unpaired data was used to compare thebaseline variables of the groups. The ANOVA test was employed todetermine significant differences, which were then analyzed withthe Newman–Keuls post-test to find intergroup differences. Dataare expressed as the mean � SD, and p < 0.05 was consideredstatistically significant. Analysis was performed with the Prism 5.0statistical package.

f tamoxifen on the vulnerability induced by hyperthyroidism to hearttp://dx.doi.org/10.1016/j.jsbmb.2014.06.006


145

146

147

148

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180

181

182

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

198

199

200

201

202

203

204

205

206

207

208

209

210

211

Fig. 2. Protective effect of tamoxifen, maintaining Ca2+ accumulation in heartmitochondria from rats with hyperthyroidism. Mitochondria (2 mg protein) wereincubated at 25 �C in 3 ml of a medium containing 125 mM KCl, 10 mM succinate,10 mM HEPES, 3 mM phosphate, 100 mM ADP, 50 mM CaCl2, 5 mg rotenone, 2 mgoligomycin and 50 mM arsenazo III. Trace a represents mitochondria isolated fromthe hearts of euthyroid rats. Trace b and trace c depict mitochondria isolated fromhyperthyroid rats treated and not treated with tamoxifen, respectively. The tracesare representative of six separate experiments.

Fig. 1. Electrocardiogram and blood pressure tracings of hearts from control (Panel A), hyperthyroid (Panel B) and hyperthyroid/tamoxifen-treated rats (Panel C).

N. Pavón et al. / Journal of Steroid Biochemistry & Molecular Biology xxx (2014) xxx–xxx 3

G Model

SBMB 4215 1–8

3. Results

The value of T3 in control rats was 56 � 7 ng/dl, in hyperthyroidrats 4008 � 1668 ng/dl, and in hyperthyroid/tamoxifen-treatedrats 4563 �1555 (p < 0.001).

Hyperthyroidism, associated with reperfusion-induced myo-cardium calcium overload, induced an increase of cardiacfrequency and arrhythmias. Fig. 1 shows ECG tracings and bloodpressure data from the control (Panel A), hyperthyroid (Panel B)and hyperthyroid/tamoxifen-treated rats (Panel C). Panel A depictsthe electrical potential profile of control hearts before beingsubjected to ischemia/reperfusion. The hearts presented a sinusrhythm (SR) and blood pressure was normal. The lower ECG tracingin Panel A shows that after 5 min of reperfusion there was elevatedventricular fibrillation and arrhythmias compared to controlhearts. Blood pressure was absent. Panel B shows ECG tracingsof hearts undergoing hyperthyroidism. An increase in ventriculartachycardia is clearly observed even before reperfusion. It shouldbe noted that blood pressure was maintained at a value similar tothat observed in control hearts. The lower ECG tracings in Panel Bshow that once blood flow was reestablished, heart frequency andblood pressure were low. Regarding the hearts of hyperthyroid ratstreated with tamoxifen (Panel C), the electrical behavior indicates asinus rhythm before the reperfusion phase, along with thepresence of ventricular tachycardia. Remarkably, blood pressureis quite similar to that of control hearts. Interestingly, a differentpicture is revealed by the ECG tracings, since tamoxifen treatmentpreserved the sinus rhythm even after blood flow was reestab-lished.

It has been reported that T3 treatment induces an abruptincrease of mitochondrial nonspecific pore opening, whichunderlies reperfusion heart damage [16]. The susceptibility tosuch a process is amplified by subjecting the heart to an ischemia/reperfusion procedure. The permeability transition is characteris-tic of mitochondrial dysfunction, and is caused by the failure toretain matrix Ca2+ content as a result of membrane leakage.

Thus, an assay was performed to determine the levels of Ca2+ inmitochondria (Fig. 2) in order to assess the possible protectiveeffect of tamoxifen on membrane leakage in hyperthyroid rats. Inmitochondria isolated from the hearts of euthyroid rats, accumu-lated Ca2+ was retained. However, the opposite occurred inmitochondria isolated from the hearts, subjected to ischemia/reperfusion, of hyperthyroid rats. After a short period of Ca2+

uptake, the cation was released due to the opening of pores and theconsequent membrane leakage. On the other hand, in mitochon-dria isolated from the hearts, also subjected to ischemia/reperfusion, of hyperthyroid rats treated with tamoxifen, Ca2+

accumulation in the matrix was retained, indicating that the non-specific pore remained closed.


Analysis of membrane energization (Dc) is useful for assessingthe intactness of the inner membrane after Ca2+ accumulation. Thedetermination of Dc (Fig. 3) in the different groups illustrates thatthis parameter was low in mitochondria isolated from hearts ofhyperthyroid rats, and that a short time after Ca2+ was added therewas a rapid drop in this value (trace a). Even with reperfusion,mitochondria from hyperthyroid rats was unable to preserve a highmembrane potential value (trace d). In contrast, mitochondriaisolated from hearts of hyperthyroid rats treated with tamoxifenmaintained a high value of Dc before and after the addition of Ca2+

(trace b).Protection of mitochondria can also be evaluated by analyzing

inflammation. Suppression of this process would indicate protec-tion against oxidative stress membrane damage. Heart mitochon-dria isolated from control rats did not suffer inflammation eitherbefore or after the addition of Ca2+ (Fig. 4, trace a). Contrarily, heartmitochondria isolated from normal rats and subjected to ischemia/reperfusion underwent inflammation after the addition of Ca2+

(trace d). Similarly, heart mitochondria isolated from hyperthyroid



212 ra213 ex214 hy215 no216

217 ov218 de219 ta220 th221 heQ3

222

223

224

225

226

227

228

229

230

231

232

233

234

235

236

237238239240241242243244245246

247

248

249

250

251

252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

275

276

277

278

279

280

281

282

283

284

285

286

287

Figfourepthb

represrat

Fig. 3. Protection by tamoxifen against hyperthyroidism and reperfusion damage,evidenced by determining the mitochondrial transmembrane electric gradient.Experimental conditions were similar to those described in Fig. 2, except that themedium contained 10 mM safranin instead of arsenazo III. Trace a and trace b showthe Dc of mitochondria from hyperthyroid rat hearts subjected to ischemia/reperfusion, untreated and treated with tamoxifen, respectively. Trace c shows theDc of heart mitochondria from euthyroid rats. Where indicated, 50 mM calciumand 0.5 mM CCCP were added.


G Model

SBMB 4215 1–8

ts and subjected to ischemia/reperfusion suffered rapid andtensive swelling after the addition of Ca2+ (trace b). Inperthyroid/tamoxifen-treated rats, the addition of Ca2+ didt induce swelling or the opening of non-specific pores (trace c).Dysfunction of the respiratory chain can also result from theerproduction of reactive oxygen species [36]. Therefore, atermination was made of the possible protective effect ofmoxifen against the damage to oxidative phosphorylation anderefore to the respiratory control induced in hyperthyroid ratarts subjected to reperfusion (Table 1).

288

289

290

291

292

293

294

295

296

297

298

299

300Q4301

302

303

304

305

306

307

308

309

310

311

. 4. Protective effect of tamoxifen against the Ca2+-induced inflammationnd in mitochondria isolated from hyperthyroid hearts subjected to ischemia/erfusion. Mitochondria (2 mg protein) were incubated in conditions similar to

ose described in Fig. 2, except that arsenazo III was not added. Trace a and traceillustrate the behavior of mitochondria of hearts subjected to ischemia/erfusion, from hyperthyroid rats untreated and treated with tamoxifen,pectively. Trace c shows the behavior of heart mitochondria from euthyroids. Where indicated, 50 mM CaCl2 was added.


Mitochondrial respiratory control is defined as the ratio betweenthe rate of oxygen consumption (nAtg O2/min/mg protein) afterthe addition of ADP and the rate of oxygen consumption withoutADP. Respiratory control with glutamate-malate in euthyroidmitochondria that were not subjected to ischemia/reperfusionattained a value of 5 � 0.5. However, hyperthyroid mitochondriasubjected to ischemia/reperfusion almost completely lost theability to establish respiratory control, evidenced by a value of 1.0,representing a significant difference (p < 0.001; n = 4). Contrarily,in mitochondria from hyperthyroid rat hearts treated withtamoxifen, the value of respiratory control was 4.8 � 0.4 after5 min ischemia and 5 min reperfusion, representing a normal level.The values for respiratory control were quite similar when usingsuccinate as the oxidized substrate.

The low value of respiratory control in hyperthyroid rat heartssubjected to ischemia/reperfusion is conceivably due to mitochon-drial leakiness that should be induced, in part, by oxidation ofmembrane polyunsaturated fatty acids. Thus, the resultingincrease in the concentration of malondialdehyde should be auseful parameter to estimate hyperthyroid-induced oxidativestress [37]. A higher amount of reactive species was generatedin reaction to thiobarbituric acid in mitochondria from hyperthy-roid than normal rats (Fig. 5). The concentration of TBARS wasabout 75% less in mitochondria from hyperthyroid rats treatedwith tamoxifen compared hyperthyroid animals not receiving thisdrug (p < 0.01; n = 6).

Apart from the analysis of TBARS concentration, aconitaseactivity is a reliable marker for assessing damage to mitochondriaby the oxidative stress of hyperthyroid rat hearts. Aconitaseactivity is induced by the superoxide formed on the matrix side ofComplex III [38,39].

Compared to control animals, after inducing reperfusion inhyperthyroid rat heart mitochondria, the activity of this enzymewas inhibited by approximately 75%, but was only inhibited byabout 25% in mitochondria from tamoxifen-treated animals,representing a significant difference (p < 0.001; n = 5).

In mitochondria isolated from hyperthyroid rat heart that isoccluded and reperfused, the increase found in oxidative stressshould be linked to a decrease in the activity of the oxyradical-scavenging system. In tamoxifen-treated animals, this decrease inanti-oxidant activity should be avoided. To test this idea, theactivity of mitochondrial superoxide dismutase (SOD) wasanalyzed (Fig. 6). Indeed, the value of SOD activity in mitochondriaisolated from hyperthyroid rat hearts (occluded and reperfused)was around 50% of that observed in mitochondria isolated from thehearts of tamoxifen-treated rats (***p < 0.001).

Both over production of thyroid hormones and reperfusion injuryare pathological states that lead to inflammation, which should beaccompanied by an increase in serum interleukins. Hence, the levelsof IL-6, IL-1 and TNFa were determined in hyperthyroid rat heartsafter ischemia/reperfusion, both with and without tamoxifentreatment. After 20 min reperfusion, tamoxifen treatment dimin-ished the level of IL-6 from 50 to 25 pg/mg of protein, and TNFa from45 to 35 pg/mg of protein, representing significant differences(p < 0.001; n = 10). However, it is worth noting that tamoxifenexercised no decrease in the level of IL-1 (Fig. 7).

Previous reports indicate that permeability transition poreopening underlies the progression of the apoptotic mitochondrialpathway, providing a mechanism for cytochrome c detachmentfrom the cytosol side of the inner membrane [12,13]. Apoptotic celldeath has been involved in post-reperfusion tissue injury and heartdysfunction. Thus, we explored the possibility that tamoxifencould protect against cytochrome c detachment after a reperfusionperiod. Indeed, treatment with this selective modulator of estrogenreceptors induced the retention of cytochrome c on the innermembrane (p < 0.05; n = 3; Fig. 8).



312

313

314

315

316

317

318

319

320

321

322

323

324

325

326

327

328

329

330

331

332

333

334

335

336

337

338

339

340

341

342

343

344

345

346

347

348

349

350

351

352

353

354

Fig. 6. Protection exerted by tamoxifen against the decrease in superoxide dismutaseExperimental conditions as described in Section 2. The bars represent the average � SD oway ANOVA with either the Student’s t or Newman–Keuls test, using Prism 5.0 softwa

Fig. 5. Evaluation of the protection induced by tamoxifen against the increasedgeneration of TBARS found in mitochondria isolated from hearts subjected toischemia/reperfusion in hyperthyroid rats. The TBARS level was determined byincubating mitochondria (2 mg protein) isolated from the hearts of control (CON),hyperthyroid-reperfused (T3 I/R) and hyperthyroid/reperfused/tamoxifen-treatedrats (TAM + T3 I/R) in 0.1 ml of basic medium during 30 min. Then 1.0 ml of 20%acetic acid and 0.8% 2-thiobarbituric acid were added. The mixture was heated inboiling water for 45 min. After cooling, TBARS were extracted in 2 ml n-butanol.After centrifugation, the butanol layer was measured at 532 nm. A standard MDAcurve was prepared with 1,3,3,3,-tertraetoxypropane. The values represent theaverage � SD of six different determinations (*p < 0.01 vs. hyperthyroid-perfusedrat hearts).

Table 1

Substrate State 4 State 3 RC ADP/O

Malate/glutamateControl 85 � 5 424 � 75.5 5 � 0.5 1.9 � 0.6T3-IR 60.2 � 1.6 60.2 � 1.6 1 –

T3-IR � tamoxifen 82.3 � 2* 400 � 87.2* 4.8 � 0.4* 2.1 � 0.1Succinate

Control 85 � 5 424.16 � 75 5 � 0.5 2 � 0.5T3-IR 60.1 � 16 60.1 � 16 1 � 0.6 –

T3-IR � tamoxifen 90.9 � 9* 350 � 54* 3.8 � 0.33* 1.7 � 0.4

Protective effect of tamoxifen against the deleterious effect of ischemia/reperfusionon the respiratory function of mitochondria isolated from the hearts ofhyperthyroid rats. Experimental conditions were similar to those described inFig. 3, except that Arsenazo III and Ca2+ were not added, and where indicated 10 mMmalate and 10 mM glutamate were added as substrates. Mitochondrial protein (0.6mg/ml) was used to analyze respiratory control. Values are expressed as the mean� SD of four different experiments.


G Model

SBMB 4215 1–8


Mitochondrial DNA is also a target of oxidative stress damage.Consequently, we examined damage to this polynucleotide inmitochondria isolated from hyperthyroid rat hearts subjected toischemia/reperfusion, with or without treatment with tamoxifen.Whereas the genetic material of mitochondria isolated fromhyperthyroid rat hearts was significantly disrupted (Fig. 9, T3 + IRtrack), it was found to be intact in control and tamoxifen-treatedrats (Fig. 9, Ctrl and T3 + IR + TAM track, respectively).

4. Discussion

Hyperthyroidism causes severe effects on the heart andcardiovascular system [1–3]. Among the several manifestationsof hyperthyroidism are increased blood pressure, atrial andventricular extra-systoles, atrial fibrillation and ventricular repo-larization [40]. The deleterious effects of thyroid hormones onheart tissue likely involve oxidative stress [7]. This process occurswhen ROS generation overrides the ability of the endogenousantioxidant enzymes.

It is generally accepted that the main generator of reactiveoxygen species is the mitochondrial respiratory chain [5,6], and T3induces an increased expression of respiratory enzymes [41].Moreover, hyperthyroidism promotes the depletion of antioxidantmolecules such as GSH [42].

Hyperthyroidism also sensitizes the rat heart to reperfusioninjury, which is characterized by severe arrhythmias and tissuedamage [16,21]. At the cellular level, L-thyroxin stimulates Ca2+

overload [43], and several publications indicate a close associationbetween this condition and alterations in the heart rhythm [44,45].Intracellular Ca2+ levels increase during myocardial ischemia [46].When blood flow is restored in the reperfusion period, a rapid andexcessive uptake of Ca2+ can occur with adverse electrophysiologi-cal effects.

Mitochondrial Ca2+ overload brings about the opening of non-specific transmembrane pore and therefore leads to mitochondrialdysfunction, which underlies the pathogenesis of heart reperfu-sion damage [15,16]. In this respect, García-Rivas et al. [46]demonstrated that Ru360, a specific mitochondrial calcium uptakeinhibitor, improves cardiac post-ischemic functional recovery.Hence, it can be inferred that protection of mitochondria from theoxidative stress-induced permeability transition should result inresistance to hyperthyroid-induced heart reperfusion injury.

In this sense, we previously demonstrated that octylguanidine,a reagent that inhibits carboxyatractyloside-induced permeabilitytransition [20], protects the heart against the deleterious effect of

found in mitochondria isolated from the hearts of hyperthyroid-reperfused rats.f 5 separate experiments statistical analysis was performed by non-parametric one-re (***p < 0.001, *p < 0.01).



355 hy356 th357 um358 ch

359Q5360

361Q6362

Fig. 7. Protection exerted by tamoxifenagainst thereleaseof proinflammatorycytokines found inthemyocardium of hearts, subjected to ischemia/reperfusion, from hyperthyroidrats. Left ventricular myocardium, frozen and ground, was homogenized in 50 mM HEPES, pH 7.5, 150 mM NaCl, 1% glycerol, 1% Triton X-100, 1.5 mM MgCl2, and 5 mM EGTAcontaining1 mMphenylmetylsulfonyl fluorideandaproteaseinhibitorcocktail. Lysates werecentrifugedat10,000gand proteinlevelwasdetermined.Cytokines weredeterminedby using specific antibodies with the ELISA method. The values represent the average � SD of 10 separate experiments. Statistical analysis was performed by non-parametric one-way ANOVA with either the Student’s t, Newman–Keuls or Dunett’s test using Prism 5.0 software (***p < 0.001, **p < 0.01 compared to the hyperthyroid-reperfused group).

Fig.

desccontn = 3


G Model

SBMB 4215 1–8

perthyroidism. On the other hand, previous reports have shownat tamoxifen impedes the harmful damage caused by myocardi-

reperfusion [47] and protects against stress-induced mito-ondrial permeability transition [48]. It should be mentioned that

8. Protection exerted by tamoxifen against the detachment of cytochrome c from thribed in Section 2. Panel A illustrates the Western blot image of cytochrome c retainedrol mitochondria (C); T3 + IR indicates the cytochrome retained in mitochondria isolat).


hyperthyroidism amplifies the susceptibility of mitochondria tothe permeability transition [8] and increases the vulnerability ofthe myocardium to oxidative stress and reperfusion damage [1–3](Table 2).

363

364

365

366

367

368

369

370

371

372

373

374

375

376

377

378

379

380

381

e cytosol side of the inner membrane of mitochondria. Experimental conditions as in the mitochondrial inner membrane. Track 1 shows the amount of cytochrome c ined from hearts, subjected to ischemia/reperfusion, from hyperthyroid rats (p < 0.05;



382383384385386387388389390391

392

393

394

395

396

397

398

399

400

401

402

403

404

405

406

407

408

409

410

411

412

413

414

415

416

417

418

419

420

421

422

423

424

425

426

427

428

429

430

431

432

433

434

435

436Q7

437

438

439440

441

442443444

445446

447448449450

Fig. 9. The protective effect of tamoxifen against hyperthyroidism-inducedoxidative damage on mitochondrial DNA. The experimental conditions aredescribed in Section 2. Mitochondria were isolated from hearts of hyperthyroidrats treated or not treated with tamoxifen. The lines show mitochondrial DNA fromcontrol (Ctrl), hyperthyroid-reperfused (T3 + IR) and hyperthyroid/reperfused/tamoxifen-treated rats (T3 + IR + TAM). The results represent data from threedifferent experiments.


G Model

SBMB 4215 1–8

In concordance with the evidence that oxidative stress plays acentral role in hyperthyroidism-induced heart damage, the presentstudy shows that tamoxifen, with known antioxidant properties,diminishes cardiac and cellular derangements.

That is, hearts from hyperthyroid rats treated with tamoxifenshowed an electrical profile with an almost total absence of cardiacarrhythmias. The electrical abnormality in cardiac rhythm is one ofthe characteristics of heart reperfusion injury [16].

Oxidative stress also exerts harmful effects on mitochondrialDNA [49] and inhibits the cis-aconitase enzyme. The currentresults indicate that treatment with tamoxifen impeded disruptionin the DNA structure and suppressed the inhibition of cis-aconitase

451

452453

454455456

457458459

460

461

Table 2

Condition nmol cis-aconitate/min/mg

Control 403 � 61T3-IR 107.3 � 30T3-IR + tamoxifen 306*� 47.1

Protective effect of tamoxifen against the deleterious effect ofhyperthyroidism and reperfusion on the cis-aconitase enzyme inmitochondria. The activity of the enzyme was determined in 150 mgof protein, as described in Section 2. Values are the mean � SD offive different mitochondrial preparations.


[48]. Additionally, the activity of superoxide dismutase is inhibitedafter acute myocardial ischemia and undergoes oxidative damageduring reperfusion. The present study demonstrated that tamoxi-fen treatment avoided oxidative damage to SOD.

Hernández-Esquivel et al. (2011) advocate the protective role oftamoxifen due to its antioxidant properties. Moreover, tamoxifenacts as an antagonist of estrogens, and these receptors make thehyperthyroid heart more susceptible to ischemia/reperfusion. It isalso important to consider that estrogens can protect the heartfrom reperfusion damage. This was demonstrated by the work ofRanki et al. [50], who reported that 17beta estradiol protectscardiac cells from hypoxia/reoxigenation by stimulating K(ATP)channel formation. In addition Ballantyne et al. [51] concluded thattestosterone, which is converted into metabolites that activateestrogen receptors, protects female embryonic H9c2 heart cellsfrom metabolic stress.

The results indicate that tamoxifen conferred protection againstpost-reperfusion arrhythmias, and diminished the concentrationof inflammation markers such as interleukins. Regarding hyper-thyroidism-induced mitochondrial oxidative stress, tamoxifenimpeded mitochondrial permeability transition and disruptionof mitochondrial DNA, and suppressed inhibition of the cis-aconitase enzyme. Furthermore, tamoxifen preserved the ability ofmitochondria to retain matrix Ca2+ and thus to maintain thetransmembrane electric gradient at a high level.

Although the protection exerted by tamoxifen against reperfu-sion damage has been reported previously [47], the currentfindings show, in a relevant manner, that tamoxifen protectsagainst the hyperthyroidism-induced vulnerability to heartreperfusion injury. Hence, these results provide robust evidencein support of the clinical use of tamoxifen as an antioxidant fordiminishing the adverse effects on the heart caused by hyperthy-roidism-induced oxidative stress.

Uncited reference

[19].

References

[1] K.A. Woeber, Thyrotoxicosis and the heart, N. Engl. J. Med. 327 (1992) 94–98.[2] I. Klein, G.S. Levey, The cardiovascular system in thyrotoxicosis, in: L.E.

Braveman, R.D. Utiger (Eds.), The Thyroid, eighth ed., Lippicott- Raven,Philadelphia, 2000, pp. 596–604.

[3] S. Danzi, I. Klein, Thyroid hormone and the cardiopvascular system, Med. Clin.North Am. 96 (2012) 257–268.

[4] E. Fernández-Vizarra, J.A. Enríquez, A. Pérez-Martos, J. Montoya, P. Fernández-Silva, Mitochondria gene expression is regulated at multiple levels anddifferentially in the heart and liver by thyroid hormones, Curr. Genet. 54(2008) 13–22.

[5] S. Drose, U. Brandt, Molecular mechanisms of superoxide productionby the mitochondrial respiratory chain, Adv. Exp. Med. Biol. 748 (2012)145–169.

[6] R. Moreno-Sánchez, L. Hernández-Esquivel, N.A. Rivero-Segura, A. Marín-Hernández, J. Nauzil, S.J. Ralph, S. Rodríguez-Enríquez, Reactive oxygenspecies are generated by the respiratory complex II-evidence for lack ofcontribution of the reverse electrán flow in complex I, FEBS J. 280 (2013)927–938.

[7] P. Venditti, S. Di Meo, Thyroid hormone-induced oxidative stress, Cell. Mol. LifeSci. 63 (2006) 414–434.

[8] P. Venditti, R. De Rosa, S. Di Meo, Effect of thyroid hormone on susceptibility tooxidants and inflammation of mitochondria from rat tissues, Free Radic. Biol.Med. 35 (2003) 485–494.

[9] H. Erdamar, H. Demirci, H. Yaman, M.K. Erbil, T. Yakar, B. Sancak, S. Elbeg, G.Biberoglu, I. Yetkin, The effect of hypothyroidism, hyperthyroidism, and theirtreatment on parameters of oxidative stress and antioxidant status, Clin.Chem. Lab. Med. 46 (2008) 1004–1010.

[10] M. Messarah, A. Boumendiel, A. Chouabia, F. Klibet, C. Abdennnour, M.S.Boulakoud, A.E. Feki, Influence of thyroid dysfunction on liver lipidperoxidation and antioxidant status in experimental rats, Exp. Toxicol. Pathol.62 (2010) 301–310.

[11] M. Crompton, A. Costi, L. Hayat, Evidence for the presence of reversible Ca2+-dependent pore activated by oxidative stress in heart mitochondria,Biochem. J. 245 (1987) 915–918.



[1462463

[1464465

[1466467

[1468469470

[1471

[1472473

[1474475

[1476477478

[2479480

[2481482

[2483484

[2485

[2486487

[2488489490

[2491492

[2493494495

[2496497498

[2499500

[3501502

[3503

504505

506507

508

509510

511512

513514

515516

517518519

520

521

522523

524525526

527528529

530531532

533534

535536

537538539

540

541542

543544545


G Model

SBMB 4215 1–8

2] F. Correa, V. Soto, C. Zazueta, Mitochondrial permeability transition relevancefor apoptotic triggering in the post-ischemic heart, Int. J. Biochem. Cell. Biol. 39(2007) 79l–787.

3] N. García, L. Hernández-Esquivel, C. Zazueta, E. Martínez-Abundis, N. Pavón, E.Chávez, Induction of mitochondrial permeability transition by the DNA-intercalating cationic dye ethidium bromide, J. Biochem. 146 (2009) 887–894.

4] Y.Y. Wang, B. Jiao, W.G. Guo, H.L. Che, Z.B. Yu, Excesive thyroxine enhancessusceptibility to apoptosis and decreases contractility of cardiomyocites, Mol.Cell. Endocrinol. 320 (2010) 67–75.

5] N. Pavón, J.C. Gallardo, L. Hernández-Esquivel, M. El-Hafidi, M. Buelna-Chontal,C. Zazueta, S. Rodríguez-Enríquez, E. Chávez, On the properties of calcium-induced permeability transition in neonatal heart mitochondria, J. Bioenerg.Biomembr. 43 (2011) 757–764.

6] N. Pavón, A. Aranda, L. García, E.E. Chávez, In hyperthyroid rats octylguanidineprotects heart from reperfusion damage, Endocrine 35 (2009) 158–165.

7] A. Fumarola, A. Di Fiore, M. Dainelli, G. Grani, A. Calvanese, Medical treatmentof hyperthyroidism: state of art, Exp. Clin. Endocrinol. Diabetes 118 (2010)678–684.

8] F. Kelestimur, A. Asku, The effect of diltiazem on the mainfestations ofhyperthyroidism and thyroid function test, Exp. Clin. Endocrinol. Diabetes 104(1996) 38–42.

9] M. Khorshidi-Behzadi, H. Alimoradi, S. Haghioo-Javanmard, M. Reza Sharifi, N.Rahimi, A.R. Dehapour, The effect of hyperthyroidism and restored euthyroidstate by methimazole therapy in rat small mesenteric arteries, Eur. J.Pharmacol. 701 (2012) 20–28.

0] E. Chávez, A. Peña, C. Zazueta, J. Ramírez, N. García, R. Carrillo, Inactivation ofmitochondrial permeability transition by octylguanidine and octylamine, J.Bioenerg. Biomembr. 32 (2000) 193–198.

1] P. Venditti, G. Napolitano, L. Di Stefano, C. Agnisola, S. Di Meo, Effect of vitaminE administration on response to ischemia-reperfusion on hearts from cold-exposed rats, Exp. Physiol. 96 (2011) 635–646.

2] J.B. Custodio, A.J. Moreno, K.B. Wallace, Tamoxifen inhibits induction of themitochondrial permeability transition by Ca2+ and inorganic phosphate,Toxicol. Appl. Pharmacol. 152 (1996) 10–17.

3] L. Hernández-Esquivel, N. Pavón, C. Zazueta, N. García, F. Correa, E. Chávez, LifeSci. 88 (2011) 681–687.

4] R.O. Ek, Y. Yildiz, S. Cecen, C. Yernisey, T. Kavak, Effects of tamoxifen onmyocardial ischemia-reperfusion injury model in ovariectomized rats, Mol.Cell. Biochem. 308 (2006) 227–235.

5] E.R. Diez, N.J. Prado, A.M. Carrión, E.R. Petrich, A.Z. Ponce-Zumino, R.M.Miatello, Electrophysiological effects of tamoxifen: mechanisms of protec-tionbagainst reperfusion arrhythmias in isolated rat hearts, J. Cardiovasc.Pharmacol. 62 (2013) 184–191.

6] E.A. Eugster, S.D. Rubin, E.O. Reiter, P. Plourde, O.H. Pescovitz, Tamoxifentreatment for precocious puberty in McCune–Albright syndrome: a multicen-ter trial, J. Pediatr. 143 (2003) 60–66.

7] V. Estienne, C. Duthoity, M. Reichert, A. Praeetor, P. Carayon, W. Hunziker, J. Ruf,Androgen-dependent expression of FcgammaRIIB2 by thyrocytes frompatients with autoimmune Graves’ disease: a possible molecular clue forsex dependence of autoimmune disease, FASEB J. 16 (2002) 1087–1092.

8] J. Seibler, B. Zevnik, B. Kuter-Luks, S. Andreas, H. Kern, T. Hennnek, A. Rode, C.Heimann, N. Faust, G. Kauselmann, M. Schoor, R. Jaenisch, K. Rajewsky, R. Kohn,F. Schwenk, Rapid generation of inducible mouse mutants, Nucleic Acids Res.31 (2003) e12.

9] D. Arteaga, A. Odor, R.M. López, G. Contreras, J. Pichardo, E. García, A. Aranda, E.Chávez, Impairment by cyclosporin A of reperfusion-induced arrhythmias, LifeSci. 51 (1992) 1127–1134.

0] E. Parra, D. Cruz, G. García, C. Zazueta, F. Correa, N. García, E. Chávez,Myocardial protective effect of octylguanidine against the damage induced byischemia-reperfusion in rat heart, Mol. Cell. Biochem. 269 (2005) 19–26.

1] O.H. Lowry, N.J. Rosebrough, A.I. Farr, R.J. Randal, Protein measurement withthe folin phenon reagent, J. Biol. Chem. 193 (1951) 262–275.


[32] N. García, J.J. García, F. Correa, E. Chávez, The permeability transition pore asa pathway for the release of mitochondrial DNA, Life Sci. 76 (2005)2873–2880.

[33] I. Pérez-Torres, P. Roque, M. El-Hafidi, E. Díaz-Díaz, G. Baños, Association ofrenal damage and oxidative stress in a rat model of metabolic syndrome.Influence of gender, Free Radic. Res. 43 (2009) 761–771.

[34] A. Hausladen, I. Fridovich, Measuring nitric oxide and superoxide: rateconstants for aconitase activity, Methods Enzymol. 269 (1996) 37–41.

[35] F.J. Neumann, I. Ott, M. Gawaz, H. Holzaptel, M. Jocum, A. Schomig, Cardiacrelease of cytokines and inflammatory response to myocardial infarction,Circulation 92 (1995) 748–755.

[36] T. Ishii, M. Miyazawa, P.S. Hartman, N. Ishii, Mitochondrial superoxide anionO2

– inducible mev-1 animal models for aging research, BMB Rep. 44 (2011)298–305.

[37] M. Lampka, R. Junik, A. Nowicka, E. Kopczynska, T. Tyrakowski, G. Odrowaz-Sypniewska, Oxidative stress markers during a course of hyperthyroidism,Endokrynol. Pol. 57 (2006) 218–222.

[38] F.L. Muller, Y. Liu, H. Van Remmen, Complex III releases superoxide to bothsides of the inner mitochondrial membrane, J. Biol. Chem. 279 (2004) 49064–49073.

[39] N. García, E. Martínez-Abundis, N. Pavón, E. Chávez, Sodium inhibitspermeability transition by decreasing potassium matrix content in rat kidneymitochondria, Comp. Biochem. Physiol. B Biochem Mol. Biol. 144 (2006) 442–450.

[40] B. Biondi, Mchanisms in endocrinology: heart failure and thyroid dysfunction,Eur. J. Endocrinol. 167 (2012) 609–618.

[41] P. Venditti, R. De Rosa, S. Di Meo, Thyroid state on H2O2 production by ratmitochondria, Mol. Cell. Endocrinol. 205 (2003) 185–192.

[42] M. Messarah, M. Saoudi, A. Boumendjel, M.S. Boulakoud, A.E. Feki, Oxidativestress induced by thyroid dysfunction in rat erythrocytes and heart, Environ.Toxicol. Pharmacol. 31 (2011) 33–41.

[43] R. Nagai, A. Zarain-Herzberg, C.J. Brandi, J. Fujii, M. Tada, D.H. MacLennan, N.R.Alpert, M. Periassamy, Regulation of myocardial Ca2+-ATPase and phospho-lamban mRNA expression in response to pressure overload and thyroidhormone, Proc. Natl. Acad. Sci. U.S.A. 86 (1989) 2966–2970.

[44] M.R. Said. Becerra, C.A. Valverde, M.A. Kaetzel, J.R. Dedman, C. Mundiña-Weilenmann, X.H. Wehrens, L. Vittone, A. Mattiazzi, Calcium-calmodulindependent protein kinase II (CaMKII): a main signal responsible to earlyreperfusion arrhythmias, J. Mol. Cell Cardiol. 51 (2011) 936–944.

[45] S. Huke, R. Venkataraman, S. Faggioni, H.S. Hwang, F. Baudenbacher, B.C.Knolimann, Focal energy deprivation underlies arrhythmia susceptibilityin mice with calcium-sensitized myofilaments, Circ. Res. 112 (2013)1334–1344.

[46] G.J. García-Rivas, K. Carvajal, F. Correa, C. Zazueta, Ru360 a specificmitochondrial calcium uptake inhibitor, improves cardiac post ischemicfunctional recovery in rats in vivo, Brit. J. Pharm. 149 (1188) (2006) 1196.

[47] Y.I.L.D.I.S. Ek, C. Yenisey, T. Kavak, Effects of tamoxifen on myocardial ischemia-reperfusion injury model in ovariectomized rats, Mol. Cell. Biochem. 308(2008) 227–235.

[48] L. Hernández-Esquivel, C. Zazueta, M. Buelna-Chontal, N. Hernández-Reséndiz, E. Chávez, Protective behavior of tamoxifen against Hg2+-inducedtoxicity on kidney mitochondria. In vivo and in vitro experiments, J. SteroidBiochem Mol. Biol. 127 (2011) 345–350.

[49] P. Mishra, L. Samanta, Oxidative stress and heart failure in altered thyroidstates, Scientific World J. 2012 (2012) 741861.

[50] H.J. Ranki, G.R. Budas, R.M. Crawford, A.M. Davis, A. Jovanovic,17Beta-estradiolregulates expression of K(ATP) channels in heart-derived H9c2cells, J. Am. Coll.Cardiol. 40 (2002) 367–374.

[51] T. Ballantyne, Q. Du, S. Jovanovic, A. Neemo, R. Holmes, S. Sinha, A. Jovanovic,Testosterone protects female embryonic heart H9c2cells against severemetabolic stress by activating estrogen receptors and up-regulating IESSUR2B, Int. J. Biochem. Cell. Biol. 45 (2013) 283–291.



antiarrhythmic effect of tamoxifen on the vulnerability induced by hyperthyroidism to heart...

Documents