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Cardiovascular Research 2002 56(1):76-85; doi:10.1016/S0008-6363(02)00502-3
© 2002 by European Society of Cardiology
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Copyright © 2002, European Society of Cardiology

Effect of ischemia–reperfusion on heart mitochondria from hyperthyroid rats

Paola Venditti, Claudio Agnisola and Sergio Di Meo*

Dipartimento di Fisiologia Generale ed Ambientale, Università di Napoli, V. Mezzocannone 8, I-80134 Napoli, Italy

* Corresponding author. Tel.: +39-08-1253-5076; fax: +39-08-1253-5090 dimeo{at}biol.dgbm.unina.it

Received 29 March 2002; accepted 28 May 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: We investigated the effect of hyperthyroidism on the functional response of mitochondria to ischemia–reperfusion and its relationship with changes in mitochondrial susceptibility to stress conditions. Methods: Hyperthyroidism was elicited by ten daily intraperitoneal injections of T3 (10 µg/100 g body weight). Mitochondria were isolated at 3000xg (M3) from homogenates of hearts perfused by the Langendorff technique after either 25 min reperfusion following 20 min ischemia or 45 min perfusion (controls). Rates of O2 consumption and H2O2 release with complex II-linked substrate, capacity to remove H2O2, extent of oxidative damage, levels of liposoluble antioxidants, such as ubiquinols and vitamin E, and susceptibility to Ca2+-induced swelling were determined. Results: During reperfusion, hyperthyroid hearts displayed a significant tachycardia together with a low functional recovery. In comparison to the respective controls, mitochondria from both euthyroid and hyperthyroid hearts subjected to ischemia–reperfusion protocol exhibited decreases in the rate of O2 consumption, capacity to remove H2O2, and concentration of antioxidants, and increases in the rate of H2O2 release, concentration of hydroperoxides and protein-bound carbonyls, and susceptibility to Ca2+-induced swelling. Such changes were higher in mitochondria from hyperthyroid hearts. The increase in the protein percent content and cytochrome oxidase activity of a mitochondrial fraction isolated at 8000xg (M8) from hyperthyroid hearts after reperfusion, suggests that the decline of mitochondrial respiration of M3 fraction could be due to the degradation of the oldest, mature mitochondria endowed of high oxidative capacity, but low antioxidant capacity, which would be lost by heavy mitochondrial fraction and recovered in the light fraction. Conclusions: The higher susceptibility to ischemia–reperfusion of the heart from hyperthyroid animals is associated with a significant increase in mitochondrial dysfunction.

KEYWORDS Free radicals; Ischemia; Mitochondria; Reperfusion; ventricular arrhythmias


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
It is known from long time that both clinical [1] and experimental hyperthyroidism [2] alter cardiac function. Recently, it has been suggested that the hypermetabolic state typical of hyperthyroidism, results in an increased production of reactive oxygen species (ROS) that can lead to oxidative injury of cardiac tissue and progressive organ dysfunction [3]. Actually, hyperthyroidism does not seem to modify H2O2 release by heart mitochondria [4], while it decreases antioxidant capacity of cardiac tissue [5]. This suggests that the increased lipid peroxidation observed in the hyperthyroid heart [5,6] is consequent to its smaller effectiveness in preventing oxidative alterations.

In a previous paper we tested this possibility using ischemia–reperfusion as a model of oxidative myocardial injury related to ROS production [7]. A significant tachycardia was observed during reperfusion by the hyperthyroid hearts, together with a low recovery of left ventricular developed pressure (LVDP) and left ventricular dP/dtmax. Conversely, the functional recovery (LVDP and dP/dtmax) of hyperthyroid hearts paced at 300 beats/min, was close to 100% and significantly higher than in euthyroid paced hearts. After ischemia–reperfusion myocardium antioxidant capacity was significantly lower, whereas lipid peroxidation and the susceptibility to in vitro oxidative stress were higher in hyperthyroid than in euthyroid rats. Vitamin E prevented the in vitro tachycardic response, the reduction in antioxidant capacity, and the increase in lipid peroxidation, suggesting that the tachycardic response to reperfusion was associated with the reduced capability of the hyperthyroid heart to face oxidative stress.

Mitochondria are a possible site of ischemia–reperfusion damage, as the loss of mitochondrial function leads to cell death, whereas an optimal energy metabolism is required to preserve cell viability. Actually, ischemia reduces in vitro mitochondrial respiration [8,9], an effect that is potentiated by reperfusion [9,10]. Furthermore, the reperfusion-induced decline of the mitochondrial function depends on the length of the oxygen deprivation period [9] and the age of the animals [11].

Experimental evidence indicates that ROS are involved in the mitochondrial functional response to ischemia–reperfusion [11,12]. Therefore, if mitochondria share the poor capacity of hyperthyroid hearts to oppose an oxidative challenge, a greater mitochondrial dysfunction should be observed in such hearts after ischemia–reperfusion. To throw light on this matter we have evaluated the changes induced by ischemia–reperfusion in the oxygen consumption of heart mitochondria from euthyroid and hyperthyroid rats. The mitochondrial function has been related with indices of lipid and protein damage, liposoluble scavenger levels, and mitochondrial ROS production rates. Finally, the relative stability of the mitochondrial preparations under stress conditions was evaluated by assessing their susceptibility to oxidative challenge and Ca2+-induced swelling.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Animals, preparation set up and equipment
Male Wistar rats (60 days old) were used in the experiments. The animals, purchased at weaning from Nossan (Correzzana, Italy), were housed in separate cages at 24±1 °C, with an artificial lighting cycle (LD 08:00–20:00 h). All animals were provided with water ad libitum and a commercial rat chow diet (Nossan). From day 50, animals were randomly assigned to one of two groups: E, euthyroid control rats (n=16, mean weight±S.E.M., 219±7 g), and H, rats made hyperthyroid by treatment with daily intraperitoneal injections of T3 (10 µg/100 g body mass) for 10 days (n=16, mean weight 224±8 g). Animals were anaesthetized by intraperitoneal injection of chloral hydrate (40 mg/100 g body weight) combined with ether and subjected to electrocardiographic recording. After heparinization, a rapid thoracotomy was performed and the aorta cannulated retrogradely. The hearts were excised and flushed to get rid of blood for 1 min with Krebs–Henseleit (KH) buffer containing (mmol/l): NaCl 118, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25, EDTA 0.5, glucose 11, pH 7.4, and gassed with 5% CO2 in O2. The same buffer was used to perfuse the isolated hearts at 37 °C and a head pressure of 70 mmHg according to Langendorff [13]. Preparations were let to stabilize for 20 min. The stabilized hearts from each group of animals were randomly assigned to two subgroups, according to the experimental protocol. Hearts from euthyroid and hyperthyroid animals were either continuously perfused for a 45-min period (EP and HP subgroups, respectively) or subjected to global normothermic ischemia for 20 min followed by 25 min of aerobic reperfusion (ER and HR subgroups, respectively).

Functional performance was determined at the end of the equilibration and perfusion periods as previously described [7].

The investigation conforms to the Guidelines for Care and Use of Laboratory Animals of the Italian Health Ministry.

2.2 Preparation of mitochondria
Mitochondria preparation was based on the procedure of Tyler and Gonze [14], which provides for proteolytic tissue treatment with nagarse. The speed used to free homogenates from debris and nuclei was lower (500xg instead of 700xg) in order to increase the mitochondrial yield. Also, the speed used to obtain the mitochondrial pellet was lower (3000xg instead of 8000xg) in order to reduce both the contamination by cytoplasmic and microsomal material and the amount of damaged mitochondria. We defined M3 this mitochondrial fraction, on which most of the present study has been performed.

In order to investigate whether T3 treatment or ischemia–reperfusion would modify the characteristics of the M3 fraction, by affecting the dynamics of the mitochondrial population, in a separated set of experiments we isolated two different mitochondrial fractions, M3 and M8, by sequential centrifugation steps at 3000 and 8000xg, respectively. On these fractions, the protein content and the activities of cytochrome oxidase (COX) and oligomycin-insensitive adenosine triphosphatase (ATPase) were determined. COX activity was determined polarographically at 25 °C, using a Gilson glass respirometer equipped with a Clark oxygen electrode (Yellow Springs Instruments, OH, USA), by the procedure of Barré et al. [15]. ATPase activity was determined according to Krieger et al. [16].

The mitochondrial protein content was determined, upon dissolving in 0.5% deoxycholate, by the biuret method [17] with bovine serum albumin as standard. Mitochondrial preparations were used for the analytical procedures described below.

2.3 Analytical procedures
Mitochondrial respiration was monitored at 30 °C by a Gilson glass respirometer equipped with a Clark oxygen electrode (Yellow Springs Instrument, Yellow Springs, OH, USA) in 1.6 ml of incubation medium (145 mM KCl, 30 mM Hepes, 5 mM KH2PO4, 3 mM MgCl2, 0.1 mM EGTA, rotenone 5 µM, pH 7.4) with 0.5 mg of mitochondrial protein per ml. Succinate (10 mM) was used as substrate, in the absence (state 4) and in the presence (state 3) of 500 µM ADP.

The rate of mitochondrial H2O2 release was measured at 30 °C following the linear increase in fluorescence (excitation at 320 nm, emission at 400 nm) due to oxidation of p-hydroxyphenylacetate by H2O2 in the presence of horseradish peroxidase [18] in a computer-controlled Jasko fluorometer; 10 mM succinate was used as substrate. Measurements in the presence of 500 µM ADP or 10 µM antimycin A were also performed. Known concentrations of H2O2 were used to establish the standard concentration curve.

The capacity to remove H2O2 (CR) was determined by comparing the ability of mitochondrial samples to reduce H2O2-linked fluorescent emission with that of desferrioxamine solutions [19]. Briefly, the p-hydroxyphenylacetate (PHPA) oxidation to the stable fluorescent product 2,2'-dihydroxy-biphenyl-5,5'-diacetate (PHPA)2 [18] by the H2O2 generated from glucose oxidase (GOX) was monitored with a Jasko fluorometer (excitation wavelength 320 nm, emission wavelength 400 nm) at 30 °C. The reaction was performed in the presence of desferrioxamine (1–12 nmol), or mitochondrial samples (0.1–1.0 mg of mitochondrial proteins). The values of fluorescence change for unit of time obtained after addition of desferrioxamine or mitochondria were converted to relative percentage of the values obtained before the addition. The desferrioxamine values were used to fit standard curves by the Fig. P program (Biosoft, Cambridge, MA, USA). The sample values were plotted on the standard curves to obtain their capacity to remove H2O2, expressed as equivalent desferrioxamine concentration.

The extent of peroxidative reactions was determined measuring the hydroperoxides (HPs) according to Heath and Tappel [20].

Quantification of protein-bound carbonyls was performed by the procedure of Schild et al. [21]. Protein recovery was estimated for each sample. Carbonyl content was calculated using the molar absorption coefficient of aliphatic hydrazones of 22 000 M/cm and expressed as nmol carbonyl/mg of protein.

Ubiquinols (CoQH2) were oxidized to ubiquinones (CoQs) with ferric chloride as the oxidation reagent. In particular, 0.5 ml of mitochondrial suspension was treated with 0.5 ml of 2% FeCl3 and 2.0 ml of ethanol. CoQs were extracted by 5.0 ml of n-hexane, which was then removed by evaporation under N2 at 40 °C. The residue was dissolved in ethanol and subjected to the mobile phase in HPLC (machine: SpectraSeries P100 isocratic pump, Thermo Separation Products, San Jose, CA; column Ultremex 5 250x4.6 mm, 5-µm particle size; Phenomenex, Torrance, CA). Eluant was a mixture of methanol/ethanol (3:7, v/v) containing 20 mM lithium perborate, and the flow rate was 1 ml/min [22]. The eluted CoQs (CoQ9 and CoQ10) were determined separately by using a SpectraSeries UV100 detector (Thermo Separation Products, San Jose, CA) (275 nm). Quantification was obtained by using external standards.

For vitamin E determination, mitochondrial preparations were deproteinized with methanol and extracted with n-hexane. The extracts were evaporated under N2 at 40 °C and the residues were dissolved in ethanol. Vitamin E content was determined using the HPLC procedure of Lang et al. [22]. Quantification was obtained by using external standard.

Mitochondrial swelling was spectrophotometrically measured by determining the apparent absorbance at 540 nm in a medium containing 125 mM sucrose, 65 mM KCl, 10 mM Hepes, pH 7.2, 2 mM succinate, 4 µM rotenone, 0.3 mg mitochondrial protein/ml, 100 µM Ca2+, and 1 mM EGTA or 1 µM cyclosporin A (CSA) where indicated.

Mitochondrial membrane potential ({Delta}{Psi}) was estimated through fluorescence changes of safranine (8 µM), recorded on the Jasko fluorometer (excitation wavelength 495 nm, emission wavelength 586 nm) in a medium containing 125 mM sucrose, 65 mM KCl, 10 mM Hepes, pH 7.2, 2 mM succinate, 6 µM rotenone, 0.3 mg mitochondrial protein/ml reaction mixture, 100 µM Ca2+. {Delta}{Psi} was calculated according to Åckerman and Wilkström [23] using a calibration curve obtained incubating mitochondria in a medium containing 200 mM sucrose, 10 mM Hepes, pH 7.2, 6 µM rotenone, 0.38 EDTA, 8 µM safranine, 38.5 ng/ml valinomycin, and KCl at concentrations from 0 to 0.96 mM.

2.4 Chemicals
All chemicals used were of the highest grade available, and were purchased from Sigma Chimica (Milano, Italy).

2.5 Statistical analysis
The data obtained from eight experiments for each experimental group are expressed as mean values±S.E.M. Data were analysed with two-way analysis of variance (ANOVA). When a significant F ratio was found, the Student–Newman–Keuls multiple-range test was used to determine the statistical significance of differences between mean values. Differences between M3 and M8 fraction in the same subgroup were statistically analysed with unpaired Student’s t-test. The level of significance was chosen as P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
The hyperthyroid state of T3-treated animals was reflected in their higher heart/body weight ratio (E=2.81±0.08 mg/g; H=3.61±0.09 mg/g, P<0.05, unpaired Student’s t-test) and in vivo heart rate (E=442±7 beats/min; H=543±8 beats/min, P<0.05).

3.1 Basal heart performance and functional recovery from ischemia–reperfusion
Unlike basal preischemic coronary flow, preischemic ventricular performance was significantly affected by T3 treatment of animals. Hyperthyroid rats displayed a higher intrinsic heart rate and lower LVPD and dP/dtmax values than euthyroid animals (Table 1). Functional recovery values (=the percent ratio between the value at the end of reperfusion period and the value of the same parameter just before the onset of the ischemic period) of the Langendorff preparations after ischemia–reperfusion are reported in Fig. 1. For comparison, the percent ratios between the values determined at the end and at the beginning of the 45-min perfusion period of the hearts from EP and HP groups are also reported.


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  		Table 1 Preischemic coronary flow and left ventricle performance (basal performance) of Langendorff preparations from euthyroid and hyperthyroid rats

 

Figure 1
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  		Fig. 1 Effect of thyroid state on functional recovery of Langendorff preparations from ischemia–reperfusion. Recovery of coronary flow (A), heart rate (B), left ventricular developed pressure (LVDP) (C), and left ventricular dP/dtmax (D) was measured after 25 min reperfusion following 20 min ischemia in euthyroid (ER) and hyperthyroid (HR) hearts, and expressed as percent of preischemic value. For comparison, the percent ratios between the values determined at the end and at the beginning of a 45-min perfusion of euthyroid (EP) and hyperthyroid (HP) hearts are also reported. Values are means±S.E.M. of eight experiments. a Significant difference for T3-treated animals vs. respective euthyroid controls (P<0.05). b Significant difference for hearts subjected to ischemia–reperfusion vs. perfused hearts (P<0.05).

 
The main effect of T3 treatment was a significant, strong tachycardia during reperfusion, with a heart rate that was 162.5% than the preischemic value at 25 min reperfusion. This tachycardia was accompanied by a recovery of ventricular contractility (LVDP and dP/dtmax) significantly lower than that of euthyroid heart. Conversely, euthyroid and hyperthyroid hearts displayed similar recovery of coronary flow.

3.2 Characteristics of mitochondrial subpopulations
As shown in Table 2, COX activities were higher in M3 fractions from hyperthyroid hearts than in those from the corresponding euthyroid controls. Furthermore, ischemia–reperfusion decreased COX activity in the hearts from hyperthyroid animals only. ATPase activity of M3 fraction was increased by ischemia–reperfusion in euthyroid hearts and by T3 treatment in hearts subjected to ischemia–reperfusion. COX activity of M8 fraction was higher in HR group than in ER group, while its protein content was higher in HR group than in ER and HP group.


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  		Table 2 Cytochrome oxidase and oligomycin-insensitive ATPase activities in mitochondrial fractions from euthyroid and hyperthyroid rat hearts subjected to ischemia–reperfusion

 
In all groups, COX and ATPase activities were higher and protein amounts were lower in M8 than in M3 fraction. Ischemia–reperfusion significantly increased the percent content of proteins in M8 fraction from hyperthyroid heart.

3.3 Mitochondrial oxygen consumption
The results concerning respiratory characteristics of the succinate-supplemented mitochondria show that the rate of state 4 oxygen consumption was not dependent on either ischemia–reperfusion or T3 treatment (Table 3). Conversely, the rate of state 3 oxygen consumption, that was higher in mitochondria from hyperthyroid hearts than in control mitochondria, was strongly reduced by ischemia–reperfusion. This reduction was higher in hyperthyroid hearts so that after ischemia–reperfusion the respiration rate was not significantly different in mitochondria from euthyroid and hyperthyroid hearts. Because of the strong fall in mitochondria respiration rate, the respiratory control ratio (RCR) was significantly lower in HR than in HP group.


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  		Table 3 Effects of ischemia–reperfusion on O2 consumption of heart mitochondria from euthyroid and hyperthyroid rats

 
3.4 Mitochondrial H2O2 release and capacity to remove H2O2
Both in the presence and in the absence of ADP, the rate of succinate-supported H2O2 release from hyperthyroid mitochondrial preparations was significantly higher than that from the corresponding euthyroid controls (Table 4). Furthermore, ischemia–reperfusion increased the rate of H2O2 release during state 3 and state 4 respiration in hyperthyroid preparations, and only during state 4 respiration in euthyroid preparations. Antimycin A-stimulated H2O2 release was also higher in the preparations from hyperthyroid hearts than in those from respective euthyroid controls. Ischemia–reperfusion did not modify such release in euthyroid preparations whereas it in hyperthyroid ones (Table 4).


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  		Table 4 Effects of ischemia–reperfusion on H2O2 release by heart mitochondria from euthyroid and hyperthyroid rats

 
Oxygen consumption and H2O2 release were measured at the same temperature, in the same buffer and using the same concentration of substrate. This allowed us to calculate the percentage of consumed oxygen released as H2O2 (Fig. 2). Statistical analysis showed a lack of significant differences between groups. The only exception was the higher fraction of O2 converted into H2O2 during state 3 respiration after ischemia–reperfusion in mitochondria from hyperthyroid heart.


Figure 2
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  		Fig. 2 Effect of ischemia–reperfusion on the percentage of O2, which is released as H2O2 by succinate-supplemented heart mitochondria from euthyroid and hyperthyroid rats. Values are means±S.E.M. of eight experiments. EP, ER, HP, and HR are euthyroid perfused, euthyroid reperfused, hyperthyroid perfused, and hyperthyroid reperfused hearts, respectively. b Significant difference for hearts subjected to ischemia–reperfusion vs. perfused hearts (P<0.05).

 
Fig. 3 reports CR values, i.e. the capacity to remove H2O2 of mitochondrial preparations. They indicate that the mitochondria from hyperthyroid non-ischemic hearts are endowed with a greater level of substances able to remove H2O2 and to prevent •OH production (H2O2-metabolizing enzymes) and/or substances able to remove H2O2 converting it into more reactive radicals via Fenton reaction (iron-ligands). Although ischemia produced an evident fall in CR, this parameter remained higher in hyperthyroid than in euthyroid preparations.


Figure 3
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  		Fig. 3 Effect of ischemia–reperfusion on the capacity to remove H2O2 (CR) of heart mitochondria. Values are means±S.E.M. of eight experiments. EP, ER, HP, and HR are euthyroid perfused, euthyroid reperfused, hyperthyroid perfused, and hyperthyroid reperfused hearts, respectively. CR is expressed as equivalent concentration of desferrioxamine (nmol/mg protein). a Significant difference for T3-treated animals vs. respective euthyroid controls (P<0.05). b Significant difference for hearts subjected to ischemia–reperfusion vs. perfused hearts (P<0.05).

 
3.5 Oxidative damage of mitochondria
As shown in Table 5, both ischemia–reperfusion and T3 treatment significantly increased oxidative damage in heart mitochondria. In fact, hydroperoxide level and protein-bound carbonyl content are both higher in hyperthyroid preparations than in the respective euthyroid controls and increase significantly after ischemia–reperfusion in both euthyroid and hyperthyroid preparations.


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  		Table 5 Effect of ischemia–reperfusion on hydroperoxide levels and protein-bound carbonyl content of heart mitochondria from euthyroid and hyperthyroid rats

 
3.6 Vitamin E and coenzyme Q levels
Mitochondrial levels of CoQ9 and CoQ10 were higher in HP than in EP hearts (Table 6). Ischemia–reperfusion significantly decreased the above coenzyme levels in hyperthyroid, but not in euthyroid hearts. In particular, ischemia–reperfusion reduced CoQ9 in the hyperthyroid hearts to the levels observed in the euthyroid hearts.


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  		Table 6 Effect of ischemia–reperfusion on coenzyme Q9, coenzyme Q10, and vitamin E concentrations in heart mitochondria from euthyroid and hyperthyroid rats

 
Mitochondrial levels of vitamin E were not different in EP and HP hearts (Table 6). Ischemia–reperfusion decreased vitamin E in hyperthyroid hearts so that the values in HR hearts were lower than in both ER and HP hearts.

3.7 Mitochondrial swelling
In order to verify whether ischemia–reperfusion or animal treatment with T3 affected susceptibility of mitochondria to Ca2+-dependent swelling, succinate-energized mitochondria were incubated in the presence of 100 µM Ca2+ and absorbance changes were monitored. Fig. 4A shows that the Ca2+-loaded mitochondrial suspensions from hearts subjected to ischemia–reperfusion suffer a more extensive decrease in absorbance measured at 540 nm, and that such a decrease was statistically significant for hyperthyroid preparations. The decreases in absorbance were compatible with a Ca2+-induced mitochondrial permeability transition (MPT). In fact, they were drastically reduced when either Ca2+ was eliminated from the reaction medium with the Ca2+ chelator EGTA or a specific inhibitor of MPT, the immune suppressor cyclosporin A, was added to the medium (Fig. 4B). Fig. 5 shows that the mitochondria undergo a rapid decrease in membrane potential ({Delta}{Psi}). Accordingly with the absorbance changes, {Delta}{Psi} was more extensive in hyperthyroid suspensions from hearts subjected to ischemia–reperfusion.


Figure 4
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  		Fig. 4 Effect of ischemia–reperfusion on Ca2+-induced swelling of heart mitochondria from euthyroid and hyperthyroid rats. EP, ER, HP, and HR are euthyroid perfused, euthyroid reperfused, hyperthyroid perfused, and hyperthyroid reperfused hearts, respectively. Mitochondria (0.3 mg/ml) were incubated in standard medium containing 100 µM Ca2+. (A) Swelling of mitochondrial preparations monitored as decrease of the absorbance at 540 nm, and expressed as percent of the initial value before Ca2+ addition. The initial absorbance values were 0.76±0.09, 0.73±0.06, 0.76±0.07, and 0.79±0.06 for EP, ER, HP, and HR preparation, respectively. Data are means±S.E.M. of eight experiments. Time course of the absorbance was significantly affected by ischemia–reperfusion (Two-way ANOVA, P<0.05). (B) Extent of mitochondrial fraction swelling in presence of 100 µM Ca2+, 100 µM Ca2+ plus 1 mM EGTA, or 100 µM Ca2+ plus 1 µM cyclosporin A (CSA). The effect of addition of EGTA or CSA on the extent of swelling induced by Ca2+ was determined by comparing the percent changes of absorbance at 540 nm obtained after a 16-min period. a Significant difference for T3-treated animals vs. respective euthyroid controls (P<0.05). b Significant difference for hearts subjected to ischemia–reperfusion vs. perfused hearts (P<0.05). c Significant vs. the same preparation in the presence of the only Ca2+.

 

Figure 5
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  		Fig. 5 Effect of ischemia–reperfusion on membrane potential dissipation induced by Ca2+. EP, ER, HP, and HR are euthyroid perfused, euthyroid reperfused, hyperthyroid perfused, and hyperthyroid reperfused hearts, respectively. Membrane potential ({Delta}{Psi}) of mitochondrial preparations (0.3 mg/ml) was estimated through fluorescence changes of safranin (8 µM) (excitation wavelength 495 nm, emission wavelength 586 nm) in a standard medium containing 100 µM Ca2+. {Delta}{Psi} was calculated using a suitable calibration curve. The decrease of {Delta}{Psi} for each preparation was expressed as percent of the initial value before Ca2+ addition. Initial values of {Delta}{Psi} were 169.5±8.9, 149.2±12.1, 154.8±15.7, and 119.4±3.6 mV for EP, ER, HP, and HR preparation, respectively. Data are means±S.E.M. from eight experiments. Time course of {Delta}{Psi} was significantly affected by ischemia–reperfusion and thyroid state (two-way ANOVA, P<0.05).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The present study on the effects of hyperthyroidism on the heart response to ischemia–reperfusion has shown that hearts from hyperthyroid rats display a significant tachycardic response, associated with an impaired inotropic recovery. This observation confirms previous reports [7,24], which also indicated that the higher susceptibility of the heart from hyperthyroid rats to ischemia–reperfusion was consequent to a higher oxidative stress on myocardial cells upon reperfusion [7].

The mechanisms of cellular and subcellular derangement and its contribution to myocardial ischemia–reperfusion injury are not well established. A primary intracellular target for oxidative stress, which may account for the reperfusion-induced tissue damage, is represented by mitochondria. In fact, mitochondria, the powerhouses of cellular life, are also the motor of cell death, and there is evidence that during reperfusion of ischemic heart, mitochondrial derangement is inversely correlated to functional recovery of the tissue [9].

Our finding that the lower functional recovery of hyperthyroid hearts after ischemia–reperfusion is associated with a higher impairment of mitochondrial respiration is consistent with the idea that heart performance is strongly conditioned by mitochondrial functionality. Besides, the analysis of other reperfusion-linked mitochondrial changes suggests that the greater functional decline of mitochondria during reoxygenation is due to an oxidative stress, which is more severe in hyperthyroid hearts.

It is well established that a major source of ROS during reperfusion of ischemic myocardium is the respiratory chain [12,25]. Under physiologic conditions, single electron transfer by reduced respiratory components to oxygen produces small quantities of the superoxide anion radical (O•–2), which is converted to H2O2 by the superoxide dismutase [26]. Hydrogen peroxide may either permeate mitochondrial membranes and enter the cytoplasm or be detoxified. In heart mitochondria, H2O2 may be removed by H2O2-metabolizing enzymes and by hemoproteins via the Fenton reaction, giving rise to •OH radicals. These are highly reactive and short-lived species, which would be expected to damage mitochondrial components at or near the site of their formation. Information on the rate of H2O2 generation by intact mitochondria can be obtained combining the determinations of the rate of mitochondrial H2O2 release and the mitochondrial capacity to remove H2O2 [19]. Because mitochondrial ROS generation depends on concentration and reduction degree of autoxidizable electron carriers [27], the higher reduction of the respiratory chain associated with ischemia is currently assumed to promote the transfer of electrons to oxygen to generate superoxide radicals upon resumption of respiration [28]. In hyperthyroid heart mitochondria, with their higher content of respiratory chain components [29], this mechanism would lead to an increase in ischemia–reperfusion ROS generation. This idea is indirectly supported by our observation that both mitochondrial rate of H2O2 release in the presence of antimycin A and capacity to remove H2O2, are higher in non ischemic hyperthyroid hearts than in the euthyroid controls. In the presence of antimycin A the respiratory chain components located between the substrate side and cytochrome b-560 become completely reduced and ROS production rate depends on their concentration only. A similar condition occurs in hearts reperfused after ischemia. Such ROS generation should end rapidly as the electron carriers of the respiratory chain are reoxidised. However, our results suggest that during reperfusion the rate of H2O2 production do not return to the preischemic level, likely because of lower electron flow across respiratory chain, and remains higher in mitochondria from hyperthyroid hearts. Indirect information on the relative mitochondrial ROS production in euthyroid and hyperthyroid hearts is also supplied by reperfusion-induced changes in the indices of oxidative damage. The high reactivity of •OH radicals makes mitochondria a likely site of reperfusion-induced oxidative damage whose severity depends on the capacity of these organelles to produce H2O2 and remove it via Fenton reaction. We have shown a wider oxidative damage to lipids and proteins parallel to a higher rate of H2O2 production during both state 4 and state 3 respiration in mitochondria from hyperthyroid non ischemic hearts than in those from euthyroid hearts. Consistently with the hypothesis of a higher increase of H2O2 production rate, ischemia–reperfusion caused the highest oxidative damage in mitochondria from hyperthyroid hearts. Furthermore, mitochondrial concentrations of important liposoluble antioxidants, such as ubiquinols and vitamin E, underwent significant decreases only after ischemia–reperfusion of hyperthyroid hearts.

The mechanisms by which ROS mediate the decline in mitochondrial function are not entirely clear. However, whatever such mechanisms may be, they must be able to explain why the extent of mitochondrial dysfunction is dependent on thyroid state. A possibility is that the decline in mitochondrial function is due, at least in part, to modification of specific mitochondrial proteins by a product of lipid peroxidation, 4-hydroxy-2-nonenal (HNE), whose concentration has been reported to increase upon reperfusion of ischemic heart [30]. modification by HNE of mitochondrial proteins only occurs in mitochondria exhibiting reperfusion-induced decline in function [11], and the reduction of COX activity by ischemia–reperfusion is associated with increases in HNE adducts with COX [31]. Although an increase of various products of lipid peroxidation has been reported in hyperthyroid heart [5,6], the effects of hyperthyroid state on heart HNE levels have not been studied.

Nitric oxide (NO) and its potent oxidative derivative peroxynitrite are also putative species responsible for altered mitochondrial function in myocardial ischemia–reperfusion. Nitric oxide synthase (NOS) stimulation upon ischemia–reperfusion [32] and inhibition of mitochondrial function by both NO [33,34] and peroxynitrite [35] have been reported. It is noteworthy that hyperthyroidism induces a significant increase in rat liver NOS [36], notwithstanding the decrease in mitochondrial NOS [37]. There is no evidence so far of NOS activity enhancement in the heart of hyperthyroid animals. On the other hand, even in absence of a hyperthyroid state-linked NO overproduction, it is possible that the concommitance of conditions, that increase the potential for oxidative stress, may have synergistic effects and enhance the probability of mitochondrial dysfunction. In particular, the concurrence of a higher oxidative stress associated with hyperthyroidism and ischemia–reperfusion should increase the extent of mitochondrial dysfunction and tissue impairment, favouring the damaging action of nitric oxide. This hypothesis would explain the extent of the fall in mitochondrial respiration and increase in H2O2 production found by us after ischemia–reperfusion of hyperthyroid hearts, and would be consistent with our previous finding that the perfusion of hearts with the NOS inhibitor, N{omega}-nitro-L-arginine (L-NNA) prevented the tachycardic response and the myocardial contractile dysfunction [24]. Whether L-NNA is also able to reduce the decline of the mitochondrial function requires further investigation.

The decline of mitochondrial respiration could also be due to the combination between oxidative stress and the increase in Ca2+ concentration that occurs in myocardial cells during ischemia–reperfusion [38]. In presence of Ca2+, oxidative alterations of mitochondrial inner membrane protein thiols promote an inner membrane permeabilization referred to as mitochondrial permeability transition (MPT) [39] that leads to mitochondrial swelling. The dependence of Ca2+-induced MPT on oxidative alteration of the inner membrane represents a link between oxidative challenge of mitochondria and their susceptibility to swelling. This is supported by our finding that mitochondria from ischemic and reperfused hearts exhibit a higher susceptibility to in vitro Ca2+-induced swelling than control mitochondria. The relevance of MPT for mitochondrial derangement can be better understood admitting that mitochondrial population of the heart consists of mitochondria of different ages and thus of varying susceptibility to ischemia–reperfusion-induced alterations.

Recent studies on liver mitochondrial fractions suggest that the light fractions, characterised by low respiratory activity, contain transitional forms in the process of development into the heavy mitochondrial structures with high respiratory activity [40,41]. The heavy fraction also exhibits the lowest antioxidant level [41,42] and the highest rates of H2O2 production and susceptibility to Ca2+-induced swelling [43]. However, not all parameters determined on mitochondrial fractions are related to the characteristics of the different mitochondrial populations. This is due mainly to the contamination of the light fraction, containing neoformed mitochondria, with microsomes and damaged mitochondria with high cytochrome content but scarce functionality, which likely come from the degradation of heavy mitochondria [41]. The amount of damaged mitochondria contaminating the light mitochondrial fraction is increased by exercise-induced oxidative stress [44]. Ischemia–reperfusion seems to induce similar modifications in mitochondrial population, as an increase in the amount of proteins recovered in the M8 fraction was observed. Moreover, ischemia–reperfusion accentuated the differences in the cytochrome oxidase activity between M8 and M3 fractions. This effect was much stronger in preparations from hyperthyroid hearts, suggesting that the light fraction from HR heart contains a greater amount of mitochondrial membranes, coming from degradation of mitochondria of the heavy fraction. This idea is supported by the fall in the capacity to remove H2O2 and antimycin-sensitive H2O2 release observed in M3 mitochondria from hyperthyroid hearts after ischemia–reperfusion. On the whole, the above changes indicate a greater loss by M3 fraction of mitochondria characterised by high oxidative capacity, and can explain the strong decline of mitochondrial function, associated to ischemia–reperfusion, of hyperthyroid heart.

Time for primary review 25 days.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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