Impairment of mitochondrial respiratory chain enzyme activities in tetralogy of fallot

Clinica Chimica Acta 377 (2007) 138 – 143 www.elsevier.com/locate/clinchim

Impairment of mitochondrial respiratory chain enzyme activities in tetralogy of fallot Santosh B. Shinde a,⁎, Vipul C. Save a , Neela D. Patil a , Kaushala P. Mishra c , Anil G. Tendolkar b a

b

Department of Biochemistry, L.T.M.M.C and L.T.M.G.H., Mumbai 400025, India Department of Cardiovascular and Thoracic surgery (CVTS), L.T.M.M.C and L.T.M.G.H., Mumbai 400025, India c Radiation Biology and Health Sciences division, BARC, Mumbai 400085, India Received 17 February 2006; received in revised form 27 August 2006; accepted 13 September 2006 Available online 26 September 2006

Abstract Background: During the last decade, disorders of the respiratory chain, so-called mitochondrial disorders, have emerged as a major clinical entity. Tetralogy of fallot (TOF) children N2 month of age are at risk for postoperative myocardial contractile failure. Myocardial ischemia is associated with a reduction in mitochondrial enzyme activity and have impaired metabolism resulting in decreased postoperative myocardial adenosine triphosphate (ATP) concentrations and increased lactate levels. With this in view, we measured the mitochondrial energy system (respiration and OXPHOS) and to study morphological changes from the right ventricular outflow tract (RVOT) muscle of patients with TOF. Methods: 30 infants with TOF were studied with age-matched control group consisted of 12 normal patients who died due to extracardiac causes. Mitochondrial respiratory chain complexes, OXPHOS, cytochrome content and ATPase activity were measured by documented standard procedure. Morphological changes examined with a transmission electron microscope. Results: In the presence of glutamate and succinate as substrates, the rate of mitochondrial oxygen consumption was significantly lower in RVOT muscles ( p b 0.001) by using with and without addition of ADP. The ADP/O ratio indices for glutamate and succinate were not significantly affected. The activities of rotenone-sensitive NADH cytochrome c reductase (complexes I + III), cytochrome c oxidase (complex IV) and the ratio of I and III to II and III complexes (complex I) were significantly lower in TOF ( p b 0.001). A significant reduction of total cytochrome content and ATPase activity ( p b 0.001) was noted in study group. Morphological changes were also seen in study group as compared with control. Conclusions: OXPHOS, mitochondrial respiratory chain complex I, I + III and IV, cytochrome content and ATPase activity are more impaired in RVOT muscles in patients with TOF. © 2006 Elsevier B.V. All rights reserved. Keywords: Oxidative phosphorylation; Mitochondrial respiratory chain; RVOT; Tetralogy of fallot

1. Introduction It is a well known fact that congenital heart disease associated with right ventricular outflow tract (RVOT) obstruction leads on to right ventricular hypertrophy, and Tetralogy of fallot (TOF) is one such entity. TOF children N 2 months of age are at risk for postoperative myocardial contractile failure and have impaired metabolism resulting in decreased postoperative myocardial Adenosine triphosphate (ATP) concentrations and increased ⁎ Corresponding author. Santosh Shinde, 9-Dhanshri CHS, Nanda Patkar Road, Vile-Parle (East), Mumbai 400057, India. Tel.: +91 22 2611 32 83. E-mail address: [email protected] (S.B. Shinde). 0009-8981/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2006.09.011

lactate concentrations [1]. Myocardial hypoxia is a less wellknown reason of ventricular dysfunction in TOF. The metabolic abnormality during reperfusion suggests that mitochondrial dysfunction may contribute to complications such as low-output syndrome and depressed ventricular function [2]. These effects are exacerbated during the postnatal period when metabolic changes occur to the myocardium, making it more reliant on oxygen than during the fetal period. It has been demonstrated that mitochondrial oxidative phosphorylation responds to physiological alterations in oxygen tension by altering the rate of cellular respiration [3]. The clinical features are rarely pathogenomic, and laboratory investigations are frequently required to confirm the diagnosis. These investigations might include indicators of cell redox status

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(e.g., lactate and lactate/pyruvate ratio), numerical or structural abnormalities of mitochondria in tissue biopsies, molecular studies, enzyme histochemistry, and studies of respiratory chain (RC) function [4]. The defects described in these studies were primarily in the activities of the electron transport system and oxidative phosphorylation (OXPHOS) pathway including complex I, I + III, II, II + III, III, IV. Notably, these enzyme complexes are partially encoded by mitochondrial DNA (mtDNA). We measured the mitochondrial energy system (respiration and OXPHOS) from RVOT muscle of patients with TOF and to study morphological changes using electron microscopy.

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(Gilson Medical Electronics Middleton, WI) with a Clark-type oxygen electrode. Oxidative phosphorylation in heart papillary muscle mitochondria was measured in a medium consisting of 225 mmol/l sucrose, 10 mmol/l potassium phosphate buffer, pH 7.4, 10 mmol/l Tris–HCl, pH 7.4, 5 mmol/l MgCl2 and 2–4 mg mitochondrial proteins. Succinate (10 mmol/l) and glutamate (10 mmol/l) were used as substrates; with succinate, 1.0 μmol/ l rotenone was also included in the reaction medium. Small aliquots of ADP (about 200 nmol in 10 μl) were added, and rate of respiration in the presence of ADP (state 3) and after its depletion (state 4) were recorded. ADP/O ratios and the respiratory control index (RCI) were calculated as described previously [6].

2. Material and methods 2.1. Subjects We examined 30 infants with TOF (21 male and 9 female with mean age 9.2 ± 0.8 years). An oxygen tension of 40 mmHg was chosen because it represents the approximate lower range of arterial oxygen tension observed in children with TOF. The age-matched control group consisted of 12 normal patients who died of extracardiac causes. Informed consent was obtained before the surgery, and the study protocol was approved by the Research Council of our institution.

2.2. Sampling Specimens from RVOT of patients with TOF were taken within 4–8 min of aortic cross clamping. RVOT specimens were divided into two parts, one was placed in ice-cold Mannitol–Sucrose–EDTA medium (MSE) for mitochondrial evaluation. The other sample was immediately minced into 1–2 mm size pieces, fixed for 12 h in cold 3% glutaraldehyde in sodium cacodylate buffer and set aside for ultra-structural evaluation. Right ventricular myocardial samples obtained from controls were also fixed in a similar manner.

2.3. Electron microscopic study Post-fixation of the glutaraldehyde fixed samples was carried out with cold 1% osmium tetroxide in Millonig's phosphate buffer for 3 h. The tissues were then dehydrated in ethanol and propylene oxide and embedded in Maraglas epoxy resin. One-micron thin sections were stained with alkaline Toluidine blue. Ultra-thin sections obtained using a diamond knife were stained with uranylacetate and lead citrate and examined with a transmission electron microscope.

3. Laboratory investigations 3.1. Isolation of RVOT muscle mitochondria RVOT muscles were washed with cold MSE medium and finely chopped with a pair of scissors and mixture was transferred to 10 volumes of MSE medium. The tissue mince was transferred to a smooth walled homogenizing vessel and disrupted with a loose fitting motor driven Teflon pestle with 15 strokes. The homogenate was centrifuged at 700 ×g for 10 min. The supernatant was centrifuged at 10,000 ×g for 10 min. to obtain mitochondrial pellet. This was rewashed with MSE medium, and recentrifuged at 10,000 ×g for 10 min. Mitochondrial pellet was resuspended to small volumes of MSE medium and store at 4 °C [5]. 3.2. Determination of oxidative phosphorylation Measurements of oxygen consumption were carried out at 25 °C and in a total volume of 1.3 ml in a Gilson Oxygraph, model KM

3.3. Determination of mitochondrial respiratory chain complex activities Succinate cytochrome c reductase (EC 1.3.2.2, complexes II and III) [7] was measured by monitoring the reduction of cytochrome c at 550 nm in the presence of succinate and enzyme. Assays for rotenone-sensitive NADH cytochrome c reductase (EC 1.6.2.1, complexes I + III) [7] were measured by monitoring the reduction of cytochrome c at 550 nm in the presence of NADH, rotenone, and the mitochondrial protein. The rotenoneresistant activity was subtracted from the total NADH cytochrome c reductase activity to yield the activity of the rotenone-sensitive cytochrome c reductase. Succinate dehydrogenase (EC 1.3.99.1, complex II) [8] was measured at 600 nm by monitoring the oxidation of succinate in the presence of the artificial electron acceptor, 2,6-dichiorophenol-indophenol, and the mitochondrial protein. Cytochrome c oxidase (EC 1.9.3.1, complex IV) was also determined spectrophotometrically by the decrease in absorbance at 550 nm of reduced cytochrome c in presence of the mitochondrial protein. [9] Reduced cytochrome c was freshly prepared before each experiment by adding a few grains of sodium borohydride to a 10 g/l solution of the pigment in 10 mmol/l potassium phosphate buffer ( pH 7.0). Addition of 0.1 mol/l HC1 stabilized the reduced cytochrome c, and the excess borohydride was removed by centrifugation at 12000 × g for 4 min. Incubation temperatures were 30 °C for complexes I + III, II + III, II and IV . Table 1 Oxidative phosphorylation in RVOT muscle in patients with TOF Heart disease

Substrate

Rate of oxidation (nmol RCI of O2/min/mg protein) +ADP

ADP: O

−ADP

Control Glutamate 15.3 ± 5.3 7.1 ± 2.0 (n = 12) TOF (n = 30) 9.2 ± 1.3⁎ 2.2 ± 0.8⁎ Control Succinate 24.8 ± 9.2 10.8 ± 3.2 (n = 12) TOF (n = 30) 12.2 ± 4.0 ⁎ 4.5 ± 0.5⁎

2.1

2.9 ± 0.1

4.1⁎⁎ 2.2

2.9 ± 0.1 (NS) 1.9 ± 0.07

2.7 (NS) 1.8 ± 0.09 (NS)

Oxygen Consumption rates in the presence and absence of ADP was determined as described in the text and ADP/O ratios and respiratory control index (RCI) were calculated according to Chance and Williams. The results are given as Mean ± S.D. Values expressed as nmol of O2/min/mg protein. ⁎P b 0.001 compared with values for controls. ⁎⁎P b 0.05 compared with values for controls.

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Table 2 Respiratory chain enzyme activities in RVOT muscle in patients with TOF

Table 4 Mitochondrial total-ATPase activity from patients with TOF

Enzymes

Sample

Total-ATPase μmole of Pi/min/mg protein

Control (12) TOF (30)

0.28 ± 0.11 0.11 ± 0.04⁎

TOF (n = 30) Control (n = 12) Nanomole of substrate/min/ mg of protein

Rotenone-sensitive NADH cytochrome c reductase (Complexes I and III) Succinate cytochrome c reductase (Complexes II and III) Ratio, Complexes I and III to Complexes II and III (Complex I) Cytochrome c oxidase (Complex IV) Succinate dehydrogenase (Complex II) Citrate Synthase

5.11 ± 1.24⁎

7.75 ± 2.0

4.22 ± 0.88

4.40 ± 1.3

1.21 ± 0.071⁎ 0.57 ± 0.088 7.31 ± 1.42⁎ 3.21 ± 0.68 22.3 ± 8.1

9.6 ± 2.0 3.25 ± 0.78 25.4 ± 7.8

Values expressed as Mean ± S.D. ⁎P b 0.001 as compared with control.

Citrate synthase (EC 4.1.3.7) [10] was measured by monitoring the change in absorbance at 412 nm caused by the reaction of 5,5′-dithiobis(2-nitrobenzoic acid) with the free coenzyme A formed by the condensation of acetyl-CoA with oxalacetate in the presence of the mitochondrial protein.

⁎P b 0.001 Compared with values for control. ATPase activities of mitral valve mitochondria were determined by estimating the amount of ATP hydrolyzed in terms of inorganic phosphorus (Pi) liberated in the supernatant [11]. The results are given as Mean ± S.D. of number of observation as indicated in bracket.

and state 4 respiration rates in the mitochondria were used. (Table 1) Oxidation with glutamate and succinate as substrates decreased significantly (P b 0.001) with and without added ADP in the study group as compared with control. The respiratory control index (RCI) was used as an indicator of coupling between oxidative phosphorylation and the mitochondrial electron transport chain. RCI was comparable for control as well as study group. Glutamate as a substrate showed

3.4. ATPase activity Mitochondrial suspension was incubated at 37 °C for 60 min in a 0.5 ml medium containing 2 mm ATP, 100 mmol/l NaCl, 20 mmol/l KCL, 5 mmol/l MgCl2, 1 mmol/l EDTA in 50 mmol/ l Tris–HCl (pH = 7.0). The tubes were chilled immediately and centrifuged at 200 ×g for 10 min. Inorganic phosphate liberated in the supernatant was estimated according to Fiske and Subbarow, [11]. Protein estimation was carried out according to Lowry [12] with crystalline bovine serum albumin as standard. 3.5. Statistical analysis Data were expressed as mean ± S.D. Comparisons between two groups were performed using the unpaired t test or Mann– Whitney U test according to parameters (Sigma Stat ver 3.0). P-value of less than 0.001 and 0.05 was considered significant. 4. Results Mitochondrial oxidative phosphorylation in RVOT muscles of patients with TOF is summarized in Table 1. Two different substrates i.e. glutamate and succinate, which involves state 3 Table 3 Mitochondrial cytochrome content in patients with TOF Cytochrome

nmoles of cytochrome/mg protein Control (n = 12)

TOF (n = 30)

a b c + c1

0.22 ± 0.09 0.17 ± 0.07 0.38 ± 0.1

0.13 ± 0.08⁎ 0.10 ± 0.03⁎ 0.21±0.10⁎

The cytochrome contents were determined in Triton X-1-00 solublized mitochondria by difference spectra of reduced and oxidized cytochromes as described in the text. The results are given as Mean ± SD. ⁎P b 0.001 compared with values for controls.

Fig. 1. A and B. Right Ventricular Outflow Tract (RVOT) muscle mitochondria in patients with TOF. The figure shows damaged mitochondrial cristae.

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Fig. 2. Control sample for Right Ventricular Outflow Tract (RVOT) muscle mitochondria. The figure shows a large number of mitochondria without damaged cristae.

significant difference (P b 0.05) as compared with control. The ADP/O ratios from mitochondria in RVOT muscle were almost similar as compared with control. However, no significant differences were seen between both the groups (study and control). Table 2 summarizes the data for all five-enzyme respiratory complex activities in RVOT muscles from patients with TOF. The activities of rotenone-sensitive NADH cytochrome c reductase (complexes I and III), cytochrome c oxidase (complex IV) and the ratio of l and III to II and III complexes (complex I) were significantly lower in patients than in control subjects (P b 0.001). However, the rest of the enzyme activities did not differ significantly between both groups. We also examined mitochondrial cytochrome content of RVOT muscles in study group (Table 3). Thus, cytochrome ‘a’, ‘b’ and ‘c + c1 showed absorption maximum at 605 nm, 560 nm and 551 nm. In the study group, mitochondrial cytochrome content such as a, b and c + c1 were significantly lower when compared with control (P b 0.001). ATPase activity in TOF patients are shown in Table 4. TotalATPase activity was decreased significantly in TOF group when compared with control. A statistically significant positive correlation was observed between ATPase and cytochrome ‘c + c1 (r = 0.70, P b 0.001), cytochrome ‘b’ (r = 0.64, P b 0.001), cytochrome ‘a’ (r = 0.64, P b 0.001) in the study group. Under these conditions, mitochondrial protein content did not show any significant changes. Changes in the RVOT muscle mitochondria represented one of the important EM findings (Fig. 1A and B). They were irregular in shape and were dilated, and in a few, they appeared ruptured. The control samples of RVOT mitochondria showed a normal ultrastructure without any defects in their cristae (Fig. 2). 5. Discussion Although ventricular dysfunction and Congestive heart failure (CHF) is a well-known consequence of long-standing hypoxia, this phenomenon is uncommon in TOF. Rowe [13] noted that

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since the right ventricle is effectively decompressed by the VSD, CHF never occurs in TOF unless there are co-existing unusual anatomic and physiologic features i.e. anaemia, infective endocarditis, systemic hypertension, viral myocarditis, aortic and pulmonic regurgitation, and other structural defects, like abnormal attachments of tricuspid or mitral leaflets causing partial or complete closure of the ventricular septal defect (VSD) [13]. Despite the morbidity and mortality, associated with right heart failure, the pathogenesis of right ventricular hypertrophy has been studied much less than left ventricular disease. Evidence from clinical observations and animal studies suggest that the right ventricle does not respond to stress in precisely the same fashion as the left ventricle. Thus, clinical evidence suggests that the genetic program of the right ventricle is not designed to face an increased work load even when the adaptation is very gradual. Several animal studies also show different responses of the right and left ventricles to pressure overload [14]. On the biochemical level, several different approaches are available for the determination of mitochondrial respiratory chain dysfunctions in patients with mitochondrial disorders. The activities of respiratory chain complexes may be analyzed spectrophotometrically as dehydrogenases and oxidoreductases or they may be examined polarographically as the oxygen consumption rate after addition of various substrates. OXPHOS and rate of respiration, a part of the free energy released during the transport of electrons from substrate to oxygen is retained in high-energy intermediates that ultimately result in the formation of ATP [15]. The decreased oxygen consumption levels of mitochondria obtained from RVOT muscles seen in the present investigation are in agreement with several authors who demonstrated that there was decrease in the oxygen consumption level of mitochondria in patients undergoing TOF. [16–19]. This implies that electron transport was lower in the diseased heart, which is consistent with a decreased capacity for these mitochondria to synthesize ATP. However, mitochondria are also capable of other energy-linked functions that complicate in vivo extrapolation from in vitro experiments. The ADP/O ratio from mitochondria in study group was almost similar as compared with control; Crestanello JA [20] reported that ADP/O ratio was unaffected during ischemia reperfusion which is similar to our observations. This implies that the quality of preparation affects the ratio, but not so much that the ratio can be off by an order of magnitude. Even best isolation will not produce ratios much above 3. Typical ratios for succinate and glutamate supported respiration approach 2 and 3, respectively. The inhibition of enzymatic complex activity has been observed in other tissues from TOF, such as left and right ventricular biopsies [21]. Several studies have previously demonstrated that the mitochondrial respiratory chain is a source of ROS [22–25]. Complex I and III are the prime sites for electron leakage to molecular oxygen resulting free radical generation in mitochondria. The rate of mitochondrial free radical production is inversely proportional to the rate of electron transport, exponentially increasing when complex I and III of the electron transport chain function at sub optimal level [25]. Our findings are shows significant decrease in the complex I + III, I and IV activity in TOF patients. However, complex II+III and II

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activity was unchanged. In addition, the specific activity of citrate synthase was similar in patients and control subjects. We attempted to correlate the foregoing observations with the mitochondrial cytochrome content in patients with TOF. The decrease in state 3 respiration could partly be correlated with their cytochrome content (r = 0.50 for c + c1; r = 0.55 for a and r = 0.49 for b); thus in patients with TOF, respiration with glutamate and succinate decreased along with a decrease in the amounts of cytochrome a, b and c + c1 similar to our observations, a significant reduction of all cytochrome has been noted by Chandel et al. [26]. Furthermore, these findings have been supported by measurement of mitochondrial ATPase activities. In agreement with Hunter JT [27], mitochondrial ATPase activity was drastically impaired in patients during hypertrophy; there is a switch of the contractible proteins to the fetal and neonatal forms. It is possible that this abnormality is related to the increased ADP levels in the failing hearts. Morphological observations on preoperatively resected right ventricular myocardium could have a predictive value in assessing the surgical outcome in the patients, although there is a paucity of such studies in literature [28,29]. Effects due to hypoxia and pressure overload on the right ventricular myocardium has been found to increase with age [29], and it has been found that the hypertrophic changes evolve into various degrees of degeneration depending on the severity and duration of the disease. The earliest study of myocardial changes in congenital heart disease was by Vtiurin et al. [30] who noted myofibrillar edema and mitochondrial changes and correlated the severity of these changes with age. Kato et al. [31] analysed the right ventricular myocardium in TOF by light microscopy and morphometry. He found a significant correlation between the diameter of the cardiac myocyte with Hb levels and the age of the patients. In a detailed ultrastructural evaluation of the crista supraventricularis in patients with congenital heart disease associated with right ventricular outflow tract obstruction, Jones and Ferrans [32] observed severe degenerative changes in patients over 30 years. They attributed these changes to the stress of prolonged right ventricular hypertrophy and hypoxia and found a correlation with clinical cardiac dysfunction. 6. Conclusion Our study shows that OXPHOS, mitochondrial respiratory chain complex I, I+III and IV, cytochrome content and ATPase activity are more impaired in RVOT muscles in patients with TOF. These enzyme changes are consistent with the hypothesis that impaired electron transport chain function contributes to the metabolic dysfunction and oxidative stress in TOF. In TOF, hypertrophy of the right ventricle progresses immediately after birth, followed soon after by atrophy of the right atrium. References [1] Saakian IR, Karapetian TD, Sherdukalova LF. Predicting heart failure by intravital study of the mitochondrial energy function in patients with heart defects. Kardiologiia 1990;30:91–4.

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