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Ann Thorac Surg 2006;82:2192-2199
© 2006 The Society of Thoracic Surgeons
a Department of Cardiac Surgery, Children’s Hospital Boston and Harvard Medical School, Boston, Massachusetts
b Department of Anesthesiology, Children’s Hospital Boston and Harvard Medical School, Boston, Massachusetts
c Department of Orthopedic Surgery, Children’s Hospital Boston and Harvard Medical School, Boston, Massachusetts
Accepted for publication June 27, 2006.
* Address correspondence to Dr del Nido, Department of Cardiac Surgery, Children’s Hospital Boston, 300 Longwood Ave, Bader-2, Boston, MA 02115 (Email: pedro.delnido{at}tch.harvard.edu).
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Abstract |
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METHODS: Hearts from 8-week-old inbred New Zealand White rabbit siblings were subjected to 2 hours of cold cardioplegic ischemia and 6 hours of reperfusion in a heterotopic transplant model. Controls consisted of the recipient rabbit’s nonischemic heart. The ischemia-reperfusion (IR) group consisted of postischemic hearts reperfused with no drugs; the IR plus dopamine (IR+D) group received dopamine (20 µg · kg–1 · min–1) continuously; the IR+D plus propofol (IR+D+P) group received dopamine (20 µg · kg–1 · min–1) plus propofol (500 to 600 µg · kg–1 · min–1); and the IR plus propofol (IR+P) group received propofol only (500 to 600 µg · kg–1 · min–1) throughout reperfusion (n = 7 to 9 in each group). Myocardial function was measured using a left ventricular balloon; terminal nick-end labeling (TUNEL) staining, DNA electrophoresis, and immunoblotting for caspase-3 cleavage were performed at the end of reperfusion.
RESULTS: Dopamine increased the number of TUNEL-positive nuclei significantly (14.0 ± 2.0/1,000 for IR+D versus 6.7 ± 2.0/1,000 for IR, p = 0.01). Propofol (IR+D+P) reduced the total number of apoptotic cells in hearts receiving dopamine (7.1 ± 1.8/1,000, p = 0.01 versus IR+D) to the extent seen in IR alone. DNA laddering and caspase-3 cleavage were observed at greater frequency in the IR+D group compared with the IR and IR+D+P groups. Propofol had no effect on dopamine-mediated increased systolic function, but improved diastolic function after ischemia.
CONCLUSIONS: Dopamine infusion has a positive inotropic effect on the postischemic heart at the expense of increased cardiomyocyte apoptosis. The addition of propofol prevents dopamine-induced apoptosis after ischemia while maintaining positive inotropy.
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Introduction |
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In previous studies using an isolated rabbit heart model of global ischemia and reperfusion, we demonstrated that mean cytosolic calcium levels are elevated after ischemia owing to impaired calcium cycling, and that catecholamine-type inotropic agents such as dopamine further augmented calcium levels. That was associated with an increase in apoptosis and a deterioration of ventricular diastolic function during reperfusion, despite improved systolic function [4]. However, the question remains whether preventing dopamine-induced apoptosis during reperfusion would maintain diastolic function and result in significant preservation of cardiomyocyte viability. This phenomenon is especially relevant to the population affected by congenital heart defects, for whom the need for multiple surgeries and prolonged use of inotropic agents can lead to myocyte loss at critical stages and contribute to late ventricular dysfunction.
Propofol (2,6-diisopropylphenol) is a lipid-soluble anesthetic agent commonly used in cardiac surgery and for postoperative sedation that has been reported to have protective effects against ischemic injury in excitable cells [5–9]. Propofol has been reported to reduce oxidative stress [10] and intracellular calcium levels in isolated cardiomyocytes [11, 12], and in isolated heart experiments, it maintained mitochondrial respiration and prevented mPTP opening after ischemia [8]. The purpose of this work was to study the effects of propofol in an in vivo model of global myocardial ischemia and reperfusion in the presence of dopamine. We hypothesized that a continuous propofol infusion during reperfusion would prevent dopamine-induced apoptosis after ischemia and preserve diastolic ventricular compliance.
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Material and Methods |
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Animal Model
Inbred 8-week-old New Zealand White (NZW) male rabbit siblings were used in a heterotopic heart transplant model. Under general anesthesia induced with an intramuscular injection of ketamine (100 mg/kg) and xylazine (5 mg/kg) and maintained with isofluorane inhalation (1% to 3%), the donor heart was harvested after cold crystalloid cardioplegic arrest (modified Krebs-Henseleit solution supplemented with KCl 22.5 mmol/L). The heart was maintained in an isotonic solution at 4°C for 2 hours, transplanted in a nonworking fashion to the recipient’s abdominal aorta and inferior vena cava, and then reperfused in vivo for 6 hours. The animals were maintained under mild general anesthesia with isofluorane (0% to 1%) throughout reperfusion, and the hearts were collected at end of reperfusion.
Absence of Rejection
Preliminary survival studies using inbred siblings were conducted to confirm the absence of acute and subacute rejection. The donor hearts were explanted after 1 and 7 days, and the tissue was stained with hematoxylin-eosin or Masson’s trichrome stain, and bright-field microscopy was used to determine rejection according to the International Society of Heart and Lung Transplant (ISHLT) criteria [13, 14]. One day after transplantation, there was no histologic evidence of rejection (n = 2) in this model. By 7 days, histologic examination showed only mild perivascular lymphocytic infiltrate without myocyte destruction or fibrosis (n = 1). Conversely, nonrelated animals showed severe rejection by day 7 (n = 3). We concluded that an inbred sibling transplant model was suitable for studies in which the reperfusion period did not exceed 1 day. In the subsequent experiments, a 6-hour reperfusion period was used, and histologic examination was performed (n = 34).
Experimental Groups
Five groups of hearts were studied after ischemia and reperfusion (IR), and nonischemic controls (C) consisted of the recipient animal’s native heart (n = 34). In postischemic groups, the transplanted heart was subjected to 2 hours of cold ischemia (4°C) after cardioplegic arrest and reperfused for 6 hours in vivo. Group IR (n = 7) received no drugs during reperfusion. For the remaining four groups, the drugs were started at the onset of, and continued throughout, reperfusion. Group IR+D (n = 8) received a continuous dopamine infusion (20 µg · kg–1 · min–1; Bristol-Myers Squibb, New York, New York). The dose was determined during a preliminary dose-response study (n = 3), and 20 µg · kg–1 · min–1 resulted in an increase in heart rate by 10% without affecting the mean arterial blood pressure. Group IR+D+P (n = 9) received a continuous infusion of dopamine (20 µg · kg–1 · min–1) and propofol (AstraZeneca, Wilmington, Delaware) during reperfusion. Propofol was started (100 µg · kg–1 · min–1) at the onset of reperfusion and increased as tolerated to 500 to 600 µg · kg–1 · min–1 within the first hour, then maintained at that rate throughout reperfusion. Based on a dose-response study, 500 to 600 µg · kg–1 · min–1 propofol was required to maintain light anesthesia and did not result in systemic hypotension. Group IR+P (n = 7) was reperfused with an infusion of propofol only, following the same protocol. Group IR+D+L (n = 3) received an infusion of dopamine (20 µg · kg–1 · min–1) and 20% soy-based lipid solution similar to the vehicle for propofol, initiated and maintained at the same volume/rate as the propofol infusion.
Myocardial Function Measurements
A separate set of animals was used to measure myocardial function (n = 7 in each group). The left ventricle (LV) intracavitary balloon was progressively inflated to keep the diastolic pressure at 5 mm Hg during the first 45 minutes of reperfusion and maintained at that volume from 60 minutes to 6 hours of reperfusion. The postischemic heart’s left ventricular systolic and diastolic pressures, heart rate, intraventricular balloon volume, as well as the recipient’s native heart rate and blood pressure were monitored every 30 minutes. Left ventricular developed pressure (LVDP) was calculated by subtracting the diastolic from the systolic pressure for each time point. Pressure-volume relationships were determined at the end of the reperfusion period by filling the LV balloon in stepwise increments of 0.1 mL and recording the diastolic and systolic pressures.
Infarct Size
At the end of the reperfusion period, the transplanted hearts (n = 7 in each group, except n = 3 in IR+D+L group) were harvested and flushed with Krebs-Henseleit solution. Then 2-mm-thick horizontal cardiac cross-sections were prepared and incubated in 1% triphenyltetrazolium chloride in phosphate-buffered saline at 37°C for 20 minutes, then placed on a flatbed scanner; the areas of viable (red) and infarcted (nonstained) regions were measured using Scion image analysis software (Scion Corp, Frederick, Maryland). Infarct size was quantified as previously described [4].
TUNEL Staining
At the end of the reperfusion period, the transplanted (n = 7 to 9 in each group, except n = 3 in IR+D+L) and control hearts (n = 34) were harvested and flushed. Two tissue sections, taken at the base and midventricular levels, were fixed in 4% paraformaldehyde in phosphate-buffered saline (pH 7.4), paraffin embedded, and mounted. The tissue was stained by terminal nick-end labeling of cleaved DNA with fluorescein-congugated nucleotides (TUNEL; FragEL Detection Kit; Calbiochem, La Jolla, California). The sections were also stained for nuclei (DAPI; Molecular Probes, Eugene, Oregon) and desmin (Sigma-Aldrich, St. Louis, Missouri). The TUNEL-positive and -negative nuclei were counted using Metamorph software (Version 6.2; Molecular Devices, Downingtown, Pennsylvania) on 7 to 10 random LV and septal fields per section, read at low magnification. Results are expressed as apoptotic nuclei (AN) per 1,000 total nuclei.
DNA Fragmentation Analysis
The transplanted and control hearts (n = 5 to 14 in each group) were flushed at the end of the reperfusion period, and LV tissue samples were snap frozen in liquid nitrogen and stored at –80°C. The tissue was homogenized in liquid nitrogen and suspended in a buffer containing 100 mmol/L Tris-HCl pH 7.8, 10 mmol/L ethylenediamine tetraacetic acid (EDTA) pH 8.0, 0.5% sarcosine, RNAse, and proteinase K and incubated at 65°C overnight. The solution was extracted with 1 vol.Tris-HCl pH 7.6 saturated phenol, centrifuged at 20,000g for 5 minutes, conserving the supernatant fraction, followed by a triple extraction with phenol:chloroform:isoamyl alcohol (25:24:1); the last extraction was performed with chloroform:isoamyl alcohol (24:1) following the same protocol. The DNA was precipitated with 0.1 vol.10 mol/L NH4Ac and 2.2 vol.100% ethyl alcohol (ETOH) followed by centrifugation at 20,000g for 10 minutes. The pellet was washed with 1 vol.70% ETOH and resuspended in 25 µL 10 mmol/LTris-HCl pH8.0 to 1 mmol/L EDTA. The concentration was determined by spectrophotometry at 260 nm. Ten micrograms of DNA was loaded on 1% Agarose tris-acetate-EDTA gel and run at 100 mA for 1 hour. The gel was stained with SYBR Green I nucleic acid gel stain (Molecular Probes, Eugene, Oregon), and photographed on Polaroid MP4 camera using SYBR photographic filter (Molecular Probes) to detect DNA fragmentation.
Immunoblot Assays
The transplanted and control hearts (n = 4 in each group) were flushed at the end of the reperfusion period, and LV tissue samples were snap frozen in liquid nitrogen and stored at –80°C. The tissue was homogenized in liquid nitrogen and suspended in cold buffer, as previously described [15]. The total protein extracts were quantified according to BCA (Pierce, Rockford, Illinois) method by spectrophotometry at 280 nm. Protein samples of 50 µg were separated by SDS-page gel electrophoresis and transferred to nitrocellulose membranes. Coomassie Brilliant Blue R-250 (Bio-Rad Laboratories, Hercules, California) staining of gels confirmed equal protein loading. The membranes were incubated with anti–human caspase-3 (polyclonal, recognizes pro–caspase-3; 32kDa and proteolytic fragment; 17 kDa; Upstate, Lake Placid, New York), followed by incubation with horseradish peroxidase-conjugated goat anti–rabbit IgG secondary antibody, and detection was done using enhanced chemiluminescence (Amersham Life Science, Arlington Heights, Illinois). Laser densitometry was performed to quantify the intensity of the respective bands using Scion Image software (Scion Corp).
Statistical Analysis
Statistical analysis was performed using the SPSS software package (version 14.0; SPSS, Chicago, Illinois), and data are expressed as the mean ± SE. Differences between the groups were tested for significance by two-way repeated measures analysis of variance (ANOVA) with post-hoc Fisher’s least significance difference correction for multiple comparisons. A two-tailed probability value of less than 0.05 was considered statistically significant. For analysis of the TUNEL data, a mixed-model regression approach was used to account for the multiple sections and fields in the same animals using a compound symmetry covariance structure to model the repeated measurements [16]. Ischemia versus control groups (IR versus C) and treatment (IR, IR+D, IR+D+P, and IR+D+L), as well as the group-by-treatment interactions were fitted into the model as between-subjects fixed effects, and sections and fields as random effects. Pearson’s 
2 was performed for analysis of DNA laddering.
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Results |
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Postischemic Left Ventricular Function
The postischemic transplanted hearts had a significantly lower heart rate than the rabbits’ native hearts throughout the reperfusion period (p < 0.001, data not shown), and there was no significant change in heart rate throughout the reperfusion period in postischemic hearts (Table 1). The balloon volume necessary to generate a diastolic pressure of 5 to 8 mm Hg at 60 minutes of reperfusion was significantly greater in the IR+D+P group than in the IR group (p = 0.03, Table 1). There was a significantly lower diastolic pressure throughout reperfusion in the group receiving propofol (IR+D+P p < 0.01 versus IR, p < 0.05 versus IR+D; Fig 1). Diastolic pressure-volume relationships determined at end of reperfusion showed a trend toward improved ventricular diastolic compliance in the IR+D+P group (p = 0.08). The hearts receiving dopamine (IR+D and IR+D+P) had a higher LVDP at early reperfusion (p = 0.04 versus IR, Table 1).
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DNA Laddering
Laddering of DNA was not seen in nonischemic controls (0 of 14 hearts; Fig 3). In postischemic hearts, the group receiving no drugs at reperfusion (IR) showed laddering in 28% (2 of 7 hearts). In the dopamine-treated group (IR+D), DNA laddering was present in 50% (4 of 8 hearts). In the group of hearts reperfused with dopamine and propofol (IR+D+P), laddering was seen in only 13% (1 of 8 hearts). In the group receiving propofol alone, 20% of hearts (1 of 5) demonstrated DNA laddering (Pearson’s 2 value 8.9, p = 0.06).
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Comment |
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Myocardial ischemia and reperfusion can lead to cell death through necrosis and apoptosis. In the context of cardioplegic arrest and perioperative ischemia, cell death through apoptosis has emerged as a factor involved in postoperative myocardial stunning along with mitochondrial dysfunction and oxygen wasting [4]. Mitochondria play a pivotal role in cell survival as energy providers and regulators of programmed cell death. The mitochondrial permeability transition pore (mPTP) is a protein complex that mediates injury to the organelle by causing an influx of water into the mitochondrial matrix, leading to outer membrane disruption and the release of proapoptotic factors that promote cell death through caspase-dependent and -independent mechanisms [17]. Crompton and colleagues [18] demonstrated that mPTP plays an important role in cardiac ischemia and reperfusion injury. Griffiths and Halestrap [19] determined that injury occurs during reperfusion, and the fate of the cardiomyocyte—for example, recovery, apoptosis, or necrosis—depends on the extent of mPTP opening. Commonly invoked triggers for opening of the mPTP include an elevated intracellular calcium, reactive oxygen species production, Bcl family proteins, and the loss of transmitochondrial membrane potential (
m) [20].
Cardiomyocyte calcium overload has been demonstrated in various models of ischemia and reperfusion [21] along with an increase in reactive oxygen species. In previous work, using an isolated crystalloid-perfused rabbit heart model [4], we found that dopamine infusion after ischemia reversed the systolic dysfunction, but also caused a rise in cytosolic calcium levels after ischemia along with increased cardiomyocyte apoptosis. The calcium-sensitizing inotropic agent ORG 30029 improved systolic function but did not result in increased cytosolic calcium or apoptosis after ischemia [4], implicating elevated cytosolic calcium as a putative agent for postischemic catecholamine-induced apoptosis.
In this study, we sought to use a drug at reperfusion that targeted mPTP transition, elevated cytosolic calcium, and reactive oxygen species production. Propofol given during ischemia has been shown to be cardioprotective against reperfusion injury in isolated heart experiments [5, 6, 8]. Javadov and associates [8] demonstrated that propofol improved functional recovery after ischemia and prevented mPTP opening, and that postischemic isolated mitochondria had improved respiratory chain activity. Propofol may also prevent the rise in postischemic cytosolic calcium in part by its ability to inhibit transsarcolemmal calcium current in ventricular myocytes [11, 12]. It has also been proposed that propofol reduces ß-receptor–induced cAMP production and L-type Ca2+ current but has no effect per se on steady-state calcium and cell shortening in nonischemic myocardium [22]. Finally, propofol is known to have antioxidant properties similar to alpha-tocopherol in vitro [10], and was shown to inhibit lipid peroxidation by oxidative stress in subcellular organelles [23]. Beneficial effects by propofol on free-radical–mediated injury in vivo have also been observed, and it is hypothesized that propofol stimulates endogenous synthesis of alpha-tocopherol [22].
In this model, propofol alone had no cardioprotective effects against IR injury when administered during reperfusion only. Although others have shown ameliorative effects by propofol, in those studies [8, 9] the drug has been administered during ischemia. Our aim in this study was to prevent the adverse effects of catecholamines on myocytes while maintaining their positive inotropic effects. Propofol administered during reperfusion appears to provide this effect.
Our model of cold cardioplegic arrest and reperfusion for 6 hours in vivo was designed to detect apoptosis beyond the period of reperfusion possible in a Langendorff model as the onset of catecholamine-induced apoptosis peaks at 3 to 6 hours and then declines [24]. Another advantage of our model is the presence of circulating endogenous catecholamines, inflammatory mediators, and cellular effectors, making it a more clinically relevant model.
Limitations of this study include the potential for immunologic reaction of the transplanted heart; however, we used inbred sibling donors and demonstrated no evidence of early rejection (6 hours, 24 hours). We did not attempt to delineate the precise mechanism leading to apoptosis or the mechanism responsible for propofol effect; nevertheless, previous work from us and others in isolated heart experiments suggests that calcium overload in the postischemic heart [4, 21], resulting from dopamine stimulation, leads to apoptosis [1, 4, 25] through opening of the mPTP [26]. Propofol has been shown to prevent mPTP opening [3, 8], at least in part by reducing intracellular calcium after ischemia [11, 12, 27]. To eliminate the possibility that the observed effect of propofol infusion was simply due to the effect of the vehicle, we demonstrated that a soy-based lipid emulsion similar to the carrier for propofol failed to reduce apoptosis in hearts exposed to dopamine.
A potential concern with our model is the relatively mild degree of cardiomyocyte loss from apoptosis compared with other models of more severe reperfusion injury. However, given that catecholamine infusion is typically continued for hours to days after the onset of reperfusion, it is likely that the cumulative effect will result in significant myocyte loss and have important clinical implications for long-term heart function.
In conclusion, in this study using a blood perfused in vivo heart model, dopamine infusion at reperfusion had a positive inotropic effect. However, the improvement in postischemic contractile function occurred at the expense of increased cardiomyocyte apoptosis. The addition of propofol at clinically relevant doses prevented dopamine-induced apoptosis without causing a negative inotropic effect, and in fact improved left ventricular diastolic compliance after ischemia.
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Acknowledgments |
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