David J. Hearse

Direct detection of free radicals in the reperfused rat heart using electron spin resonance spectroscopy.

The purpose of this study was to use a direct method, that of electron spin resonance (ESR) spectroscopy, to demonstrate that reperfusion after a period of ischemia results in a sudden increase in the production of free radicals in the myocardium. The isolated buffer-perfused rat heart was used with N-tert-butyl-alpha-phenylnitrone (PBN) as a spin-trapping agent. Samples of coronary effluent were taken and extracted into toluene for detection of radical adducts by ESR spectroscopy. After 15 minutes of total, global ischemia, aerobic reperfusion resulted in a sudden burst of radical formation that peaked at 4 minutes. When hearts were reperfused with anoxic buffer, no dramatic increase in radical production was observed. Subsequent reintroduction of oxygen, however, resulted in an immediate burst of radical production of a similar magnitude to that seen in the wholly aerobic reperfusion experiments. The ESR signals obtained (aN = 13.60 G, aH = 1.56 G) are consistent with the spin-trapping by PBN of either a carbon-centered species or an alkoxyl radical, both of which could be formed by secondary reactions of initially-formed oxygen radicals with membrane lipid components.

The oxygen paradox and the calcium paradox: two facets of the same problem?

An isolated rat heart preparation was used to study and compare the sudden tissue damage which may result from calcium repletion after a period of calcium-free perfusion with the effects of reoxygenation after a period of anoxic perfusion and with reperfusion after a period of ischaemia. Striking similarities in morphological and enzyme release characteristics exist for all three phenomena. Our results suggest that the primary lesion in the calcium paradox is the separation of the basement membrane from the sarcolemma and the energy-dependent loss of intracellular calcium to the extracellular space during the phase of calcium depletion. These changes, coupled with separation of opposing faces of the intercalated disc predispose the cell to the major damage which results from an energy independent, sudden, cellular influx of calcium during the phase of calcium repletion. In the case of the oxygen paradox the primary lesion would be the accumulation of cytoplasmic calcium and the loss of cellular and subcellular membrane control. While these anoxia or ischaemia-induced processes are related to the cellular availability of energy, the sudden damage induced by oxygen repletion would appear to be initiated by an energy-independent mitochondrial calcium uptake which is triggered by the reoxidation of the electron transport chain. Studies of such factors as the critical duration of calcium or oxygen depletion, the effects of stepwise calcium or oxygen repletion or the striking effects of hypothermia, reinforce the association between the calcium and oxygen paradoxes. While the basic principles underlying the cause of damage may be similar in both cases, fine differences exist in the progression, the nature and the extent of damage.

Reperfusion-induced arrhythmias and oxygen-derived free radicals. Studies with "anti-free radical" interventions and a free radical-generating system in the isolated perfused rat heart.

We have assessed (1), whether six agents, that either inhibit free radical formation or scavenge free radicals once they are produced, can reduce the incidence of reperfusion-induced arrhythmias, (2) whether a free radical-generating system (FeCl3 ± adenosine diphosphate) can increase the incidence of reperfusion-induced arrhythmias, and (3) whether “anti-free radical interventions can reduce reperfusion rhythm disturbances caused by the addition of FeCl3-adenosine diphosphate.” With the isolated, perfused rat heart (n = 15 in each group), inclusion of L-methionine (1 and 10 mM), superoxide dismutase (2.5 × 104 and 1 × 105 U/liter), catalase (5 × 104, 5 × 105, and 1 × 106 U/liter), mannitol (50 mM), glutathione (10 μM), or desferrioxamine (150 μM) significantly reduced the incidence of reperfusion-induced ventricular fibrillation and, in many cases, the incidence of reperfusion-induced ventricular tachycardia. The mean duration of sinus rhythm during reperfusion was also increased significantly. Perfusion of hearts with boiled superoxide dismutase (1 × 105 U/liter) or boiled catalase (1 × 106 U/liter) did not decrease arrhythmias. Conversely, under conditions where, in the control group, the incidence of reperfusion arrhythmias was lowered by increasing perfusate potassium to 6.5 mM, the addition of the free radical-generating system FeCl3· adenosine diphosphate (0.1 μM-1μM) to the perfusion fluid increased dramatically the incidence of reperfusion-induced ventricular fibrillation and tachycardia. Simultaneous perfusion with FeCl3 · adenosine diphosphate and superoxide dismutase (1 × 105 U/liter), catalase (1 × 106 U/liter), mannitol (50 μM), methionine (10 mM), or desferrioxamine (150μM) again reduced the incidence of reperfusion-induced arrhythmias and increased the duration of normal sinus rhythm during the reperfusion phase. Thus, addition of six “anti-free radical” interventions reduced the incidence of reperfusion-induced arrhythmias, addition of a free radical-generating system increased the incidence of reperfusion-induced arrhythmias, and simultaneous perfusion of the hearts with FeCl3 ± adenosine diphosphate and “anti-free radical” interventions again reduced reperfusion rhythm disturbances. These results are further evidence supporting the hypothesis that oxygen-derived free radicals play an important role in the genesis of reperfusion-induced arrhythmias.

Ischemic Contracture of the Myocardium: Mechanisms and Prevention

Ischemic contracture of the left ventricle (“stone heart”) occurs in the rat heart after a period of global ischemia at 37° C. The time of onset of contracture can be delayed by various interventions that reduce myocardial energy demand, increase myocardial energy supply or reduce cellular calcium influx. Conversely, the time of onset of contracture may be advanced by interventions that reduce the energy supply. Analysis of myocardial energy metabolism, and particularly of adenosine triphosphate (ATP) availability in relation to the onset and duration of contracture suggests that contracture is initiated when the cellular ATP content decreases to approximately 12 μmoles/g dry weight. Furthermore, the progression of contracture is characterized by an acceleration in the rate of ATP degradation and the completion of contracture coincides with the reduction of ATP to 3 to 4 μmoles/g dry weight. The results of this study suggest that the development of ischemic contracture represents the accumulation of rigor complexes arising as a result of an ATP deficiency. These rigor complexes are able to exacerbate the contracture by promoting uncontrolled ATP hydrolysis. Although the development of these complexes and the onset of contracture is calcium-insensitive the results indicate that calcium availability may influence the magnitude of the developed contracture.

Ischemia and reperfusion-induced arrhythmias in the rat. Effects of xanthine oxidase inhibition with allopurinol.

We have investigated the possibility that xanthine oxidase-linked free radical production has a role in the genesis of arrhythmias during ischemia and reperfusion. In this study, rats were treated with allopurinol (20 mg/kg, orally, 24 hours before study, plus 20 mg/kg, iv, 15 minutes prior to study). Using an anesthetized open-chest preparation with either coronary artery occlusion for 30 minutes, or 5 minutes followed by 10 minutes reperfusion, we monitored and compared the rhythm disturbances in experimental vs. placebo-treated rats (n = 18 in each group). Allopurinol treatment reduced the incidence of ventricular tachycardia during ischemia from 88% to 50% (P less than 0.05) and the number of premature ventricular complexes from 471 +/- 120 to 116 +/- 46 (P less than 0.02), but the treatment had no effect upon the incidence or duration of ventricular fibrillation or upon mortality. In contrast, far more dramatic protection was observed during reperfusion after 5 minutes of ischemia. Allopurinol treatment reduced the incidence of ventricular fibrillation from 67% to 11% (P less than 0.01), reduced the mean duration of fibrillation from 230 +/- 70 to 14 +/- 1 seconds (P less than 0.05), and reduced mortality by half (10/18 to 4/18), although this did not reach a level of statistical significance. In addition, the mean duration of tachycardia was reduced from 83 +/- 26 to 38 +/- 8 seconds (P less than 0.05). Allopurinol pretreatment thus affords some protection against ischemia-induced arrhythmias, but a higher degree of protection against reperfusion-induced arrhythmias. Allopurinol inhibits xanthine oxidase activity, and, in turn, this inhibits superoxide radical production.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxygen deprivation and early myocardial contractile failure: A reassessment of the possible role of adenosine triphosphate☆

The precise mechanism responsible for early contractile failure after the onset of myocardial anoxia or ischemia has attracted speculation and controversy. The simple and attractive hypothesis that adenosine triphosphate (ATP) deficiency is responsible for this failure has often been dismissed on the basis of claims that there is only a small reduction in cell ATP content at a time when contractile activity is severely reduced. The premise of this article is that the changes in cell ATP content and distribution that theoretically should occur after oxygen depletion may not have been adequately considered and that previous measurements of cell ATP content may not have been carried out at the correct time. Using an isolated rat heart preparation and high speed freeze-clamping techniques it has been possible to demonstrate that a substantial decrease in myocardial ATP and creatine phosphate content occurs after the onset of anoxia but before the onset of contractile failure. Thus, during the first 5 seconds of anoxia contractile activity remains constant whereas ATP decreases by 25 percent and creatine phosphate by 50 percent. Thereafter, contractile failure occurs and the rate of utilization of high energy phosphates declines with the cell content at a plateau or possibly increasing. These results are assessed in the light of the dynamic changes in energy metabolism occurring in early anoxia and suggest that ATP depletion in a specific cell compartment may be the primary trigger for early contractile failure.

Ischaemic preconditioning and contractile function: studies with normothermic and hypothermic global ischaemia.

A significant reduction in the extent of cell necrosis or the incidence of reperfusion-induced arrhythmias can be achieved with ischaemic preconditioning. If preconditioning was also found to be effective in protecting against global ischaemia, then this may have significant implications for the preservation of the heart during cardiac surgery. We therefore investigated this phenomenon in relation to recovery of contractile function after global ischaemia in the isolated rat heart. Isolated working rat hearts (n = 6 per group) were perfused aerobically at 37 degrees C for 20 min and contractile function recorded. This was followed by 10 min of aerobic Langendorff perfusion (control hearts) or 5 min global ischaemia (37 degrees C) + 5 min Langendorff reperfusion (preconditioned hearts). The hearts were then subjected to 10, 15, 20 or 25 min of global ischaemia (37 degrees C) and reperfusion (15 min Langendorff + 20 min working) after which function was again assessed. Preconditioning improved functional recovery after all durations of ischaemia. Thus aortic flow after 10, 15, 20 and 25 min of ischaemia and 35 min of reperfusion recovered to 84, 58, 16 and 5%, respectively, in controls and 88, 74, 55 and 20%, respectively, in the preconditioned groups. To assess whether preconditioning was effective in a surgically relevant model of hypothermic ischaemia, the experiments were repeated with longer periods (45, 70, 90, 115, 135 and 160 min) of ischaemia at 20 degrees C. Under these conditions, normothermic preconditioning increase the post-ischaemic recovery of aortic flow after 115, 135 and 160 min of ischaemic (from 36, 20 and 10%, respectively, in controls to 57, 39 and 26%, respectively, in preconditioned hearts). There was no consistent correlation between tissue high energy phosphate content and enhanced post-ischaemic recovery. Thus, we have demonstrated that ischaemic preconditioning can improve contractile function after global ischaemia in the isolated rat heart, we have defined the duration of ischaemia for which it is operative, and we have shown that this protection is additive to that of hypothermia-induced protection during ischaemia. This may have clinical implications for cardiac surgery.

Improved functional recovery by ischaemic preconditioning is not mediated by adenosine in the globally ischaemic isolated rat heart

The experiments described in this paper were designed and performed between 1991 and 1992 when I was in the final year of my Ph.D studentship. I was introduced to preconditioning at my first American Heart Association meeting in 1989 and was fascinated that evolution had devised an endogenous protective mechanism for the heart, which was far more effective than any intervention developed by man. Not surprising really, considering that evolution has been working on stress adaptation for a couple of a million years! Since the aim of my PhD project was to extend the window of preservation for donor hearts prior to transplantation, after the AHA meeting, I began experiments to explore whether ischaemic preconditioning could provide protection over and above that provided by cardioplegia. For the transplanted heart, we felt the most important parameter for assessment was contractile function. For a heart to be weaned off cardiopulmonary bypass, contractile function must recover sufficiently to support the body. It would be somewhat irrelevant for the transplanted heart if preconditioning reduced cell necrosis but left the heart so stunned that it could not maintain a sufficient cardiac output. However, in 1989, there were no published studies specifically investigating preconditioning-induced protection against contractile dysfunction. Therefore, my initial work characterised the phenomenon of preconditioning using the isolated ejecting rat heart and contractile function as the end-point of assessment [1] before proceeding to investigate whether preconditioning could …

XANTHINE-OXIDASE IS NOT A SOURCE OF FREE-RADICALS IN THE ISCHEMIC RABBIT HEART

The xanthine oxidase pathway has been proposed as a source of oxygen-derived free radicals in ischemic and reperfused myocardium. A spectrophotometric assay was employed to measure the xanthine oxidase activity of rat and rabbit hearts exposed to varying durations of global ischemia. In the rat 24.6 +/- 4.8 mIU/g wet wt of xanthine dehydrogenase + xanthine oxidase activity were detected in both ischemic and normally perfused myocardium. In the non-ischemic state only 6% of this activity was associated with the free radical-producing oxidase form. After 5 min of ischemia however about 25% of the enzyme was in the oxidase form, a value which remained unchanged over the following 25 min. Neither xanthine dehydrogenase nor xanthine oxidase could be detected in the rabbit heart. Failure of allopurinol, an inhibitor of xanthine oxidase, to limit infarct size in a rabbit model of ischemia/reperfusion provides further evidence that this species has insignificant amounts of xanthine oxidase in its heart. Anesthetized rabbits were subjected to coronary artery ligation for 45 min and 3 h of reperfusion. The volume of the zone of underperfusion was assessed with fluorescent microspheres and infarct size was assessed by tetrazolium staining. In control animals 67.5 +/- 3.8% of the zone of underperfusion became necrotic. In rabbits given superoxide dismutase (15000 IU/kg) + catalase (50,000 IU/kg) for 90 min starting 15 min before occlusion, infarct size was only 35.4 +/- 3.3% of the zone of underperfusion. However, in rabbits pretreated with allopurinol (75 mg p.o. 24 h before study + 30 mg/kg 5 min before occlusion) infarct size was 65.8 +/- 8.7%.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the “border zone” in acute regional ischemia in the dog☆

Abstract A biopsy instrument with multiple drill heads was used to retrieve simultaneously, and freeze within seconds, 29 individual transmural tissue samples from the left ventricular wall of the dog heart. Studies were undertaken 21 to 26 minutes after coronary arterial ligation to define the border zone between ischemic and nonischemic tissue using the following criteria: (1) metabolic changes (adenosine triphosphate, creatine phosphate, glycogen, lactate, potassium, sodium, water); (2) electro-cardiographic S-T changes; and (3) blood flow distribution (microspheres). The existence, position and sharpness of the gradients for each variable were defined in relation to the edge of the visible area of cyanosis. Adenosine triphosphate, creatine phosphate and lactate values in the nonischemic tissue were essentially constant until 2 to 3 mm from the edge of visible cyanosis. The high energy phosphate content of the tissue then decreased sharply across a zone 8 to 15 mm wide that spanned the visible edge. Across this zone lactate content increased sharply as did S-T segment elevation, and coronary flow decreased to approximately 20 percent of the control value. Multiple cross-correlation studies revealed that changes in the tissue content of adenosine triphosphate, creatine phosphate and lactate were accurately reflected by S-T segment changes, and further that all of these variables were directly related to the degree of ischemia, as indicated by the reduction in coronary flow. In addition to confirming the existence of a clearly defined border zone, these studies suggest that therapeutic salvage of this zone could considerably influence the extent of the ultimate infarct.