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Ann Thorac Surg 2005;79:1620-1626
© 2005 The Society of Thoracic Surgeons

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Original articles: Cardiovascular

Cyclosporin A But Not FK-506 Protects Against Dopamine-Induced Apoptosis in the Stunned Heart

^a Department of Cardiac Surgery, Children's Hospital and Harvard Medical School, Boston, Massachusetts
^b Department of Anesthesiology and Pain Medicine, Children's Hospital and Harvard Medical School, Boston, Massachusetts

Accepted for publication October 19, 2004.

^_* Address reprint requests to Dr del Nido, Department of Cardiac Surgery, Children's Hospital Boston, Harvard Medical School, 300 Longwood Ave, Bader 279, Boston, MA 02115 (E-mail: pedro.delnido{at}tch.harvard.edu).

	Abstract

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BACKGROUND: Dopamine given at moderate doses for inotropy to postischemic hearts has been shown to augment myocyte apoptosis in association with elevated cytosolic calcium. We hypothesize that dopamine-mediated apoptosis occurs through calcium-induced opening of the mitochondrial permeability transition (mPT) pore. We also hypothesize that cyclosporin A (CSA), a calcineurin inhibitor known to block mPT pore opening, would prevent dopamine-induced apoptosis primarily by inhibiting pore opening (cyclophilin D binding).

METHODS: Isolated perfused rabbit hearts (n = 6/group) were subjected to 30 minutes of 37°C cardioplegic arrest followed by 120 minutes reperfusion (ischemic injury that produces < 3% infarct by triphenyl-tetrazolium chloride [TTC] staining). Four groups were studied: (1) control; (2) dopamine (10 µmol/L) postischemia (dopa); (3) dopamine+CSA (0.2 µmol/L) (CSA+D) group; (4) dopamine+FK-506 (0.2 µmol/L) (FK+D) group. Left ventricular developed pressure and oxygen consumption were measured preischemia and postischemia. Bax, caspase-3 and caspase-9, and poly-ADP-ribose polymerase (PARP) activation were measured by Western blotting. Apoptotic nuclei were quantified by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining.

RESULTS: Dopamine postischemia improved contractile function and heart rate and this was not affected by CSA or FK. However, TUNEL positive nuclei, Bax, caspase-3 and caspase-9 activation, and PARP cleavage were all increased in dopa and FK+D groups, but not in CSA+D.

CONCLUSIONS: Cyclosporin is effective in preventing dopamine-induced apoptosis in the postischemic heart. The mechanism is likely due to inhibition of mPT pore opening since FK-506, a potent calcineurin inhibitor that does not bind to cyclophilin, did not prevent this. Low dose cyclosporin may prove useful to prevent dopamine-induced apoptosis resulting in long-term preservation of cardiac function.

	Introduction

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Dopamine is an inotrope that is commonly used in the postoperative period to treat low output states resulting from ischemia reperfusion-induced myocardial dysfunction. The positive inotropic effect of catecholamines like dopamine is by induction of cyclic adenosine monophosphate (cAMP) dependent protein kinase-mediated phosphorylation of plasma membrane calcium channels [1]. Dopamine increases calcium entry, and thus calcium binding to contractile proteins. Calcium is also essential for the final step of excitation contraction coupling. It is well-documented that there is significant calcium overload in the postischemic heart [2]. The mechanism of calcium overload is multifactorial. There is increased calcium entry secondary to membrane damage, decreased uptake into the sarcoplasmic reticulum, and increased calcium entry in exchange for intracellular sodium by the sodium-calcium exchanger. While calcium is essential for maintaining contractility, calcium overload has deleterious effects especially on mitochondrial energetics and function. When dopamine is given postischemia it has the potential for further elevation of calcium. This sustained elevation of cytosolic calcium can result in mitochondrial calcium overload and myocyte apoptosis through the intrinsic pathway.

Mitochondria play a role in cardiomyocyte apoptosis in one of two ways. Recent work by Halestrap and colleagues [3] indicates that the mitochondrial permeability transition pore (mPT pore) is an inner membrane pore composed of adenine nucleotide translocase (ANT) and cyclophylin D. Several proteins, hexokinase, voltage-dependent anion channel (VDAC), Bax, and benzodiazepine receptor (BDR) interact with ANT and modulate the pore. The pore is a mitochondrial megachannel, a multiprotein complex formed at the contact site between mitochondrial inner and outer membrane usually at a site where Bax, Bcl-2, and Bcl-XL are particularly abundant. The mPT pore regulates matrix calcium, pH, and matrix volume and is a calcium, pH, voltage, and redox-gated channel with several levels of conductance but poor ion selectivity [4].

Pathologic mPT pore opening occurs in response to high matrix calcium, oxidative stress, high inorganic phosphate levels (> 10 mmol/L), and adenosine triphosphate (ATP) depletion (< 1 mmol/L), and this pore opening can be reversed by removal of calcium [4]. The mPT pore is large with nonspecific conductance of all ions and solutes less than 1.5 kDa. Thus, pore opening results in the rapid influx of solutes and water into the hyperosmolar matrix resulting in matrix swelling. This swelling can result in outer membrane rupture and release of several proapoptotic factors such as cytochrome c, procaspase 9, Smac/Diablo, and apoptosis inducing factor (AIF) [5]. Of note, mitochondria can induce apoptosis even in the absence of mPT pore opening. Bax and Bid can interact with outer membrane and alter its permeability with release of intermembrane contents, which then initiate the apoptotic cascade [6].

Previous work has shown that in a model of postischemic myocardial stunning, dopamine leads to an increase in cytosolic calcium and thus activation of proapoptotic factors and increased cardiomyocyte apoptosis [7]. Based on these findings, we hypothesized that the mechanism of dopamine mediated apoptosis is through calcium-induced opening of the mPT pore initiating the apoptotic cascade. We also hypothesized that cyclosporin A (CSA), a calcineurin inhibitor known to block mPT pore opening by binding to cyclophilin D, would prevent dopamine-induced apoptosis while FK-506 which does not bind to cyclophilin D and has no effect on the pore, would not. The CSA would therefore result in improved contractile function in the dopamine-treated postischemic hearts.

	Material and Methods

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Animal Care
All animals received humane care in compliance with the "Principles of Laboratory Animal Care" recommended by the National Society for Medical Research, and the "Guide for the Care and Use of Laboratory Animals" formulated by the National Academy of Science and published by the National Institutes of Health (National Institutes of Health Publication No. 85 to 23, revised 1996). The protocol was reviewed and approved by the Investigational Animal Care and Use Committee at Children's Hospital.

Perfusion Protocol
New Zealand White rabbits (1.5 to 2.5 kg) (7 to 8 weeks old) were euthanized by intravenous injection of ketamine (100 mg/kg), Xylazine (5 mg/kg), and heparin 500 U/kg. The hearts were rapidly excised, the aorta cannulated, and the hearts were perfused retrograde in the Langendorff mode at 80 mm Hg constant perfusion pressure with modified Krebs-Henseleit buffer (KH: 115 mmol/L of NaCl, 26 mmol/L of NaHCO₃, 11 mmol/L of glucose, 1.8 mmol/L of MgSO₄, 1.8 mmol/L of KH₂PO₄, 4.7 mmol/L of KCl, 1.25 mmol/L of CaCl₂, and 10 U/L of insulin) at 37°C and equilibrated with a 95% O₂/5% CO₂ gas mixture passed through a 0.2 µ filter. In addition, the hearts were placed in a heated chamber filled with humidified room air to keep the temperature constant. All chemicals used were obtained from Sigma-Aldrich (St. Louis, MO). The venae cavae and pulmonary veins were sutured closed and the pulmonary artery was cannulated for collection of venous effluent into an air sealed container for measurement of myocardial oxygen consumption (MVO₂) at set time points, and a timed collection volume was used to estimate coronary flow. The MVO₂ was derived from the difference in oxygen tension between aortic perfusate and coronary effluent (Stat Profile Plus 9, Nova Biochemical, Waltham, MA) multiplied by coronary flow and divided by dry weight of heart. A fluid filled balloon, connected to a micromanometry catheter, was placed in the left ventricle (LV) through the left atrium (Millar Instruments, Houston, Texas) for isovolumic left ventricular function measurement. Temperature was monitored continuously with a thermistor probe placed in the right ventricle.

After 30 minutes stabilization, the hearts were subjected to 30 minutes warm ischemia (37°C) with cardioplegic arrest (KH buffer +22.5 mmol/L KCl) followed by reperfusion for 120 minutes. The following groups of hearts were studied as per the following protocol.

• Ischemia-reperfusion (control) group (n = 6): 30 minutes stabilization, warm cardioplegic ischemia for 30 minutes, reperfusion with KH buffer containing 0.05% dimethyl sulfoxide (DMSO).

• Ischemia-reperfusion-dopamine (dopa) group (n = 6): 30 minutes stabilization, warm cardioplegic ischemia for 30 minutes, and reperfusion with KH buffer containing 0.05% DMSO. Dopamine (10 µmol/L) was added to the perfusate 5 minutes postischemia and given throughout the entire reperfusion period.

• Ischemia-reperfusion-dopamine-cyclosporin A (CSA+D) group (n = 6): 30 minutes stabilization, warm cardioplegic ischemia for 30 minutes, 0.2 µmol/L of CSA mixed in DMSO (final concentration of DMSO 0.05%) in KH buffer at reperfusion. Dopamine (10 µmol/L) was added to the perfusate 5 minutes postischemia and given throughout the reperfusion period.

• Ischemia-reperfusion-dopamine-FK-506 (FK+D) group (n = 6): 30 minutes stabilization, warm cardioplegic ischemia for 30 minutes, 0.2 µmol/L of FK mixed in DMSO (final concentration of DMSO 0.05%) in KH buffer at reperfusion. Dopamine (10 µmol/L) was added to the perfusate 5 minutes postischemia and given throughout the reperfusion period.

Assessment of Infarct Size
In a separate group of hearts, at the end of the respective perfusion protocols, 2-mm-thick slices of myocardium were prepared and incubated with 1% triphenyl tetrazolium chloride (TTC) in phosphate-buffered saline (PBS) at 37°C for 20 minutes. The stained slices were placed on a flatbed scanner and electronic images were obtained. The TTC negative area, which represents the infarct area, was measured using Scion Image analysis software (Scion Corp, Frederick, MD). Infarct size was quantified for each slide using the formula negative stained area-positive stained area x 100 and the mean value of all slices obtained from one ventricle was calculated.

Western Blotting
Left ventricular tissue was homogenized in cold buffer containing 150 mmol/L NaCl, 2 mmol/L tris HCl pH 7.6, 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 0.1 mmol/L Na orthovanadate, Tergitol (Union Carbide Corp) 1 mL/100 mL, 1.5 mmol/L deoxycholic acid, 70 mmol/L sodium fluoride, 0.1 mmol/L phenylmethylsulphonyl fluoride (PMSF), complete tablet 2/100 mL, allowed to sit on ice for 10 to 20 minutes and centrifuged at 1,000g for 5 minutes (supernatant = unfractionated tissue extract). For detection of poly-ADP-ribose polymerase (PARP) cleavage, nuclear protein extracts were prepared by suspending homogenized LV tissue in cold buffer A (10 mmol/L N -2-hydroxyethylpiperazine- N -2-ethanesulfonic acid [HEPES]-KOH pH 7.9 at 4°C, 1.5 mmol/L MgCl₂, 10 mmol/L KCl, 0.5 mmol/L dithiothreitol, 0.2 mmol/L PMSF) on ice for 10 minutes, then vortexed for 10 seconds. Samples were centrifuged for 10 seconds and the supernatant fraction discarded. The pellet was resuspended in 20 to 100 µL of cold buffer B (20 mmol/L HEPES-KOH pH 7.9 at 4°C, 25% glycerol, 420 mmol/L NaCl, 1.5 mmol/L MgCl₂, 0.2 mmol/L EDTA, 0.5 mmol/L dithiothreitol, 0.2 mmol/L PMSF) and incubated on ice for 20 minutes for high salt extraction. Cellular debris was removed by centrifugation for 2 minutes at 4°C and the supernatant fraction (containing DNA binding proteins) was used for detection of PARP cleavage

Protein samples of 50 µg each of homogenates (nuclear extract for PARP) were separated by SDS-Page gel electrophoresis and transferred to nitrocellulose membranes. Coomassie Brilliant Blue R-250 staining (Bio-Rad Laboratories, Hercules, CA) of gel was performed to confirm equal protein loading. The membranes were incubated with the following primary antibodies to caspase 9 (Calbiochem, San Diego, CA), active caspase 3 (Calbiochem, San Diego, CA), Bax (Upstate, Lake Placid, NY), and PARP (ABR, Golden, CO). This was followed by incubation with horseradish peroxidase conjugated secondary antibody. The blots were detected using enhanced chemiluminescence (Amersham Biosciences, Buckinghamshire, UK). Laser densitometry was performed to quantify the intensity of respective bands.

TUNEL Staining
Terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was performed in tissue sections. The slides were deparaffinized and rehydrated. Fluorescent detection was performed by fragment end labeling of cleaved double stranded DNA using the Fluorescein-FragEL kit (Oncogene, San Diego, CA). Sections were also stained for nuclei (DAPI; Molecular Probes, Eugene, OR) and desmin (Sigma-Aldrich, St. Louis, MO). Both total nuclei and TUNEL positive nuclei were counted electronically in 20 random fields of vision per tissue section using Metamorph software (Universal Imaging Corp, Downington, PA) and results were expressed as the number of TUNEL positive nuclei/1,000 total nuclei.

Statistical Analysis
Data are expressed as the mean ± standard error and statistical analysis was performed using SPSS software package version 9.0 (SPSS Inc, Chicago, IL ). Differences between the groups were tested for significance by one-way analysis of variance using Bonferroni's correction for multiple comparisons. If normal distribution and equal variance testing was passed, the Student t test was used to compare individual data set. A two-tailed probability value of less than 0.05 was considered statistically significant.

	Results

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Contractile Function
Postischemia there was a significant fall in systolic blood pressure in the control group at all points compared to preischemia. However, in the dopa, CSA+D, and FK+D groups, left ventricular contractility improved on initiation of dopamine treatment with a slow gradual fall to preischemic levels by 120 minutes (Fig 1A). The diastolic pressure showed a gradual increase in all four groups with time, indicative of worsening relaxation (Fig 1B). There was no significant difference in the four groups studied. These changes were reflected as a fall in developed pressure over time (Fig 1C) with the most significant decrease seen in the control group (p < 0.05 vs control). Heart rate remained unchanged in the control group and the coronary flow and oxygen consumption gradually declined with time. All three dopamine-treated groups had a significant increase in heart rate reflecting the chronotropic effect of dopamine. Similarly, the coronary flow and oxygen consumption significantly increased in these groups at 30 minutes reperfusion (coronary flow data not shown). There was a gradual fall in coronary flow to baseline levels at 120 minutes in all three treated groups although oxygen consumption remained elevated when compared to control, indicating energy wasting associated with dopamine treatment (Fig 1D).

View larger version (25K): [in this window] [in a new window]   Fig 1. Systolic pressure, diastolic pressure, developed pressure and oxygen consumption, preischemia and postischemia. Data are mean ± standard error of the mean, *p < 0.05 versus dopamine, CSA+D, and FK+D. There was no significant difference between the three dopamine treated groups. (A) Change in systolic blood pressure; (B) change in diastolic blood pressure; (C) change in developed pressure; (D) change in oxygen consumption. = control; = dopamine; = CSA+D; x = FK+D. (CSA+D = cyclosporin A + dopamine; FK+D = FK-506 + dopamine.)

View larger version (50K): [in this window] [in a new window]   Fig 2. Representative fluorescent and confocal photomicrographs of tissue sections, with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) positive nuclei in green, total nuclei in blue (4',6-diamidino-2-phenylindole [DAPI]), and cardiomyocytes stained red (Desmin). The numbers of TUNEL positive nuclei are expressed as n/1,000 total nuclei. Data are mean ± standard error of the mean, *p < 0.05 versus control; #p < 0.05 versus CSA+D. (A) TUNEL staining, fluorescent microscope; (B) TUNEL staining, confocal microscope; (C) quantitative data. = control; = Dopa; = CSA+D; = FK+D. (CSA+D = cyclosporin A + dopamine; dopa = dopamine; FK+D = FK-506 + dopamine.)

View larger version (22K): [in this window] [in a new window]   Fig 3. Representative Western blots for Bax, active caspase-3 (from tissue extracts), and poly-ADP-ribose polymerase (PARP) (from nuclear extracts). Laser densitometry data are presented in the bar graph. Data are mean ± standard error of the mean, *p < 0.05 versus CSA+D. (A) Bax; (B) caspase-3; (C) PARP. = control; = dopa; = CSA+D; = FK+D. (CSA+D = cyclosporin A + dopamine; dopa = dopamine; FK+D = FK-506 + dopamine.)

	Comment

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Calcineurin is a calcium-calmodulin activated protein phosphatase, which is ubiquitous in all cells [8]. Calcineurin is activated by binding of calcium to its regulatory subunit and calmodulin to its catalytic subunit. Shibasaki and colleagues [9, 10] proposed that calcineurin plays a role in calcium-induced apoptosis in mammalian cells and that this effect is attenuated by Bcl-2 expression. Wang and colleagues [11] have shown that calcineurin-mediated desphosphorylation of Bad results in apoptosis and that protein kinase B (Akt)-mediated phosphorylation of Bad reverses this effect. However, Lotem and colleagues [12] have demonstrated that calcineurin activation can activate opposing pathways that can either induce or suppress apoptosis. Saito and colleagues [2] have shown that calcineurin plays a role in isoproterenol-induced apoptosis in neonatal cardiac myocytes. They attributed this to calcineurin-mediated desphosphorylation of Bad. Molkentin and colleagues [13, 14], Wilkins and Molkentin [15], and De Windt and colleagues [16] have demonstrated that calcineurin plays an important role in induction of cardiac hypertrophy through its effects on the transcription factor NF-AT3. Further work by these groups [14–16] has demonstrated that this calcineurin-mediated hypertrophy had a protective effect against apoptosis in both in vivo and in vitro studies. Thus, there are conflicting mechanisms for calcineurin, where under some circumstances it can be proapoptotic and under others, antiapoptotic.

There is extensive work that has implicated the mPT pore in calcium-mediated apoptosis [17, 18]. This includes seminal work by Suleiman and colleagues [19], Halestrap and colleagues [20] and Crompton and colleagues [6], who have elucidated the signaling pathways involved in mPT pore mediated apoptosis. Griffiths and Halestrap [21–23] have clearly shown that cyclosporin has a protective effect on mPT pore and thus inhibits apoptosis in ischemia-reperfusion injury. They have also shown that the best protection was afforded by low dose cyclosporin (0.2 µmol/L) [23] and that high doses (1 µmol/L) actually reversed this effect. Recent work by Borutaite and Brown [24] and Borutaite and colleagues [25] has confirmed this. Photolabeling studies by Andreeva and colleagues [26] and Tanveer and colleagues [27] have elucidated that cyclophilin D presumably serves as the CSA receptor during pore blockade. Cyclophilin D represents the mitochondrial isoform of a family of cis-trans isomerases; ie, the cyclophilins which play an important role in protein folding. Cyclosporin binds to the active site of cyclophilin D and thus inhibits pore formation by preventing cyclophilin D binding to ANT [3, 4, 28, 29]. The conflicting reports of both induction of apoptosis and protection against it by calcineurin was the key factor in our experimental model, which was designed to elucidate if calcineurin inhibition protected against calcium-mediated dopamine-induced apoptosis in the postischemic heart. We found that cyclosporin had a protective effect while FK-506 did not, indicating that mPT pore inhibition, rather than calcineurin inhibition, is a key factor in protecting against dopamine-induced apoptosis in the stunned heart.

Cardiomyocytes are terminally differentiated cells incapable of regeneration. Cardiomyocyte apoptosis leads to cumulative loss of cells, which is believed to have an impact on long-term cardiac function and reserve [30, 31]. This is especially significant in the setting of congenital heart disease, where multiple procedures involving varying lengths of intraoperative ischemia are required for correction of complex intracardiac defects. Inotropic support of the postischemic heart is often an essential step but care should be taken to better protect the heart compromised by impaired calcium handling. Therefore, the findings of this study may have therapeutic potential for long-term preservation of myocardial function in the setting of postischemic inotropic support with catecholamines.

	Acknowledgments

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This work was supported in part by NIH Grant No. HL-46207 (PdN). Dr Nathan was supported by a grant from the Thoracic Surgery Foundation (Nina Braunwald Research Fellowship Award).

	References

Top Abstract Introduction Material and Methods Results Comment Acknowledgments References

 

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