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Ann Thorac Surg 2001;71:1296-1303
© 2001 The Society of Thoracic Surgeons
a Crafoord Laboratory of Experimental Surgery, Karolinska Hospital, Stockholm, Sweden
b Department of Thoracic Surgery, Karolinska Hospital, Stockholm, Sweden
c Department of Clinical Chemistry, Huddinge University Hospital, Huddinge, Sweden
Accepted for publication October 18, 2000.
Address reprint requests to Dr Valen, Crafoord Laboratory of Experimental Surgery L6:00, Karolinska Hospital, S-17176 Stockholm, Sweden
e-mail: guro.valen{at}cmm.ki.se
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Abstract |
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Methods. To investigate if atherosclerosis would influence the preconditioning response, Apolipoprotein E/low density lipoprotein (LDL) receptor double knockout mice (ApoE/LDLr-/-) were fed an atherogenic diet (21% fat, 0.15% cholesterol) for 6 to 8 months. At that time, extensive atherosclerotic lesions throughout the coronary tree were seen in transverse sections stained with Oil Red-O. Hearts of ApoE/LDLr-/- mice were Langendorff-perfused with 40 minutes of global ischemia and 60 minutes reperfusion, and compared with C57BL/6 controls. Preconditioning with two episodes of 2 minutes of ischemia and 5 minutes reperfusion, or exposing the mice to a hyperoxic environment (O2 > 98%) for 60 minutes before heart perfusion, was performed.
Results. Hearts of mice with coronary atherosclerosis had worse postischemic function, and increased infarct size and troponin T release compared to hearts of C57BL/6 mice. Ischemic preconditioning improved postischemic ventricular function, and reduced myocardial infarct size and troponin T release in both normal and ApoE/LDLr-/- mice. The effects were most pronounced in ApoE/LDLr-/- hearts. Exposure to hyperoxia exerted a similar protection of function and cell viability of ApoE/LDLr-/- mice hearts.
Conclusions. These findings suggest that the severely atherosclerotic heart may be protected by preconditioning induced by ischemia or hyperoxia.
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Introduction |
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One factor which makes man different from experimental animals and may contribute to the controversial results of clinical preconditioning, is the occurrence of coronary atherosclerosis which profoundly influences vascular and myocardial biology. Possibly atherosclerosis is accompanied by myocardial metabolic adaption secondary to chronic or intermittent hypoxia, making it difficult to achieve protection of such hearts [1]. No previous investigation has evaluated preconditioning in the severely atherosclerotic experimental animal, although one factor contributing to atherosclerosis, hypercholesterolemia, has been studied. Pacing-induced preconditioning was attempted in hearts of rats or rabbits fed a high-cholesterol diet (1% to 2% cholesterol) for 8 to 24 weeks. Hypercholesterolemia blocked pacing-induced immediate, but not delayed, protection, and this was reversed by reducing cholesterol to normal in the presence or absence of mild atherosclerotic lesions [7–9]. However, the importance of coronary atherosclerosis per se, rather than hypercholesterolemia, for the preconditioning response has not been investigated.
With the development of recombinant DNA-technology, new tools for atherosclerosis research are emerging with mice knocked out for or overexpressing specific lipoproteins [10]. One species employed is the mouse double knocked out for Apolipoprotein E and the low density lipoprotein (LDL) receptor (ApoE/LDLr-/-) [10, 11]. The distribution and contents of the atherosclerotic lesions, including in the coronary arteries, are comparable to those of man, and the process is accelerated when the animals are fed an atherogenic diet [10, 11]. Thus, the ApoE/LDLr-/- mouse is suitable as a model to represent the severe atherosclerosis of patients needing revascularization.
The clinical applicability of ischemic preconditioning is also hampered by problems in determining the mechanisms of action, and thus pharmacologically to evoke the protection in patients. While waiting for universal mechanisms of ischemic preconditioning to be determined for pharmacological targeting, we have recently established a clinically acceptable way of inducing the preconditioning response by pretreating rats with hyperoxia (> 98% oxygen) before perfusing their hearts [12]. Hyperoxia induces a systemic low-grade oxidative stress, and profoundly protects the function and necrosis development in hearts subjected to global ischemia [12].
The aim of the present study was twofold: (1) To investigate if ischemic preconditioning would reduce infarct size and improve cardiac function in the isolated hearts of mice with severe atherosclerosis; and (2) To investigate if exposure to a hyperoxic environment might evoke myocardial protection in the severely atherosclerotic mouse heart analogous to that previously observed in rats with healthy coronary vessels.
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Material and methods |
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ard (Bomholt, Denmark). When the animals were weaned and approximately 6 weeks old, feeding with an atherogenic diet containing 21% fat/0.15% cholesterol commenced (R683, AnalyCen, Linköping, Sweden). After 6 to 8 months on the diet and water ad libitum, the animals were used for experiments after evaluating the extension of coronary atherosclerosis (see below). Male C57BL/6 mice 3 to 4 months old (B&K Universal AB, Sollentuna, Sweden) were employed as control animals.
Isolated heart perfusion
Mice were anesthetized by intraperitoneal injections of midazolam (Dormicum, Hoffman-La Roche, Lausanne, Switzerland, 25 mg/kg) and 1 mg/kg fentanyl, 50 mg/kg fluanisone (Hypnorm, Janssen Pharmaceutica, Beerse, Belgium). Heparin (500 IU/mouse) was injected into the peritoneum. The hearts were rapidly excised, and placed in ice-cold Krebs-Henseleit buffer (NaCl 118.5 mmol/L, NaHCO3 25.0 mmol/L, KCl 4.7 mmol/L, KH2PO4 1.2 mmol/L, MgSO4 7H2O 1.2 mmol/L, glucose H2O 11.1 mmol/L, CaCL2 H2O 1.8 mmol/L). The aorta was secured onto a grooved 20-gauge stainless steel cannula, and transferred to a Langendorff perfusion apparatus. The hearts were retrogradely perfused with gassed (5% CO2, 95% O2) Krebs Henseleit buffer at a constant pressure of 55 mm Hg. The apparatus was water-jacketed to maintain a core temperature of the heart of 37°C. Global ischemia was achieved by clamping the inflow tubing.
Assessment of cardiac performance
A balloon made of polyethylene plastic was inserted into the left ventricle through the left atrium for isovolumetric recordings of left ventricular systolic (LVSP) and end-diastolic (LVEDP) pressures. The volume of the balloon was large enough to be inflated with 30 µL saline without producing a pressure of more than 1 mm Hg. The balloon was coupled to a graded threaded microsyringe (Hugo Sachs Electronik, March-Hugstetten, Germany), and inflated to obtain a LVEDP of 4 to 7 mm Hg during stabilization (approximately 20 µL saline). For recordings of electrocardiogram (ECG), the tips of two thin teflon-insulated platinum wires were scratched and inserted into the apex of the left ventricle and to the right atrium as ECG electrodes, and a metal clip attached to the 20-gauge steel cannula as the reference electrode. The ECG was imported into a computer system (PCLAB, Astra Hässle AB, Mölndal, Sweden), which calculated heart rate (HR) and percentage of arrhythmias. Every 30 seconds, 3 seconds of ECG were registered, and the waves of all beats during the registration were averaged into one QRS-complex. Arrhythmias were evaluated by the computer as all beats with abnormal QRS complexes or irregular R-R intervals. Coronary flow (CF) was continuously measured using a funnel coupled to a force transducer with an automated air valve deflating every 30 seconds, and is presented as µl/min/mg heart. The maximal and negative value of the first derivative of pressure (dP/dt max and negative dP/dt) as well as left ventricular developed pressure (LVDP = LVSP - LVEDP) and rate pressure-product (RPP = HR x LVSP) were calculated by PCLAB.
Experimental protocol
Hearts were allowed to stabilize for 21 minutes. Only hearts with CF between 1.6 and 4.0 ml/min (C57BL/6 1.6 to 3.1, ApoE/LDLr-/- 1.8 to 3.9), HR 280 to 480 beats/min, LVSP greater than 60 mm Hg, and LVEDP 4 to 7 mmHg were included. After stabilization and the interventions listed below, hearts were subjected to 40 minutes of global ischemia and 60 minutes reperfusion.
C57BL/6 mice were randomized into 2 groups:
ApoE/LDLr-/- mice were divided into 3 groups:
A corresponding hyperoxic group of C57BL/6 mice are not included in the present study. A shorter period of hyperoxia (30 minutes) gives optimal protection in those mice and, for the simplicity of presentation, the data are not shown.
Measurement of infarct size
After reperfusion for 60 minutes, hearts were immediately perfused with a total volume of 3 ml 10% triphenyl tetrazolium chloride (TTC, Sigma Chemical Co, St. Louis, MO) delivered at 100 cm H2O. The hearts were fixed in 4% formaldehyde for 24 hours, thereafter preserved in 10% sucrose in phosphate-buffered saline (PBS). The hearts were cut manually into 0.8 to 1.0 mm transverse slices. The sections were visualized in a computer imaging system, and infarct size marked and calculated (LEICA Qwin, Leica Imaging Systems Ltd, Cambridge, UK). An unstained epicardial ring appeared in all hearts. After calculating the size of this ring in various experimental groups and finding it constant, independent of intervention, the outer ring was evaluated as an artifact and excluded from the infarct size calculation. The area of unstained (infarcted) myocardium was calculated as the percentage of total ventricular area minus cavities, and the mean value of all sections in one heart was treated as one value and used for statistics.
Measurement of cardiac troponin T
At the end of stabilization and after 30 minutes of reperfusion, 2 ml samples of the coronary effluent were collected in precooled tubes, stabilized with addition of 4% albumin, and rapidly frozen at -80°C until analysis of cardiac troponin T (cTnT). cTnT was measured with the third generation of the TnT test on the Elecsys 2010 immunoassay analyzer (Roche Diagnostics, Mannheim, Germany). The third generation TnT test uses the same monoclonal antibodies (M11.7 and M7) as the second generation test, but is standardized with human recombinant cTnT instead of bovine cTnT (Troponin T STAT package insert, Roche Diagnostics, Mannheim, Germany). cTnT in the coronary effluent was calculated as the amount released per minute [cTnT (ng/ml)] x CF (ml/min) = ng/min. The delta cTnT release was calculated as release after 30 minutes reperfusion minus release at stabilization, and employed for statistical evaluation.
Microscopic analysis of coronary atherosclerosis
The extension of coronary atherosclerosis was evaluated in hearts of mice fed an atherogenic diet for 6, 7, or 8 months (n = 3 of each). Hearts were harvested and fixed in 4% formaldehyde for 24 hours, thereafter kept on 10% sucrose in PBS until they were cryomounted in OCT. Each heart was divided in three to visualize lesions in the aortic root, the middle section, and the apex area of the heart. Consecutive sections (10 µm thick, n = 30 to 50) from each of the three areas were air-dried, dehydrated, and stained with Oil Red-O for 15 minutes. Counter-staining was performed with hematoxylin. After rinsing with water, the slides were mounted with Kaiser’s glycerol gelatin (Merck, Darmstadt, Germany) under coverslips. The Oil Red-O stained cryosections were observed under microscopy.
Statistical analysis
Data are expressed as mean ± standard error of mean. A two-way analysis of variance was used to compare hemodynamic parameters, with a Scheffe’s post hoc test to verify or falsify apparent differences in comparison between the three groups. Comparisons of infarct size and cTnT release were performed by unpaired t test for independent samples. p less than 0.05 was considered significant.
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Results |
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The atherosclerotic lesions in the hearts of ApoE/LDLr-/- mice after 6 to 8 months on an atherogenic diet were widespread. In the aortic root, advanced fibrofatty lesions were observed, with lipid deposits in the proximal coronary arteries (Fig 1A). The coronary atherosclerosis could be observed distally in all hearts. A representative section showing five Oil Red-O stained branches of coronary arteries in the right ventricle in the middle section of the heart is shown in Figure 1B. Figure 1C illustrates a coronary artery caught longitudinally in the apex region of the heart, where lipid deposits can be observed.
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First derivative of pressure
In C57BL/6 hearts, maximum and negative dP/dt were impaired at the start of reperfusion, without being influenced by ischemic preconditioning (Fig 3A). The depression of max dP/dt and increase of neg dP/dt was more apparent in atherosclerotic hearts, and was attenuated by both ischemic and hyperoxic preconditioning (Fig 3B).
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Heart rate and arrhythmias
There were no intergroup differences of heart rate before interventions. Heart rate tended to increase, then decrease during reperfusion of C57BL/6 control hearts, and was not influenced by ischemic preconditioning. The rate of ApoE/LDLr-/- mice was not different from C57BL/6 mice. Hyperoxic preconditioning did not influence heart rate in ApoE/LDLr-/- mice, whereas ischemic preconditioning reduced it during reperfusion (at the end of reperfusion, 305 ± 32 beats per minute versus 347 ± 22 in controls; p < 0.006).
The frequency of arrhythmias increased during reperfusion of C57BL/6 hearts, and this was modified by ischemic preconditioning (p < 0.004). For instance, after 15 minutes reperfusion, 30 ± 7% of control hearts had arrhythmias, versus 17 ± 5% of preconditioned hearts. Hearts of ApoE/LDLr-/- mice had less arrhythmias than C57BL/6 hearts during reperfusion (15 minutes reperfusion, 7 ± 3%, p < 0.0001). Neither ischemic nor hyperoxic preconditioning influenced the occurrence of arrhythmias in these hearts.
Rate pressure-product
RPP was not significantly different when comparing C57BL/6 groups, when comparing the three ApoE/LDLr-/- groups, or when comparing C57BL/6 to ApoE/LDLr-/-.
Myocardial infarct size
At the end of reperfusion, hearts were stained with TTC, sectioned, and unstained areas were imaged and calculated. In C57BL/6 hearts, approximately 28% of myocardial tissue was calculated as necrotic after 60 minutes reperfusion (Fig 4A).
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Troponin T release
Basal cTnT release was not different between groups. cTnT release into the coronary effluent increased during reperfusion in all hearts. When the increase in release from stabilization to 30 minutes of reperfusion was calculated, ischemic preconditioning reduced cTnT release in C57BL/6 hearts (Fig 4B). Like infarct size, cTnT release from ApoE/LDLr-/- mice was higher than from C57BL/6 mice, and the cTnT release was attenuated by both hyperoxic and ischemic preconditioning (Fig 4B).
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Cardiac function of normal and ApoE/LDLr-/- mice was similar during the stabilization period, but during reperfusion, after global ischemia, the atherosclerotic hearts had more depressed left ventricular performance, increased infarct sizes, and increased cTnT release. Studies performed in hearts of rabbits fed a high-cholesterol diet for a much shorter time than that employed here have found cardiac performance deteriorated [13], unchanged [14], or improved [15] during reperfusion. However, the effects of hypercholesterolemia and atherosclerosis are not necessarily comparable. Hypercholesterolemia before induced infarction in rabbits [14, 16] or LDLr-/- mice [17] has increased infarct size compared to control animals.
In the present study, hearts of ApoE/LDLr-/- mice were larger than those of normal mice. This was due to the fact that the whole mouse was bigger due to the atherogenic diet. It may be a limitation of the present study that the mice were not diet and age-matched. When correcting coronary flow for differences in heart weight, coronary flow was reduced to almost half in hearts of ApoE/LDLr-/- mice. This may be due to obstructed or stenotic coronary vessels, or impairment of endothelium-dependent relaxation associated with atherosclerosis [18]. Indeed, in vitro vessel reactivity of ApoE/LDLr-/- mice of the same age and diet as ours was altered, and this appeared partially dependent on nitric oxide [19]. A surprising finding was that hearts of ApoE/LDLr-/- mice had reduced incidence of arrhythmias during reperfusion compared to C57BL/6 mice. We have not found similar references in the literature, and are at the moment at a loss to explain this finding.
In the present study, classic ischemic preconditioning improved cardiac function, and reduced myocardial infarct size and cTnT release after 40 minutes global, normothermic ischemia in both atherosclerotic and normal mouse hearts. Ischemic preconditioning has previously been shown to reduce infarct size and protect the function of murine hearts [20, 21]. In the present study, the clearest protection on both function and cell survival was afforded ApoE/LDLr-/- mice. The most likely explanation is that the ApoE/LDLr-/- mice had larger infarcts and deteriorated cardiac performance, and therefore benefited more from endogenous protection. Clinical studies employing ischemic preconditioning during coronary artery bypass surgery have controversial results, and it has been questioned whether metabolic alterations due to hypercholesterolemia and atherosclerosis make hearts with chronic ischemic disease difficult to precondition [1]. Rapid pacing induced myocardial protection in hearts of rabbits fed high cholesterol for 8 weeks [9], but not in hearts of rats fed high cholesterol for 24 weeks [8]. Although the preconditioning models and degree of hypercholesterolemia/atherosclerosis are different in the previous and present studies, the present study shows that the severely atherosclerotic heart can be preconditioned. The inconsistent protection found in clinical studies is probably due to other reasons, such as preconditioning of patient groups with short surgical procedures where loss of function and necrosis is negligible.
ApoE/LDLr-/- mice were exposed to 60 minutes of hyperoxia before heart isolation and Langendorff-perfusion. This model has been established in rats to induce both immediate and delayed myocardial protection through inducing a systemic oxidative stress evident as increased serum lipid peroxidation products after 60 minutes [12]. In the present study, hyperoxia protected postischemic function, reduced infarct size, and reduced release of cTnT analogous to classic preconditioning. Although the mediators of hyperoxic cardioprotection are not determined, the mechanism of protection is dependent on activation of nuclear factor kappa-B (NF
B). After 60 minutes hyperoxia, NFB is activated in cardiac tissue, and pharmacological inhibition of its activation abolishes the functional effects [22]. Hyperoxia is a mode of myocardial protection which potentially may be of direct clinical applicability, particularly since it protects the function and inhibits infarct development of the atherosclerotic heart.
Although the mechanisms of preconditioning in the mouse heart are largely unknown, we may speculate on a recently discovered factor. Maulik and colleagues [23] found that classic ischemic preconditioning in the rat was dependent on NFB, which was activated during preconditioning, and inhibition of this activation abolished the functional effects as described above for hyperoxia in rats [22]. Two NFB-regulated genes, inducible nitric oxide synthase and inducible cyclooxygenase, have recently been suggested to mediate delayed preconditioning in studies of knockout animals [24, 25]. However, it remains to be determined whether the mechanisms are analogous in classic preconditioning.
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Acknowledgments |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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ke Wiberg, Ragnhild and Einar Lundströms Memory, and Aga Gas. Guohu Li has been supported by the Wenner-Gren Foundations, while Peeter Tähepôld has received a grant from the Karolinska Institute. |
References |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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B-activation. J Mol Cell Cardiol 2000;32:A20.
B in tyrosine kinase signaling of p38 MAP kinase regulation of myocardial adaptation to ischemia. FEBS Lett 1998;429:365-369.[Medline]
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TnT) shown as the difference between release after 30 minutes of reperfusion and release at end of stabilization (mean ± standard error of mean of 7 to 10 animals in each group). (* IPC, C57 versus C, C57, p < 0.05; # IPC, ApoE versus C, ApoE, p < 0.04; § HPC, ApoE versus C, C ApoE, p < 0.03;