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J Thorac Cardiovasc Surg 1996;112:778-786
© 1996 Mosby, Inc.
CARDIOPULMONARY BYPASS, |
Supported by National Institutes of Health grants HL-43696, HL-44186, and GM-08315. D.R.M. is a recipient of the National Institutes of Health National Research Service Award.
Received for publication Nov. 1, 1995 Revisions requested Dec. 6, 1995; revisions received Jan. 5, 1996; Accepted for publication Feb. 13, 1996. Address for reprints: Daniel R. Meldrum, MD, Department of Surgery, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., C-320, Denver, CO 80262.
Abstract
Cardiac preconditioning is mediated by protein kinase C. Although endogenous calcium is a potent stimulus of protein kinase C, it remains unknown whether preischemic administration of exogenous calcium can induce protein kinase C–mediated myocardial protection against ischemia-reperfusion injury. To study this, calcium chloride was administered retrogradely through the aorta at a rate 5 nmol/min for 2 minutes to isolated perfused rat hearts 10 minutes before a 20-minute ischemia and 40-minute reperfusion insult. Calcium-mediated cardioadaptation was then linked to protein kinase C by means of the protein kinase C inhibitor chelerythrine (20µmol · L-1 · 2 min-1). To determine whether exogenous calcium administration induces protein kinase C translocation and activation, immunohistochemical staining for the calcium-dependent protein kinase C isoform
was performed on adjacent 5µm myocardial sections with and without calcium chloride treatment. Results indicated that preischemic calcium chloride administration improved myocardial functional recovery, as determined by enhanced developed pressure, improved coronary flow, reduced end-diastolic pressure, and decreased creatine kinase leakage during reperfusion. Beneficial effects of calcium chloride were eliminated by concurrent protein kinase C inhibition. Immunohistochemical staining for theisoform of protein kinase C demonstrated that calcium chloride induces translocation of this isoform from the cytoplasm to the sarcolemma, indicating that exogenous calcium administration activates this isoform. These results suggest that calcium chloride, a safe and routinely administered agent, can induce protein kinase C–mediated cardiac preconditioning. Calcium-induced cardioadaptation to ischemia-reperfusion injury may be promising as a clinically feasible therapy before planned ischemic events such as cardiac allograft preservation and elective cardiac operations. (J THORAC CARDIOVASC SURG 1996;112:778-86)
Cardiac preconditioning, first termed "ischemic preconditioning", confers myocardial protection against ischemia-reperfusion injury.
1-11 Several apparently disparate stimuli, including adenosine, norepinephrine, and bradykinin, simulate the protective effects of ischemic preconditioning. 1-4,7,10 Although cardiac preconditioning with either ischemia or pharmacologic agents is possible in animal models, clinical application has been limited for two reasons: (1) ischemic preconditioning stimuli are clinically unappealing, and (2) cardiac preconditioning requires planned ischemic events. Elective cardiac operations and heart transplantation may represent the extent of cardiac preconditioning's clinical application.
The protective effects of different cardiac preconditioning stimuli appear to rely on protein kinase C (PKC) as a common intracellular effector. 3,4,11-15 Indeed, we 3 and others 11,12 have directly activated PKC with phorbol esters and diacylglycerol, simulating the protection afforded by ischemic preconditioning. 3,11,12 Calcium is an important endogenous activation stimulus of calcium-dependent PKC isoforms (cPKC). 16 It is unknown whether the administration of exogenous calcium chloride (CaCl2), which is both relatively safe and routinely administered clinically, can induce PKC-mediated myocardial functional protection. To study this, exogenous CaCl2 was administered as a preconditioning stimulus before ischemia-reperfusion to determine the following: (1) whether preischemic CaCl2 administration can induce endogenous myocardial functional protection, (2) whether CaCl2 reduces myocellular damage (as marked by creatine kinase [CK] leakage), (3) whether CaCl2 results in translocation and activation of the cPKC isoform (PKC), and (4) whether CaCl2-induced myocardial protection is mediated by PKC.
Materials and methods
Materials.
Male Sprague-Dawley rats (weight 325 to 350 gm; Sasco, Omaha, Neb.) were fed a standard diet and acclimated in a quiet quarantine room for 2 weeks before the experiments. The animal protocol was reviewed and approved by the Animal Care and Research Committee of the University of Colorado Health Sciences Center. All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" prepared by the National Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH publication No. 86-23, revised 1985). The PKC inhibitor chelerythrine was obtained from L C Laboratories (Woburn, Mass.). Antibody to PKC was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.). All other chemicals and reagents were obtained from Sigma Chemical Co. (St. Louis, Mo.).
Experimental design and groups.
Each ischemia-reperfusion experiment totaled 80 minutes, beginning with a mandatory equilibration period. Stability control hearts (with and without 0.25 mmol/L CaCl2 and 20 µmol/L chelerythrine treatment) underwent 80 minutes of oxygenated perfusion without any ischemia to ensure preparation stability. Injury control hearts were perfused for 8 minutes (equilibration) and then received 2-minute infusions of each respective vehicle, followed by a 10-minute washout period, and a standard ischemia-reperfusion insult (20 minutes of 37º C global ischemia and 40 minutes of reperfusion). CaCl2-treated hearts received 8 minutes of perfusion followed by a 2-minute retrograde aortic infusion of CaCl2 at 5 nmol/min for 2 minutes (based on a 20 ml/min coronary flow [CF] rate). This was followed by a 10-minute washout period of standard perfusion. The CaCl2 dose was based on dose-response curves generated in our laboratory, which demonstrated that this dose provided optimal protection compared with both higher and lower doses. To determine whether the observed protective effects of CaCl2 were mediated by PKC, the PKC inhibitor chelerythrine was administered with and without CaCl2 after equilibration, 10 minutes before the standard ischemia-reperfusion protocol. All drug solutions were prepared in deionized water and infused through a port above the aortic root at 0.1 ml/min (not recirculated). Hemodynamic parameters were continuously recorded before, during, and after infusion.
Ischemia-reperfusion of the isolated rat heart: developed pressure (DP), end-diastolic pressure (EDP), CF, and heart rate measurements.
The isolated, crystalloid-perfused rat heart model described elsewhere was used. 2-4,17,18 In brief, after anesthesia and heparinization (60 mg/kg intraperitoneal sodium pentobarbital and 500 units intraperitoneal heparin sodium) hearts were excised into 4º C Krebs-Henseleit solution and perfused with oxygenated buffer within 45 seconds. Hearts were retrogradely perfused in the isolated, isovolumetric Langendorff mode (70 mm Hg) with modified Krebs-Henseleit solution (5.5 mmol/L glucose, 1.2 mmol/L calcium ion [Ca2+], 4.7 mmol/L potassium chloride, and 25.0 mmol/L sodium bicarbonate) and saturated with 92.5% oxygen and 7.5% carbon dioxide atmosphere to achieve an oxygen tension of 440 to 460 mm Hg, a carbon dioxide tension of 39 to 41 mm Hg, and a pH of 7.39 to 7.41 (ABL-4 blood gas analyzer; Radiometer Medical A/S, Copenhagen, Denmark). Pulmonary arteriotomy and left atrial resection were performed before insertion of a water-filled latex balloon through the left atrium into the left ventricle. The balloon was then adjusted to a left ventricular EDP of 6 mm Hg during the initial equilibration. This preload volume was held constant during the entire experiment to allow continuous recording of the ventricular pressure during ischemia-reperfusion. Pacing wires were fixed to the right atrium and pulmonary outflow tract and hearts were paced at approximately 6 Hz (355 beats/min) for 1 minute to ensure a standardized heart rate during the time points at which DP was measured. During the remainder of the experiments, the pacer was off. A unipolar electrocardiogram was obtained through a lead placed on either the right or left ventricular free wall to allow continuous recording of an electrocardiographic signal.
Measured indexes of myocardial function were left ventricular DP, EDP, CF, and native heart rate. Data were continuously recorded with a computerized MacLab 8 preamplifier/digitizer (AD Instruments Inc., Milford, Mass.) and an Apple Quadra 800 computer Inc., Cupertino, Calif.). The electrocardiographic signal was amplified with a DP-301 differential amplifier (Warner Instrument Corp., Hamden, Colo.) and digitized at 400 Hz on the MacLab 8 preamplifier/digitizer. Paced hearts that did not produce 105 ± 25 mm Hg DP at 6 mm Hg EDP were discarded. A three-way stopcock above the aortic root was used to create global ischemia, during which time the heart was placed in a 37º C degassed organ bath. CF was measured in graduated cylinders at the end of equilibration, immediately after drug infusion, and at the end of reperfusion. At 10, 20, 30, and 40 minutes of reperfusion, 1 ml coronary effluent was collected and frozen at -70º C. Coronary effluent CK activity was determined within 2 weeks.
Coronary effluent CK activity.
Coronary effluent (1 ml) was collected at 10, 20, 30, and 40 minutes of reperfusion and then frozen at -70º C until assay. All assays were performed within 2 weeks of effluent collection. The assay was performed with Sigma diagnostic kit no. 47-UV on an automated spectrophotometer (Centrifichem 500 discrete autoanalyzer; Union Carbide Chemicals & Plastics Co. Inc., Bound Brook, N.J.) in cuvettes maintained at 37º C. Samples and reagents were maintained at 4º C before assay. Solutions were prepared in distilled, deionized water. Results are presented as CK activity in units per liter.
Subcellular localization of PKC.
Myocardial subcellular localization of the PKC was performed with immunofluorescence staining. These hearts were obtained immediately after 2-minute CaCl2 infusion (0.25 mmol/L) in CaCl2-treated hearts and after 10-minute equilibration in control hearts. Ventricular tissue was excised from isolated beating hearts, blotted, embedded in tissue-freezing medium, rapidly frozen in dry ice–cooled 2-methylbutane, and stored at -70º C. Transverse 5 µm cryosections were prepared with a cryostat (2800 Frigocut E; Reichert-Jung, Germany) and collected on slides coated with poly-l-lysine. All sections were fixed for 10 minutes in a 70% acetone–30% methanol mixture at -20º C. Normal goat serum (5% in phosphate-buffered saline solution [PBS] and 5% normal sheep serum) was applied as a blocking agent and washed three times in PBS for 3 minutes. Sections from the experimental groups were then incubated for 1 hour with diluted primary antibody (rabbit polyclonal antibody against PKC) at room temperature with mouse antisarcomeric -actin antibody. For all groups, individual PKC isoform staining was performed on adjacent sections. After the sections were washed three times with PBS for 3 minutes, they were incubated with Cy-3 conjugated goat antirabbit IgG for 45 minutes. During this step, sections were also exposed to antisarcomeric -actin antibody coincubated with fluorescein isothiocyanate–conjugated sheep antimouse immunoglobulin G antibody. Sections were then washed three times with PBS for 3 minutes. Slides were mounted with a glycerol-based antiquenching agent (O-phenylene diamine-D:HCl) and stored at 4º C. To test for nonspecific fluorescence, adjacent sections of each experimental group were incubated with nonimmune purified rabbit immunoglobulin G instead of primary antibodies. Sections were viewed and photographed with a microscope equipped with fluorescence optics (Axioskop with MC-100 camera; Carl Zeiss, Oberkochen, Germany). Multiple sections per heart were examined. Three different hearts were examined per group. Representative sections are presented.
Presentation of data and statistical analysis.
All reported values are mean (± standard error of the mean, n = 6 to 8 per group). Differences at the 95% confidence level were considered significant. Functional recoveries—DP (in millimeters of mercury), left ventricular EDP (in millimeters of mercury), CF (in milliliters per minute), heart rate (in beats per minute), and CK activity (in units per liter)—were compared at the corresponding time points between groups with one-way analysis of variance with post hoc Bonferroni-Dunn test (StatView 4.0; Abacus Concepts, Inc., Berkeley, Calif.).
Results
Twenty minutes of normothermic (37º C) global ischemia was chosen as the ischemia-reperfusion insult because this reproducibly establishes a 50% decrease in functional recovery of untreated hearts. 2-4,17-19 Final function in CaCl2-pretreated hearts was greater than 75%, which provided a dynamic range within which cardiac functional protection could be studied. Stability control hearts for vehicle, CaCl2, and chlerythrine-treated hearts retained more than 95% of baseline DP, heart rate, and CF.
Effects of CaCl2 pretreatment, with and without concurrent PKC inhibition, on DP.
The effect of preischemia CaCl2 on DP after ischemia-reperfusion was explored in a manner similar to that in previous regional and global ischemia isolated rat heart models. 2-4,17-19 DP did not differ between the groups during equilibration and measured 112 ± 9, 113 ± 7, 117 ± 8, and 115 ± 7 mm Hg in the control, CaCl2, CaCl2 plus chelerythrine, and chelerythrine groups, respectively (Fig. 1). After agent or vehicle administration, DP increased transiently in the CaCl2 and the CaCl2 plus chelerythrine groups to 124 ± 9 and 125 ± 8 mm Hg, respectively. DP did not change after vehicle (control) or chelerythrine administration alone. After washout, DP returned to baseline in all groups. At 40 minutes of reperfusion, DP was increased in the CaCl2 group (89 ± 6.1) with respect to control, CaCl2 plus chelerythrine, and chelerythrine groups, which measured 60.2 ± 6.8, 69 ± 9, and 58 ± 7, respectively (p < 0.05).
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PKC translocation. 3,4,16 Indirect immunofluorescent microscopy was used to verify Ca2+-induced activation of the calcium-dependent PKC. Although this technique primarily provides qualitative data, the use of isoform-specific anti-PKC antibodies allows the assessment of both isoform-selective activation and compartmentalization. Fig. 5 (A) demonstrates control heart sections with diffuse cytoplasmic distribution of PKC; after CaCl2 treatment, however, PKC translocates to the sarcolemma (Fig. 5, B). Results represent multiple sections from three different hearts per group.
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Ischemic preconditioning has emerged as a means of inducing endogenous myocardial protection after ischemia-reperfusion injury. 1-4,9,12-14 The protective effects of different cardiac preconditioning stimuli appear to rely on PKC as a common intracellular effector. 3,4,11-15 Ca2+ is an important endogenous activation stimulus of cPKC. 16 Although increased intracellular Ca2+ concentration ([Ca2+]i) represents the final common denominator of cell dysfunction and death after ischemia-reperfusion, 8,9,20-29 it is possible (although counterintuitive) that a preischemic Ca2+ stimulus might incite endogenous events that culminate in myocardial protection against ischemia-reperfusion. A calcium stress thus may provide cardioprotection, analogous to transient ischemic preconditioning, against sustained ischemia-reperfusion. Steenbergen and coworkers 9 have demonstrated that ischemic preconditioning limits alterations in various intramyocellular ions after ischemia-reperfusion. Ischemic preconditioning therefore appears to induce, or alternatively to "prime," the cellular machinery required to maintain ion homeostasis during a subsequent ischemic episode. These beneficial effects appear to explain, at least in part, preconditioning's protective effects. Although the intermediaries have yet to be completely elucidated, PKC activation is required for all preconditioning stimuli examined to date. 3,4,11-14 Indeed, direct PKC activation with phorbol esters and diacylglycerol results in functional and viability protection after ischemia-reperfusion. 3,11,12 Although [Ca2+]i is an important endogenous activator of PKC, it is unknown whether exogenous Ca2+, a clinically accessible and acceptable stimulus, can activate PKC and thereby induce myocardial functional protection.
Our observations suggest that exogenous CaCl2 administration can induce PKC-mediated myocardial functional protection. Exogenous CaCl2 administration before ischemia-reperfusion improved DP (Fig. 1) and CF (Fig. 2) after ischemia-reperfusion and decreased EDP (Fig. 3) and CK leakage (Fig. 4) after ischemia-reperfusion. Furthermore, PKC inhibition with chelerythrine abolished these effects (Figs. 1 through 4), thus implicating PKC activation in the mechanism of CaCl2-induced protective effects. Further substantiating the hypothesis that CaCl2 can induce PKC-mediated myocardial functional protection is the observation that CaCl2 results in PKC translocation to the sarcolemma (Fig. 5). Because PKC was the only isoform examined, it is possible that other isoforms inhibited by chelerythrine are involved. The translocation of this isoform may be coincidental, and not responsible for the protection observed. Indeed, different preconditioning stimuli may effect preconditioning through the activation of different isoforms. 3,4 Nevertheless, Ca2+ preconditioning was indeed prevented by concurrent PKC inhibition. It is of interest that the improved functional recovery observed is derived almost entirely from a reduction in EDP, without appreciable increase in systolic pressure. This suggests that the protective effects may be caused by reduction of irreversible injury, rather than of stunning.
Brief ischemia can itself condition the heart against a more prolonged ischemic insult. 1-3,7,9,10,12 In an effort to analyze the mechanisms of ischemia-induced functional protection, several investigators established a role for ischemic stress hormones 2,4,24,30,31 and suggested a role for [Ca2+]i.*). Ashraf and coworkers 20 focused on manipulating the extracellular Ca2+ concentration ([Ca2+]o) and report that exposing the heart to a transient depletion and repletion of Ca2+ protects the cell against a subsequent, more severe depletion and repletion of Ca2+ (calcium paradox). Calcium paradox injury indeed approximates the damage inflicted during ischemia-reperfusion injury. It therefore seemed possible that a preischemic Ca2+ stimulus would also provide functional protection against ischemia-reperfusion injury. The mechanisms by which an antecedent stress (ischemia, stress hormones, or elevated [Ca2+]i) results in cardioadaptation to a subsequent insult are only beginning to be elucidated; however, PKC appears to act as a common intracellular effector. 3,4,12,14,15
The signal transduction cascades of a large number of receptors are linked to PKC. 16 Consequently, many different stimuli that activate PKC could theoretically lead to cardiac functional protection against ischemia-reperfusion injury. Indeed, transient ischemic, purinergic, and 1 adrenergic stimuli, all of which provide myocardial functional protection, appear to act in common through PKC.* PKC regulates intracellular processes both by direct phosphorylation and by initiating the induction of other protein kinase cascades. 16 Multiple extracellular and intracellular ischemia–responsive stimuli may provide common communication by PKC. Direct PKC activation induces myocardial functional protection, and PKC inhibition can block this protection. 3,12,14 Endogenous [Ca2+]i is an important activator of PKC, 16 and elevation of [Ca2+]i is also coincident with the action of these ischemic stress stimuli 32; however, it is unknown whether exogenous Ca2+ administration can activate PKC and thereby induce myocardial functional protection similar to that observed after ischemic preconditioning. Our data suggest that preischemic CaCl2 administration can induce functional protection after ischemia-reperfusion and limit myocellular CK leakage by means of a PKC-dependent mechanism.
The exact mechanism by which PKC provides myocardial functional protection is unknown. Because PKC translocation and activation appear to be central to the functional protection afforded by several different stimuli, it is possible that PKC translocation to various intracellular sites regulates upward the cellular machinery required to adapt to the ensuing ischemia-reperfusion insult. For example, translocation of PKC to the sarcolemma may signify the priming of cellular machinery at this location; phosphorylation and activation of the sodium ion–hydrogen ion exchanger or sodium ion–Ca2+ exchanger may prepare the heart to better regulate ion gradients after ischemia-reperfusion. Similarly, sarcoendoplasmic reticulum Ca2+ adenosine triphosphatase activity may be upregulated, allowing the heart to adapt to the subsequent Ca2+ load associated with ischemia-reperfusion injury. This, however, remains to be determined. Indeed, preconditioning's link with PKC may only be an association. Although PKC may be operative in some forms of preconditioning, other kinases may be operative after other preconditioning stimuli. It is therefore possible according to the work of Asimakis and Conti, 31 that Ca2+ preconditioning is independent of PKC. These authors reported that dobutamine, which increases [Ca2+]i but is not known to activate PKC, also produced functional protection in isolated rat hearts. Furthermore, the mechanism by which exogenous Ca2+ activates PKC remains unknown. It is possible that increasing [Ca2+]o increases [Ca2+]i by either calcium-induced-calcium release, voltage-operated channels, or both, and that this increase in [Ca2+]i is sufficient stimulus to activate PKC.
The ultimate benefit of preischemic induction of endogenous functional protection relates to its clinical applicability. Transient ischemia, which is a potent protective stimulus in animals, is less appealing clinically. Because CaCl2 is both clinically accessible and acceptable, stimulating PKC-mediated myocardial functional protection with preischemic CaCl2 infusion may even provide a means of enhancing cardiac function after transplantation and bypass operations. It must be emphasized, however, that these studies were performed with normal rat hearts. Both the salutary and the deleterious effects of Ca2+ preconditioning of diseased human ventricles remain to be determined.
Acknowledgments
We thank Drs. David A. Fullerton, James M. Brown, Robert C. McIntyre, Jr., and Xianzhong Meng for their insightful criticisms and suggestions; Dr. Daniel Chan for use of the fluorescent microscope; and Mr. James Walther for suggestions and expert technical assistance.
Footnotes
*References 3,4,8,9,14,15 and 20. 
*References 1,3,4,7,12,15 and 30.
References
1-Adrenergic agonists precondition rabbit ischemic myocardium independent of adenosine by direct activation of protein kinase C. Circ Res 1994;75:576-85.
synthesis and decreases host susceptibility to sepsis following hemorrhage. J Surg Res 1991;51:158-64.[Medline]This article has been cited by other articles:
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  K. L. Schreiber, L. Paquet, B. G. Allen, and H. Rindt Protein kinase C isoform expression and activity in the mouse heart Am J Physiol Heart Circ Physiol, November 1, 2001; 281(5): H2062 - H2071. [Abstract] [Full Text] [PDF]
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 M. Loubani and M. Galinanes {{alpha}}1-Adrenoceptors during simulated ischemia and reoxygenation of the human myocardium: Effect of the dose and time of administration J. Thorac. Cardiovasc. Surg., July 1, 2001; 122(1): 103 - 112. [Abstract] [Full Text] [PDF]
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 C. Stamm, I. Friehs, D. B. Cowan, H. Cao-Danh, S. Noria, M. Munakata, F. X. McGowan Jr., and P. J. del Nido Post-ischemic PKC inhibition impairs myocardial calcium handling and increases contractile protein calcium sensitivity Cardiovasc Res, July 1, 2001; 51(1): 108 - 121. [Abstract] [Full Text] [PDF]
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K. Lu, H. Otani, T. Yamamura, Y. Nakao, R. Hattori, H. Ninomiya, M. Osako, and H. Imamura Protein kinase C isoform-dependent myocardial protection by ischemic preconditioning and potassium cardioplegia J. Thorac. Cardiovasc. Surg., January 1, 2001; 121(1): 0137 - 148. [Abstract] [Full Text] [PDF]
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 L. P. Perrault and P. Menasche Preconditioning: can nature’s shield be raised against surgical ischemic-reperfusion injury? Ann. Thorac. Surg., November 1, 1999; 68(5): 1988 - 1994. [Abstract] [Full Text] [PDF]
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 B. S. Cain, D. R. Meldrum, K. S. Joo, J.-F. Wang, X. Meng, J. C. Cleveland Jr., A. Banerjee, and A. H. Harken Human SERCA2a levels correlate inversely with age in senescent human myocardium J. Am. Coll. Cardiol., August 1, 1998; 32(2): 458 - 467. [Abstract] [Full Text] [PDF]
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B. S. Cain, D. R. Meldrum, X. Meng, B. D. Shames, A. Banerjee, and A. H. Harken Calcium Preconditioning in Human Myocardium Ann. Thorac. Surg., April 1, 1998; 65(4): 1065 - 1070. [Abstract] [Full Text] [PDF]
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 D. R. Meldrum Tumor necrosis factor in the heart Am J Physiol Regulatory Integrative Comp Physiol, March 1, 1998; 274(3): R577 - R595. [Abstract] [Full Text] [PDF]
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D. R. Meldrum, J. C. Cleveland Jr, B. S. Cain, X. Meng, and A. H. Harken Increased Myocardial Tumor Necrosis Factor-{alpha} in a Crystalloid-Perfused Model of Cardiac Ischemia-Reperfusion Injury Ann. Thorac. Surg., February 1, 1998; 65(2): 439 - 443. [Abstract] [Full Text] [PDF]
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 K. Przyklenk, K. Hata, and R. A. Kloner Is Calcium a Mediator of Infarct Size Reduction With Preconditioning in Canine Myocardium? Circulation, August 19, 1997; 96(4): 1305 - 1312. [Abstract] [Full Text]
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