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Ann Thorac Surg 2006;81:1817-1823
© 2006 The Society of Thoracic Surgeons

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Original article: Cardiovascular

Preservation of Mitochondrial Structure and Function After Cardioplegic Arrest in the Neonate Using a Selective Mitochondrial K_ATP Channel Opener

Division of Cardiovascular Surgery, Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada

Accepted for publication November 4, 2005.

^_* Address correspondence to Dr Caldarone, Division of Cardiovascular Surgery, The Hospital for Sick Children, 555 University Ave, Toronto, Ontario, Canada, M5G 1X8 (Email: christopher.caldarone{at}sickkids.ca).

	Abstract

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BACKGROUND: Mitochondrial dysfunction may contribute to early postoperative neonatal heart dysfunction. Diazoxide, a mitochondrial-selective adenosine triphosphate–sensitive potassium-channel opener, is associated with mitochondrial preservation after cardioplegic arrest. We evaluated the mitochondrial-protective effect of diazoxide in terms of mitochondrial structure and function after neonatal cardioplegic arrest.

METHODS: Newborn piglets (age, approximately 14 days) underwent cardiopulmonary bypass and 60 minutes of cardioplegic arrest using cold crystalloid cardioplegic solution (CCP, n = 5) or cold crystalloid cardioplegic solution with diazoxide (CCP+D, n = 5). After 6 hours of recovery, myocardium was harvested. Control myocardium from piglets that did not undergo cardiopulmonary bypass (non-CPB, n = 5) was obtained.

RESULTS: Cardioplegic arrest was associated with translocation of Bax to the mitochondria, which was not prevented by diazoxide. Nevertheless, by electron microscopy, CCP-associated remodeling of mitochondrial structure was subjectively diminished in CCP+D hearts. In addition, CCP-associated mitochondrial permeabilization and cytochrome c release into the cytosol were prevented with CCP+D (p < 0.05). In vitro oxygen consumption of isolated mitochondria demonstrated deficient function of mitochondrial complex I in CCP, but it was preserved in the CCP+D myocardial mitochondria (p < 0.05). Complex II and IV activity was not different among groups. In parallel with impaired complex I function, the cardiac adenosine triphosphate content was diminished in CCP hearts, but well maintained in CCP+D hearts (p < 0.05).

CONCLUSIONS: Although early apoptotic signaling events (Bax translocation) are not prevented by diazoxide, addition of the mitochondrial-selective adenosine triphosphate–sensitive potassium-channel opener to the cardioplegic solution is associated with protection of mitochondrial structural and functional integrity in a clinically relevant model of neonatal cardiac surgery. The mitochondrial-protective effects of diazoxide may contribute to improved postoperative myocardial function in the neonate.

	Introduction

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The early postoperative period after neonatal cardiac surgery can be notable for a period of myocardial dysfunction reaching a nadir approximately 6 to 8 hours after surgery [1–3]. Neonatal models of cardioplegic arrest have demonstrated mitochondrial ultrastructural remodeling and respiratory depression within this time frame, suggesting that preservation of mitochondrial architectural and functional integrity may be an important cardioprotective strategy in the postoperative neonatal heart [4].

Modulation of adenosine triphosphate (ATP)-sensitive potassium channels (K_ATP) by potassium-channel openers has been associated with myocardial protection against ischemia–reperfusion injury [5]. Consequently, K_ATP channel openers have been examined as additives to cardioplegic solution to enhance myocardial protection [6–8]. Although initial studies suggested that cardioprotection by potassium-channel openers was afforded by opening sarcolemmal K_ATP channels, selective modulation of mitochondrial K_ATP channels has been associated with prevention of mitochondrial injury induced by extramitochondrial calcium, inorganic phosphate, and oxidant stress [9, 10], and enhancement of myocardial preservation during cardioplegic arrest [6, 11]. Because mitochondrial electron transport capacity and ATP synthesis are inextricably linked with cardiac function, the observation that addition of diazoxide, a mitochondrial-selective K_ATP channel opener [12, 13], to cardioplegic solution can maintain mitochondrial functional and structural integrity after cardioplegic arrest [11] suggests an important role for a mitochondrial-protective component in a myocardial preservation strategy.

Our previous animal studies have demonstrated that cardioplegic arrest and 6 hours of reperfusion is associated with mitochondrial permeabilization, cytochrome c release, and deficits in electron transport [4]. Of note, neonatal myocardium is more susceptible to these alterations in mitochondrial permeabilization when compared with mature myocardium [14]. Therefore, in the present study, we investigated the influence of the mitochondrial-protective effect of diazoxide, a mitochondrial-selective K_ATP channel opener, on mitochondrial structure and function in a neonatal model of cardioplegic arrest.

	Material and Methods

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Surgical Preparation
Newborn piglets (age, approximately 14 days) were anesthetized with intravenous sodium pentothal, intubated, and maintained with 1% isoflurane throughout the experiment. The piglets underwent cardiopulmonary bypass, 60 minutes of cardioplegic arrest using cold crystalloid cardioplegic solution (Plegisol; Abbott Laboratories, Chicago, IL; CCP, n = 5) or cold crystalloid cardioplegic solution containing 50 µmol/L diazoxide (Sigma Chemical Co, St. Louis, MO; CCP+D, n = 5). Diazoxide was only delivered during the initial administration with cardioplegia and was not included with subsequent cardioplegic administration to prevent systemic hypotension as described by McCully and colleagues [6]. Diazoxide was dissolved in dimethyl sulfoxide (Fisher Scientific Co) before being added to the cardioplegia in CCP+D experiments. Dimethyl sulfoxide alone was added as a sham vehicle control to CCP. Control myocardium from piglets that did not undergo cardiopulmonary bypass (non-CPB, n = 5) was also obtained.

For the CCP and CCP+D protocols, atrial and aortic cannulas were placed through a median sternotomy. Cardiopulmonary bypass was initiated with a blood prime and passive cooling to 28° to 30°C core temperature. The ductus arteriosus was ligated if patent. The aorta was then cross-clamped, and cardioplegic solution was delivered at 4°C into the aorta with 50 to 70 mm Hg perfusion pressure to a total initial dose of 20 mL/kg body weight. Subsequent cold cardioplegic doses of 15 mL/kg were delivered at 20-minute intervals. Between cardioplegic doses, topical cooling was applied, and the aortic root and pulmonary artery were vented. After 60 minutes of arrest, the cross-clamp was removed, the animal was rewarmed, and after a 10- to 20-minute stabilization period, cardiopulmonary bypass was terminated.

General anesthesia was maintained for 6 hours after reperfusion, during which time carotid artery blood pressure, electrocardiogram, and heart rate were displayed. The arterial blood gases, electrolytes, and hematocrit were maintained in a physiologic range, and pump blood was infused as necessary to maintain arterial pressure. Inotropic agents were not administered. After 6 hours, the heart was quickly excised and perfused through the coronary arteries with 250 mL of ice-cold Dulbecco's phosphate-buffered saline (Invitrogen, Burlington, VT). Full-thickness left ventricular myocardium was cut and snap-frozen in liquid nitrogen and stored at –80°C. For mitochondrial oxygen consumption and cytochrome c release measurements, another piece of tissue (approximately 1 g) was immediately fractionated as previously described [4]. The freshly isolated mitochondrial fractions were used for oxygen consumption measurement, and randomly selected fractions were fixed for electron microscopic examination. The purity of mitochondrial fractions was confirmed by visual inspection of electron micrographs as well as batch process evaluation of porin and citrate synthase activity as described below. The cytosol was stored at –80°C for further analysis of mitochondrial permeability change. For the non-CPB protocol, after appropriate anesthesia for 6 hours, a thoracotomy was performed, and myocardial tissue was harvested in an identical fashion.

All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" prepared by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC; March 1999).

Electron Microscopy
The mitochondrial fraction was resuspended and fixed with 2% glutaraldehyde in 0.1 mol/L cacodylate buffer overnight. Mitochondria were postfixed with 1% OsO₄ in 0.2 mol/L cacodylate buffer (pH 7.3), dehydrated, and embedded in Epon. Ultrathin sections were stained with lead citrate and uranyl acetate and examined with Hitachi H 7000 transmission electron microscope at x10,000 magnification.

Mitochondrial Fractionation for Biochemical Analysis
For mitochondrial Bax and Bid analysis, frozen ventricular myocardium samples were mechanically homogenized in 5 mL of ice-cold lysis buffer (250 mmol/L sucrose, 50 mmol/L Tris-HCl, pH 7.6, 1 mmol/L MgCl₂, 1 mmol/L dithiothreitol, 1 mmol/L phenylmethylsulfonyl fluoride). After centrifugation at 800g for 15 minutes, the supernatant was centrifuged at 7,500g for 20 minutes at 4°C. The resulting pellet was washed twice in lysis buffer (7,500g twice) and then dissolved in hypotonic buffer (10 mmol/L HEPES (N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]), pH 7.9, 1 mmol/L phenylmethylsulfonyl fluoride). After incubation on ice for 30 minutes, the suspension was sonicated and recentrifuged at 13,000g for 30 minutes. The soluble mitochondrial proteins were stored at –80°C.

Cytochrome c Release
Mitochondrial outer membrane permeability was evaluated by measurement of cytochrome c release into the cytosol. After subcellular fractionation, cardiac cytosolic samples were standardized for protein concentration, denatured, and subjected to 12% denatured sodium dodecylsulfate–polyacrylamide gel and subsequently transferred to nitrocellulose membranes by 100V for 2 hours at 4°C. The membrane was blocked with 5% nonfat milk in TBS (25 mmol/L Tris, 137 mmol/L NaCl, and 2.7 mmol/L KCl) containing 0.1% Tween-20 and incubated overnight at 4°C with specific anti-mouse cytochrome c (1:2,000 dilution; BD Biosciences Pharminutegen). For the protein loading control, the membranes were stripped and reprobed with anti-goat ß-actin (1:2,000 dilution; Santa Cruz Biotechnology). Secondary antibodies were coupled to horseradish peroxidase (anti-mouse immunoglobin G 1:5,000 dilution; anti-goat immunoglobin G 1:5,000 dilution; Santa Cruz Biotechnology). The resulting autoradiographs were scanned and quantified with densitometry (Fluorchem 8000, Alpha Innotech Corporation, San Leandro, CA). The amount of cytosolic cytochrome c was normalized by cytosolic citrate synthase activity. Citrate synthase is an inner mitochondrial membrane enzyme and can be used to correct for disruption of the inner mitochondrial membrane during the isolation process as described by Schmitt and coworkers [15].

Mitochondrial Bax and Bid Translocation
Translocation of Bax and Bid from cytosol to mitochondria was examined by Western blotting analysis of mitochondrial protein fractions. Briefly, after transblotting protein onto nitrocellulose membranes, the membranes were probed with anti-rabbit Bax (N-20, 1:500 dilution; Santa Cruz Biotechnology) and anti-rabbit Bid (SC-11423, 1:1000 dilution; Santa Cruz Biotechnology). For protein loading control, the membranes were stripped and reprobed with anti-mouse mitochondrial porin (1:20,000 dilution; Molecular Probes, Inc). Secondary antibodies were coupled to horseradish peroxidase (anti-mouse immunoglobin G 1:5,000 dilution; anti-rabbit immunoglobin G 1:1,000 dilution, Santa Cruz Biotechnology). The resulting autoradiographs were scanned and quantified. The amount of mitochondrial Bax and Bid was normalized by porin (a mitochondrial protein) to control for variation in mitochondrial protein loading.

Clark-Electrode Oxygen Consumption Measurement
Mitochondrial complex I, II, and IV respiration was measured by the method of Ricci and associates [16] using a Clark-type oxygen electrode (Instech Laboratories Inc, Plymouth Meeting, PA). After loading 0.2 g of mitochondrial protein in the chamber, oxygen consumption was measured in the presence of sequential administration of substrates and inhibitors (glutamate–malate for complex I, rotenone with succinate for complex II, antimycin with N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD)–ascorbate for complex IV). State 2 oxygen consumption was defined as the mitochondrial oxygen consumption in the presence of substrate but before the addition of adenosine diphosphate (ADP). State 3 was defined as the mitochondrial oxygen consumption in the presence of substrate after the addition of ADP. Mitochondrial respiratory state 4 (oxygen consumption of mitochondria after depletion of substrate and ADP) was not available because an excess of ADP was needed to drive mitochondrial respiration during sequential administration of substrate and inhibitors. Therefore, the respiratory control index in this experiment was calculated as the ratio of state 3 to state 2 oxygen consumption as previously described [17].

The mitochondrial suspension (0.2 mg) was injected into a water-jacketed sample chamber. The respiratory buffer (130 mmol/L KCl, 20 mmol/L HEPES-KOH, pH7.2, 2.5 mmol/L MgCl₂, 0.5 mmol/L EDTA) was preequilibrated with air at 37°C. Substrates and inhibitors for mitochondrial complex I, II, and IV were added in the following order and final concentration: 2.5 mmol/L glutamate, 2.5 mmol/L malate, 2 mmol/L ADP, 2 µmol/L rotenone, 5 mmol/L succinate, 1 µmol/L antimycin A, and 1 mmol/L ascorbate with 0.4 mmol/L TMPD. All the substrates and inhibitors were purchased from Sigma-Aldrich. The voltage signal was amplified and digitalized by a computer-supported PowerLab ADInstruments System (ADInstruments Pty Ltd, Castle Hill, Australia). Respiration rates are expressed as nanomoles of oxygen per milligram of mitochondrial protein.

Cardiac Adenosine Triphosphate Content Measurement
Tissue samples (0.5 g) from free left ventricular wall were homogenized in 2 mL of 2.5% trichloroacetic acid for 30 seconds. After centrifugation (1,000g x 15 minutes), supernatants were transferred to 2-mL tubes and centrifuged (1,000g x 10 minutes); supernatants were transferred to 2-mL tubes and neutralized (1 mol/L Tris-base to pH 7.0). Neutralized supernatants were centrifuged (1,000g x 5 minutes) and stored at –80°C. Pellets from the first spin were homogenized in 2 mL of 0.5 mol/L NaOH and centrifuged (1,000g x 5 minutes). Supernatants were transferred to 2-mL tubes and centrifuged (14,000g x 20 minutes). Protein concentrations were determined using the Lowry method [18]. Tissue ATP content was measured per manufacturer's instructions using the Adenosine 5'-triphosphate Bioluminescent Assay Kit (Sigma).

Statistics
Data are expressed as mean ± standard error of the mean, and group comparisons were made with Fisher's least significant difference analysis of variance. A Tukey test was used for multiple post hoc comparisons. A p value less than 0.05 was considered statistically significant.

	Results

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Hemodynamics
At 6 hours of reperfusion, systolic blood pressure and central venous pressure were not different between CCP, CCP+D, and non-CPB groups. Heart rate was increased in the CCP and CCP+D groups when compared with non-CPB (138 ± 6 and 145 ± 20 versus 113 ± 10 beat/min, respectively; p < 0.01 for each comparison). In addition, diastolic blood pressure was lower in the CCP and CCP+D groups when compared with non-CPB (30 ± 4 and 26 ± 6.6 versus 44 ± 4 mm Hg, respectively; p < 0.01 for each comparison). There were no differences in any of these hemodynamic measurements when comparing CCP and CCP+D groups.

Electron Microscopy
A group of representative electron microscopic images are shown in Figure 1. In contrast to the normal appearance of the mitochondrial cristae in the non-CPB mitochondria, there is extensive alteration in cristae architecture in the CCP mitochondria, including swelling and disruption of the inner mitochondrial membranes and electron-dense mitochondrial granules. In contrast, the CCP+D mitochondria have preservation of cristae architecture.

View larger version (72K): [in this window] [in a new window]   Fig 1. Representative electron micrographs of mitochondria isolated from the control (non-CPB) hearts (a), cold crystalloid cardioplegic solution (CCP) perfused hearts (b), and cold crystalloid cardioplegic solution with diazoxide (CCP+D) perfused hearts (c). (X 10,000.) Note the distortion of the cristae in the CCP mitochondria (arrowhead), which is absent in the CCP+D mitochondria, and the presence of electron-dense granules in the CCP mitochondria (arrow).

View larger version (35K): [in this window] [in a new window]   Fig 2. (A) Immunoblots of cytosolic cytochrome c and ß actin in control (non-CPB), cold crystalloid cardioplegic solution (CCP), and cold crystalloid cardioplegic solution with diazoxide (CCP+D) perfused hearts. (B) Densitometric measurements of cytosolic cytochrome c in non-CPB, CCP, and CCP+D hearts. Increased concentrations of cytochrome c in the cytosolic fractions normalized by citrate synthase activity are indicative of mitochondrial permeabilization and release of cytochrome c. *p < 0.05 versus non-CPB and p < 0.05 versus CCP. (Prot = protein.)

View larger version (39K): [in this window] [in a new window]   Fig 3. (A) Immunoblots of mitochondrial Bax, Bid and porin in control (non-CPB), cold crystalloid cardioplegic solution (CCP), and cold crystalloid cardioplegic solution with diazoxide (CCP+D) perfused hearts. (B) Densitometric measurements of mitochondrial Bax and Bid in non-CPB, CCP, and CCP+D hearts. Increased concentrations of Bax and Bid in the mitochondrial fractions normalized by porin content are indicative of translocation of Bax and Bid to the mitochondria. For Bid, the difference between CCP and non-CPB was not statistically significant (p = 0.08). **p < 0.01 versus non-CPB.

View larger version (12K): [in this window] [in a new window]   Fig 4. Cardiac tissue adenosine triphosphate (ATP) levels in control (non-CPB), cold crystalloid cardioplegic solution (CCP), and cold crystalloid cardioplegic solution with diazoxide (CCP+D) perfused hearts. All results are shown as mean ± standard error of the mean. **p < 0.01 versus non-CCP; p < 0.01 versus CCP.

	Comment

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Recent studies have demonstrated that activation of the mitochondrial K_ATP channels by a variety of stimuli can preserve mitochondrial integrity after ischemia–reperfusion injury in vivo [20, 21]. Mitochondrial K_ATP channel opening is proposed to play an important role both as a trigger and as an end-effector in cardioprotection against ischemia–reperfusion injury [22–24]. The data from this study support the hypothesis that diazoxide preserves mitochondrial structural integrity by preventing outer mitochondrial membrane permeabilization, release of cytochrome c, and mitochondrial cristae distortion. In the present study, diazoxide-associated maintenance of mitochondrial structural integrity was accompanied by preservation of mitochondrial functional integrity observable at three sites: complex I, maintenance of intramitochondrial sequestration of cytochrome c, and maintenance of myocardial ATP concentrations.

Interestingly, diazoxide did not prevent translocation of Bax to the mitochondria or a trend toward Bid translocation, which are important apoptosis-signaling events [25]. Although the mechanism of action is not well defined, Bax may induce outer mitochondrial membrane permeabilization through formation of multimers with pore-forming capacity [26] or through some other undefined direct interaction with the outer mitochondrial membrane [27]. In the present study, the translocation of Bax suggests initiation of an apoptotic signal. Although the signal is translated into permeabilization of the outer mitochondrial membrane and loss of cytochrome c into the cytoplasm in the CCP myocardium, addition of diazoxide is associated with suppression of this signal and preservation of outer mitochondrial membrane integrity. Consequently, these data suggest that the mitochondrial-protective effect of diazoxide is downstream from apoptosis-signaling events such as Bax (or Bid) translocation.

The localization of the deficit in electron transport after cardioplegic arrest to complex I is consistent with previous reports of protected ischemia [17] and unprotected myocardial ischemia [28]. In our previous report [4], we found postcardioplegic mitochondrial Bax translocation was correlated with mitochondrial permeabilization, cytochrome c release, and mitochondrial dysfunction and described this phenomenon as apoptosis-related mitochondrial dysfunction. The present study supports the concept of apoptosis-related mitochondrial dysfunction in the early postoperative neonate and localizes the deficit in electron transport to complex I.

Although the cause of the deficit in electron transport is not determined in this study, potential mechanisms include the following: (1) mitochondrial Bax translocation directly uncouples and inhibits mitochondrial respiration [27]; (2) Bid translocation jeopardizes mitochondrial respiration through Bid-induced mitochondrial remodeling and cytochrome c release [29]; (3) cytochrome c release through a permeabilized outer mitochondrial membrane allows cytosolic-derived mediators to induce isolated dysfunction of electron transport complexes [16, 30]; and (4) ischemia-induced deficiency of complex I function is associated with localized production of reactive oxygen species, which creates a positive feedback loop inducing further complex I dysfunction through reactive oxygen species–mediated cardiolipin damage [28].

The present data do not allow quantitative comparison of the relative contribution of the two lesions in electron transport chain (complex I dysfunction and loss of cytochrome c) to the overall deficiency of myocardial ATP concentration noted after 6 hours of reperfusion. Incomplete reversal of defective electron transport with exogenous cytochrome c suggests that complex I retains respiratory control of electron transport after protected ischemia and reperfusion [17]. It is plausible, therefore, that maintenance of mitochondrial architecture with diazoxide prevents access of cytosolic inhibitors to the outer mitochondrial space and subsequent direct inhibition of complex I function [16].

Cardioplegic arrest in the neonate is associated with alterations in mitochondrial structure and function, which can be ameliorated with diazoxide, a mitochondrial-specific K_ATP channel opener. Prevention of mitochondrial permeabilization is associated with preservation of function at the level of complex I in the electron transport chain and maintenance of myocardial ATP. This mitochondrial-protective effect is downstream of apoptosis-signaling events (Bax or Bid translocation), suggesting the potential for myocardial-protective strategies specifically designed to ameliorate apoptosis-related mitochondrial dysfunction.

	The Society of Thoracic Surgeons: Forty-Third Annual Meeting

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Please mark your calendars for the Forty-Third Annual Meeting of The Society of Thoracic Surgeons, to be held in San Diego, California, from January 29–31, 2007. The program will provide in-depth coverage of thoracic surgical topics selected to enhance and broaden the knowledge of cardiothoracic surgeons. Attendees will benefit from traditional Abstract Presentations, as well as Surgical Forums, Breakfast Sessions, Surgical Motion Pictures, and Town Hall Meetings on specific topics.

Advance registration forms, hotel reservation forms, and details regarding transportation arrangements, as well as the complete meeting program, will be mailed to Society members this fall. Also, complete meeting information will be available on the Society's Web site at www.sts.org. Nonmembers who wish to receive information on the Annual Meeting may contact the Society's secretary, Douglas E. Wood.

Douglas E. Wood, MD

Secretary

The Society of Thoracic Surgeons

633 N. Saint Clair St, Suite 2320

Chicago, IL 60611-3658

Telephone: (312) 202-5800

Fax: (312) 202-5801

e-mail: mailto:sts{at}sts.org

website: www.sts.org

	Acknowledgments

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Supported by a Scientist Development Grant from the American Heart Association (CAC).

	References

Top Abstract Introduction Material and Methods Results Comment The Society of Thoracic... Acknowledgments References

 

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