Improved 4- and 6-Hour Myocardial Preservation by Hypoxic Preconditioning
Background A brief hypoxic episode can precondition myocardium against a subsequent ischemic-reperfusion injury. The present study sought to determine whether intracellular ionic alterations, induced expression of heat-shock proteins (hsps), and/or catalase are involved in the cellular mechanisms by which hypoxic preconditioning can preserve postischemic function in a model of prolonged hypothermic storage.
Methods and Results Two groups of isolated working rat hearts were studied: control (CON) and hypoxically preconditioned (HP) hearts. Hearts were arrested at 4°C with St Thomas’ cardioplegic solution and immersion-stored for either a 4- or 6-hour period. Myocardial function (ie, heart rate, aortic flow, coronary flow, developed pressure, and its first derivative dP/dtmax) was determined at baseline, after preconditioning, and during reperfusion. At similar time points, myocardial [Na+]i, [K+]i, [Mg2+]i, and [Ca2+]i were measured using an atomic absorption spectrophotometer, and the induction of hsp 70 and catalase mRNAs was assayed using Northern blot analysis. After 4 and 6 hours of hypothermic storage, aortic flow, dP/dtmax, and [K+]i were increased, whereas [Na+]i and [Ca2+]i were decreased significantly in the HP group compared with the CON group. Steady state mRNA levels of catalase and hsp 70 were increased from baseline levels only in the HP group, with a peak (2.8- and 2.4-fold versus baseline) after 4 hours of storage.
Conclusions Our results indicate that intracellular ionic alterations and upregulation of catalase and hsp 70 gene expression may contribute to the mechanisms underlying hypoxic preconditioning, leading to improved postischemic function during prolonged hypothermic storage of hearts.
In 1986, Murry et al1 noted that brief periods of ischemia render the heart more tolerant to a subsequent ischemia-reperfusion injury. This phenomenon is known as ischemic preconditioning. We have demonstrated that a brief hypoxic perfusion can substitute for ischemia as a preconditioning stimulus in an isolated working normothermic rat heart.2 To extend the clinical applicability of this phenomenon, we have shown that HP can preserve postischemic function and reduce cellular necrosis in a model of prolonged hypothermic storage. This protection was afforded after both the 4- and 6-hour time periods and was not associated with improved high-energy phosphate content.3 In this study we have attempted to more clearly delineate the mechanisms underlying this protective effect.
Oxygen-derived free radicals are an integral component in the pathogenesis of myocardial reperfusion injury.4 HP has been shown to strengthen the endogenous antioxidant defense system after a period of global normothermic ischemia.5 It has also been shown that hearts from rats subjected to heat shock are more resistant to subsequent hypothermic arrest6 and prolonged storage.7 Hence, we hypothesized that preconditioning may act to stimulate the induction of hsps and the antioxidative enzyme catalase, thereby improving myocardial preservation. To test this hypothesis, we compared catalase and hsp expression in HP and CON hearts before and after a period of prolonged hypothermic storage.
Ischemic preconditioning has been found to result in reduced ionic derangements during ischemia.8 After ischemia-reperfusion, increased intracellular calcium [Ca2+]i may be responsible for increased cell injury.9 Similarly, increased intracellular acidification [H+]i results in increased [Na+]i via Na+-H+ exchange and increased [Ca2+]i via Na+-Ca2+ exchange.8 In this study, we therefore also measured [Na+]i, [K+]i, [Mg2+]i, and [Ca2+]i to assess whether ionic alterations may contribute to improved myocardial preservation.
Male Sprague-Dawley rats weighing 320 to 360 g were anesthetized with an injection of sodium pentobarbital (Nembutal, 80 mg/kg IP) and heparin (500 IU/kg IV). All animals received humane care in compliance with the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health (NIH publication 85-23, revised 1985). After thoracotomy, the hearts were excised and placed in ice-cold perfusion buffer. The aorta was then cannulated, and the hearts were perfused by the Langendorff method at a constant perfusion pressure of 100 cm H2O.10
The perfusion medium consisted of a modified KHB (mmol/L concentration: NaCl 118, NaHCO3 24, KCl 4.7, KH2PO4 1.2, MgSO4 1.2, CaCl2 1.7, and glucose 10) gassed with 95% O2/5% CO2, pH 7.4, at 37°C. This solution was filtered through a 5-μm filter to remove any particulate contaminants. The pulmonary vein was then cannulated and the Langendorff perfusion discontinued. The preparation was then converted to the working heart as described by Tosaki et al.11 Essentially, it is a left-heart preparation in which oxygenated KHB at 37°C enters the cannulated left atrium at a filling pressure of 17 cm H2O. The perfusion fluid then passes to the left ventricle from which it is ejected spontaneously through the aortic cannula against a pressure equivalent of 100 cm H2O. In this mode, myocardial function could then be measured.
The experimental protocol is illustrated in Fig 1⇓. After conversion to the working heart mode, the hearts were perfused for 5 minutes with KHB gassed with a 95% O2/5% CO2 mixture (Po2 ≥600 mm Hg). Baseline contractile function was then measured. Langendorff perfusion was then begun for the HP group with 10 minutes of KHB gassed with a 95% N2/5% CO2 mixture (Po2 ≤50 mm Hg) followed by 5 minutes of aerobic perfusion. The CON group underwent 15 minutes of continuous Langendorff perfusion with the 95% O2/5% CO2 (aerobic) mixture. After conversion to the working heart mode for an additional 5 minutes, contractile function was measured.
Cardioplegic arrest was then obtained by clamping the aortic and atrial cannulas and infusing cardioplegic solution into a sidearm of the aortic cannula at a perfusion pressure of 60 cm H2O for a period of 2 minutes. The hearts were then immersed in cardioplegic solution maintained at 4°C for either a 4- or a 6-hour period. The hearts were randomly assigned to each protocol. After both 4- and 6-hour arrest, all hearts were reperfused with oxygenated KHB at 37°C via the Langendorff method for 10 minutes and then converted to the working mode for 20 minutes. Contractile function was obtained after 10 and 20 minutes of working heart reperfusion.
A single infusion of St Thomas’ cardioplegic solution (mmol/L concentration: NaCl 110, KCl 16, MgCl2 16, CaCl2 1.2, NaHCO3 10) was used for myocardial preservation. This solution was filtered through a 5-μm filter to remove any particulate contaminants before infusion.
Indexes of Myocardial Function
Myocardial function was assessed in hearts that were stored in cardioplegic solution for either a 4- or 6-hour period (n=11 and n=6 for both groups, respectively). The aortic flow rate was measured by a calibrated rotameter, and the coronary flow rate was measured by timed collection of the coronary effluent. Direct measurements of heart rate, developed pressure (defined as the aortic end-systolic pressure minus the end-diastolic pressure), and the first derivative of the aortic pressure (dP/dtmax) were made at each time point. All data were recorded and analyzed in real time using the Cordat II data acquisition, analysis, and presentation system (Data Integrated Scientific Systems; Triton Technologies, Inc).
Measurement of Myocardial [Na+]i, [K+]i, [Mg2+]i, and [Ca2+]i
Electrolyte contents in the myocardium were measured in a separate group of hearts, as previously described.12 Six nonischemic hearts were perfused in the working mode for 5 minutes as described above, and then rapidly cooled to 0° to 5°C by submersion in, and perfusion with, an ice-cold ion-free solution containing 100 mmol/L Tris-HCl and 220 mmol/L sucrose (pH adjusted to 7.4 by HCl; Po2 and osmolality were 0.0 to 4.0 kPa and 300 to 330 mOsm/g, respectively) to wash out ions from the extracellular space.13 These hearts were used for subsequent baseline ion measurements. Six other hearts were similarly perfused in the working mode for 5 minutes, hypoxically preconditioned for 10 minutes, and reoxygenated for 5 minutes before extracellular ion flushing. These hearts were used for preischemic measurements after the preconditioning stimulus. After both 4 and 6 hours of cardioplegic arrest, separate HP hearts (n=6 per group) and CON hearts (n=6 per group) were reperfused for 5 minutes in the Langendorff mode and similarly flushed.
Myocardium (left and right ventricles) was dried for 48 hours at 100°C and ashed at 550°C for 20 hours. The ash was dissolved in 5 mL (3 mol/L) nitric acid (Suprapur, Merck) and diluted 10-fold with deionized water. The [Na+]i was measured at a wavelength of 330.3 nm, [K+]i was measured at 404.4 nm, [Ca2+]i at 422.7 nm, and [Mg2+]i at 286.0 nm, in an air-acetylene flame, using a Perkin-Elmer atomic absorption spectrophotometer.
Assessment of HSP and Catalase Expression
Total RNA was extracted from left ventricular tissue samples, in a separate group of hearts (n=4 per group), at time points similar to those of the intracellular ion measurements (Fig 1⇑) by the acid guanidinium-thiocyanate-phenol-chloroform method as described previously.14 The isolated RNAs had OD260-OD280 ratios between 1.94 and 2.05 indicating the purity of the preparation. This preparation was further verified by running RNA on 1% agarose gel stained with ethidium bromide. Ten micrograms of RNA from each sample was subjected to 1% agarose–2.2 mol/L formaldehyde gel electrophoresis and transferred to nylon membrane (Gene Screen Plus, NEN Products) by capillary blot. The membranes were baked under vacuum at 80°C for 2 hours. Prehybridization was performed for 10 to 20 minutes at 68°C followed by hybridization for 2 hours in a hybridization oven (Hybaid, Labnet) using 10 mL QuickHyb aqueous exclusion rate–enhancing solution (Stratagene) per membrane per roller bottle. Each membrane was covered with a thin film of the QuickHyb solution during both prehybridization and hybridization.
The probes used in our experiments were (1) HSP 70 (inducible) (Stress Gen), which was a 4-kb EcoRI fragment of the human cDNA; (2) catalase, which was a 1.8-kb Xba I fragment of the mouse cDNA; and (3) β-actin (Oncor), a 770-bp fragment of chicken cDNA, which was used as a housekeeping gene. cDNA probes were labeled with α-32PdCTP using a random-primed DNA labeling kit (Boehringer Mannheim Biochimica). The specific activity was approximately 0.7×109 disintegrations per minute/μg.14
After hybridization, each membrane was washed twice for 15 minutes at room temperature with 2×SSC-0.1% SDS buffer followed by 30 minutes of high-stringency wash at 60°C with 0.1 SSC-0.1% SDS buffer. The membrane was exposed to Kodak X-OMAT AR film with an intensifying screen at −80°C for 24 hours. Each hybridization was repeated at least three times using different membranes. After each hybridization, the residual cDNA was removed and rehybridized with the β-actin cDNA probe. The autoradiogram was quantitatively evaluated by computerized gel scanner. The results of densitometric scanning were normalized relative to the signal obtained for β-actin.
The values for myocardial function, HSP 70 and catalase mRNA expression, and ion concentrations are expressed as the mean±SEM. A two-way ANOVA (Sheffé’s) was first carried out to test for any differences between groups. If differences were established, the values were compared using a Student’s t test. Significance was considered at a value of P<.05.
For the combined groups, baseline heart rate was 317.6±6.0 beats per minute, baseline aortic flow was 43.0±1.6 mL/min, baseline coronary flow was 23.2±1.1 mL/min, baseline developed pressure was 78.6±2.7 mm Hg, and baseline dP/dtmax was 2920.0±113.7 mm Hg/s. There were no significant differences between groups for any of these baseline functions. Ten minutes of hypoxic perfusion (preconditioning) had little effect on these hemodynamic measurements, as reported in the Table⇓. Immediately after the hypoxic preconditioning stimulus, there was a transient decline in cardiac function. However, only the dP/dtmax was significantly decreased in the preconditioned group because after reoxygenation myocardial function had largely returned to baseline values by the 10-minute measurement. There was no significant difference between 20 and 30 minutes of reperfusion for aortic flow, coronary flow, heart rate, developed pressure, or dP/dtmax in either the 4- or 6-hour cardioplegic arrest groups. Data are therefore presented only for the 30-minute reperfusion measurement. After both 4 and 6 hours of hypothermic storage, the HP hearts had a significant (P<.05) improvement in both aortic flow and force of contraction (dP/dtmax) compared with CON hearts. Heart rate and coronary flow were not significantly different between CON and HP hearts.
It is of interest to note that despite improved mechanical function after the 4- or 6-hour storage period, HP hearts were shrunken, were discolored, and had the characteristics of a “stone heart.” On the other hand, CON hearts were soft and pink. This observation, though not scientifically documented, may be a “preconditioning paradox” that follows ischemia-reperfusion in preconditioned hearts and deserves further attention.
Myocardial [Na+]i, [K+]i, [Mg2+]i, and [Ca2+]i
Myocardial [Na+]i, [K+]i, [Mg2+]i and [Ca2+]i are shown in the Table⇑. Ten minutes of hypoxic perfusion followed by 10 minutes of reoxygenation had no significant effect on [Na+]i, [K+]i, or [Mg2+]i. After hypoxia and reoxygenation, however, [Ca2+]i was significantly increased in the preconditioned group. After both 4 and 6 hours of storage, HP hearts had significantly decreased [Na+]i and [Ca2+]i and increased [K+]i compared with CON hearts. There was no significant difference in [Mg2+]i between CON and HP hearts at any time point.
As shown in Figs 2⇓ and 3⇓, the brief hypoxic perfusion prior to ischemia led to minimal expression of HSP 70 and catalase mRNAs. After 4 and 6 hours of storage, the CON group similarly expressed only a small amount of these mRNAs. However, a significant induction of the expression of both HSP 70 and catalase mRNAs was observed after both 4 and 6 hours of storage in the HP hearts. Both HSP 70 and catalase mRNA expression were significantly increased in HP hearts compared with CON hearts after 4 hours of storage.
This study has shown that HP can preserve postischemic function, prevent intracellular ionic alterations, and induce catalase and HSP 70 mRNA expression in a rat heart model of prolonged hypothermia, which simulates heart storage before transplantation. This protection was afforded after both the 4- and 6-hour time periods. The mechanisms underlying hypoxic preconditioning remain ill-defined. Using a rat heart model of normothermic ischemia and reperfusion, we have previously demonstrated that hypoxic preconditioning preserves myocardial antioxidant stores and reduces calcium overload.2 Other researchers have shown that the beneficial effects of preconditioning may be due to an attenuation of ionic alterations during ischemia.8 In this experiment, we have confirmed that similar protective mechanisms may operate in a model of hypoxic preconditioning followed by prolonged hypothermia.
The fundamental protective mechanisms of preconditioning may involve the prevention of ischemia-reperfusion–induced ion shifts. Using a model of ischemic preconditioning, it has been stressed by Tani and Neely15 that in hearts subjected to intermittent ischemia, there is less accumulation of [Ca2+]i, a reduced magnitude of Ca2+ uptake on reperfusion, and improved recovery of systolic function during reperfusion than during a similar period of continuous ischemia. In cardiac cells, this rise in [Ca2+]i results from increased Na+-H+ exchange and the resultant Na+-Ca2+ exchange.16 In addition, there is increased influx via the slow Ca2+ channels, which in turn triggers release of [Ca2+]i from the sarcoplasmic reticulum. An elevation in [Ca2+]i can also activate Ca2+-dependent protein kinases, which can alter K+ homeostasis, leading to [K+]i loss during the postischemic period. Furthermore, the maintenance of a relatively low [Na+]i in intermittently ischemic myocardium (due to enhanced function of the Na+-K+ ATPase), reduces Ca2+ overload occurring by Na+-Ca2+ exchange on reperfusion. During prolonged ischemia, [Ca2+]i increases markedly, which may result in part from impaired Na+-K+ exchange.17 Although the critical aspect of Ca2+ overload occurs in the cytosolic compartment, our washout technique cannot dissociate between the cytosolic versus sarcoplasmic reticular Ca2+ components.
The maintenance of the ionic balance across the cell membrane is essential for prevention of lethal tachyarrhythmias during ischemia-reperfusion. Our studies provide a basis for inquiry but do not dissociate the different pathways involved in postischemic ionic accumulation or loss during the storage of the myocardium. It is of interest to note that [Mg2+]i did not show a significant change between the nonpreconditioned and preconditioned myocardium. This may be explained by the high Mg2+ content of the cardioplegic solution that may mask the protective effects of preconditioning during hypothermic storage. In addition, the [Mg2+]i levels may be similar because ATP catabolism is similar in the two groups after prolonged storage.3
Exposure of cells to environmental stresses such as heat, anoxia, and hypoxia has been shown to be associated with the induction of mRNAs for HSPs.18 Most prominent and widely studied of these HSPs is HSP 70, which is highly conserved both structurally and functionally. HSP 70 is a molecular chaperone that may function to mediate protein conformations and prevent irregular folding interactions after ischemia and reperfusion.19 Previous studies have demonstrated that even a transient ischemic episode may induce the expression of HSP 70 mRNA in the mammalian heart.20 This HSP gene can also be expressed during myocardial adaptation to ischemia and may play a role in the development of myocardial tolerance to subsequent severe ischemia.14 21 In this study, hypoxic preconditioning resulted in a significant increase in the HSP 70 mRNA expression in conjunction with enhanced myocardial preservation. This may suggest a role for the HSP 70 gene mediating this beneficial effect.
Intracellular antioxidants also play a major role in the cellular protection against injury.22 Similar to the HSPs, a variety of stresses can induce the expression of antioxidant mRNAs. Both hyperthermia and ischemia have been shown to induce the expression of catalase as well as HSP 70 mRNA in the mammalian heart.14 23 Catalase is responsible for the removal of the cytotoxic reactive oxygen metabolite, H2O2.24 In view of the fact that hypoxia-reoxygenation and ischemia-reperfusion lead to the generation of oxygen-free radicals,4 the induction of catalase mRNA expression by hypoxic preconditioning appears to reflect the heart’s upregulation of the endogenous antioxidant defense system, enabling it to survive a subsequent ischemic stress by reducing an oxidative insult.
The time course of HSP 70 and catalase protein production can only be surmised in this experiment because we only measured mRNA expression. However, HSP 70 has been previously detected at the protein level as rapidly as 2 hours after brief myocardial ischemia.25 HSP 70 and catalase mRNA may have been translated into functional protein after the hypoxic stimulus. While the 4- or 6-hour interval of hypothermic storage would be expected to significantly slow the metabolic activity of the heart, we speculate that protein synthesis may still continue at a reduced level, especially immediately before and after hypothermic storage.
On the basis of our results, we cannot prove a cause-and-effect relation between the maintenance of ionic balance or the upregulation of HSP 70–catalase mRNA and improved myocardial protection. The hypoxic preconditioning response may result in many cellular changes, including the preservation of endogenous antioxidants and upregulation of adenosine pathways, Gi proteins, or KATP channels.5 Each of these mechanisms may be responsible for improved myocardial preservation. Future studies are necessary using specific protein synthesis inhibitors to delineate the contributions of HSP 70 and catalase in this experimental model.
Hypoxic preconditioning has previously been shown to be cardioprotective after ischemic periods that are much shorter (ie, 30 minutes) than would allow for catalase–HSP 70 translation.5 Therefore, HSP 70–catalase protein upregulation cannot account for the beneficial effects in these studies. We propose that the heart possesses a two-stage intracellular response to stress. The early effect is manifested by the preconditioning phenomenon and is mediated by an unknown endogenous compound. As the preconditioning effects begin to disappear, a number of molecular and cellular events take place mediated by transcriptional and/or posttranscriptional alterations of genes and are referred to as myocardial adaptation to stress. This response is associated with, though may not be mediated by, the upregulation of HSP 70 and catalase mRNAs. This experiment may be a unique example of a single stress (hypoxia) inducing a combined preconditioning-HSP response with simultaneous myocardial protection.
While the precise mechanism(s) responsible for hypoxic preconditioning remains speculative, the maintenance of ionic homeostasis and the preservation of endogenous antioxidants are possible components. While a brief hypoxic perfusion to donor hearts is feasible, a direct manipulation of endogenous protective mechanisms through molecular biology would be preferable. The induction of HSPs may be a marker for this protection or may be directly involved in myocardial protection.19 Although the significance of these inducible genes has not been resolved, it appears likely that the heart is capable of a uniform response to stress that may underlie all preconditioning phenomena and, ultimately, myocardial adaptation.
In this study, stress preconditioning induced by hypoxia led to improved myocardial preservation and upregulation of HSP 70 and catalase mRNA, after a subsequent severe ischemic insult. This provides one of the mechanisms by which hypoxic preconditioning may stimulate a prolonged period of relative myocardial resistance to ischemia. The goal for the future is to use this knowledge to harness the power of preconditioning for use in a clinical setting, such as improved heart storage before transplantation.
Selected Abbreviations and Acronyms
|KHB||=||Krebs-Henseleit bicarbonate buffer|
This study was supported by National Institutes of Health Grants HL 22559-14 and HL 34360-07.
- Copyright © 1995 by American Heart Association
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