Stuart M. Humphrey

The relationship of ischemic contracture to vascular reperfusion in the isolated rat heart

Abstract Isolated Langendorff rat heart preparations were used to measure ischemic contracture of the left ventricle by means of an intraventricular balloon catheter connected to a pressure recorder. After various times of global ischemia, the extent of reflow to the ventricles was visualized by perfusion with 1% fluorescein solution. The onset of ischemic contracture was accelerated and its magnitude increased by preischemic perfusion with 0.5 m m iodoacetate (IAA). Conversely, its onset was delayed and magnitude decreased by preischemic perfusion with buffer containing only 0.05 m m calcium. Compared with control hearts, those pretreated with the low calcium buffer showed more extensive reflow, whereas IAA-treated hearts showed a greater loss of vascular competence in the early stages of reperfusion. Although the reperfusion defect developed after ischemic contracture, its ultimate extent correlated closely with the force of contracture. It is proposed that subendocardial blood vessels closed by the tension generated in ischemic contracture, eventually lose their ability to dilate and allow reperfusion as compliance of the myocardium is reduced by the development of rigor mortis.

Reverse Phase HPLC For Rapid, Comprehensive Measurement of Nucleotides, Nucleosides and Bases of The Myocardial Adenine Pool

Abstract A novel reverse phase HPLC method is described for the simultaneous measurement of adenosine tri-, di- and monophosphates (ATP, ADP, AMP), inosine monophosphate (IMP), adenosine, inosine, hypoxanthine, nicotinamide adenine dinucleotide (NAD) and uric acid in cardiac tissues and coronary effluent. The use of a simplified perchloric acid extraction procedure and ODS columns easily modified with Mq++, Tris and phosphate buffer, allows considerable saving in analysis time together with extremely good resolution, particularly for ATP and ADP, and provides a very practical tool for the routine assessment of changes in adenine pool metabolites.

Changes in vascular morphology associated with the no-reflow phenomenon in ischaemic myocardium

To investigate the pathogenesis of the reperfusion defect which develops in ischaemic myocardium, intravascular casts were prepared by injection of methyl methacrylate into the coronary arteries of isolated heparinised rat hearts. Using a scanning electron microscope, the vascular morphology following 60 min of global ischaemia at 37° C was compared to that of non-ischaemic control hearts injected immediately after stopping perfusion with oxygenated Krebs-Henseleit buffer. Complete casts were obtained from control hearts and from all parts of ischaemic hearts except the subendocardial half of the left ventricular wall of ischaemic hearts where the blood vessels were not filled. At the border between the perfused subepicardial and unperfused left subendocardial regions, the resin which filled the radial penetrating arteries and their branches projected from the filled capillary plexus to an extent proportional to their diameter. Intravascular events such as erythrocyte plugging and thrombosis were excluded as causative factors by the use of a cell-free perfusate. Also, there was no morphological evidence that endothelial cell swelling or constriction of any particular population of vessels was involved. The observed pattern of vascular occlusion suggests that, during global ischaemia, blood vessels in the endocardial half of the left ventricular myocardium lose their ability to be reperfused because of extravascular compression.

Critical early metabolic changes associated with myocardial recovery or failure after total ischaemia in the rat heart

SummaryIsolated rat hearts were used to measure tissue levels of the adenine nucleotides plus their degradation products and the ‘intracellular’ electrolytes after 15 or 25 min of global ischaemia alone and also after 5 or 60 min of reperfusion. Following 15 min of ischaemia, hearts showed near complete recovery of cardiac function (aortic flow rate =92±7% of control), but recovery was severely depressed following 25 min of ischaemia (aortic flow rate =18±15% of control). Similarly, after 5 min of reperfusion, hearts made ischaemic for 25 min had a reduced tissue content of ATP (10.5 vs 18.9 μmol·g·dry wt), and NAD (4.30 vs 4.75 μmol·g·dry wt) and a 3–4-fold increase in AMP, adenosine and oxypurines, as compared with hearts ischaemic for 15 min. However, the extent of loss of oxypurines during 5 min of reperfusion was essentially similar in both groups (21% vs 18% of total purine pool). After this initial period of reperfusion a significant (p<0.01) difference in potassium content was seen between the two groups; hearts which recovered function gained, whilst failing hearts lost, potassium. Changes in the other electrolytes were essentially similar in the two groups of hearts. Extending reperfusion from 5 to 60 min did not change ATP levels in either group but in the functionally depressed group it was associated with a 5-fold increase in calcium and a 30% reduction in potassium, together with a further loss of oxypurines. Thus, loss of nucleotide precursors does not appear to be a critical event in the relatively sudden transition from reversible to irreversible functional injury in ischaemia. There may, however, be a reduction in the activity of the ATP synthesizing processes.

The influence of the no-reflow phenomenon on reperfusion and reoxygenation damage and enzyme release from anoxic and ischaemic isolated rat hearts

Eighty isolated rat heart preparations were used to study relationships among creatine kinase (CK) release, the loss of vascular competence (no-reflow), and the distribution of morphological changes across the left ventricular wall which occur during 60 min global ischaemia or anoxia and following subsequent oxygenated reperfusion. Hearts were either fixed with glutaraldehyde for light and electron microscopy or were injected with 1% fluorescein to define the distribution of perfusable vessels. The extent of no-reflow in half of the hearts was reduced experimentally by maintaining the diastolic volume of the left ventricular lumen during ischaemia and anoxia with a water-filled balloon. The amount of CK released during 20 min of reoxygenation or reperfusion was inversely proportional to the extent of the no-reflow area observed just prior to reoxygeneration, and also reflected the transmural extent and the severity of myocardial cell damage. Extensive contraction band necrosis was only observed in reperfused regions of anoxic hearts. In isovolumic hearts reoxygenation caused no-reflow to develop in the ventricular myocardium, and this appeared to be associated with hypercontraction. Thus the no-reflow phenomenon has a profound effect on the transmural distribution of myocardial cell damage and enzyme release which follows post ischaemic reperfusion and post anoxic reoxygenation.

Adenine pool catabolism in the ischemic, the calcium-depleted ischemic, and the substrate free anoxic isolated rat heart: Relationship to contracture development

Metabolic changes in the myocardial adenine and hypoxanthine pools of isolated rat hearts subjected to global ischemia, hypocalcemic global ischemia, and global substrate-free anoxia were compared. At timed intervals between 0 and 60 min separate aliquots of extracts of the ventricles were used to determine either tissue pH, or the components of the adenine pool and their catabolites by reverse phase high performance liquid chromatography (HPLC). The coronary perfusate draining from anoxically perfused hearts was collected over perchloric acid, neutralised and chromatographed by HPLC. The development of left ventricular resting tension (contracture) was recorded in the three groups of hearts. After 60 min ischemia the major catabolites, (AMP, inosine and hypoxanthine) comprised 70% of the total pool (11, 7 and 4 mumol/g dry wt, respectively). After the same period of anoxia 50% of the total pool, comprising adenosine, inosine, hypoxanthine and uric acid in approximately equal proportions, was recovered from the coronary perfusate. The major products remaining in the tissue were IMP and, to a lesser extent AMP (8 and 5 mumol/g dry wt, respectively). Left ventricular contracture developed at different rates in the three groups of hearts but always correlated closely with the maximum rate of adenine pool catabolism. The loss of components from the tissue and the divergence in pathway from adenosine to IMP production which occurs during anoxic perfusion should possibly be considered when assessing the biochemical events occurring in regionally ischemic heart muscle with significant residual flow.

Catecholamine-depletion and the no-reflow phenomenon in anoxic and ischaemic rat hearts

Abstract The effect of depleting catecholamines in myocardium on the extent of the no-reflow phenomenon was investigated in isolated Langendorff rat heart preparations. Catecholamines were depleted by pretreating the rats with reserpine. After ischaemia or anoxia the proportion of the ventricular walls perfused by 1% fluorescein was assessed, and compared with the changes in glycogen content, lactate production, ATP levels and general morphology. After 60 min of global ischaemia, 60% of the ventricular walls of both catecholamine-depleted and untreated hearts was not reperfused. A similar period of anoxia produced 30% no-reflow in untreated controls, but only 16% in catecholamine-depleted hearts. Reserpine pretreatment caused a doubling of glycolytically derived ATP in anoxic hearts. However, in catecholamine-depleted ischaemic hearts there was no increase in ATP because of an inhibition of glycolysis, presumably due to the fall in pH. These findings indicate that, in anoxia, catecholamine depletion delays the onset of no-reflow by prolonging anaerobic ATP generation due to increased glycogen stores, and in global ischaemia at least, the no-reflow phenomenon is not due to catecholamine-induced sustained contraction of coronary arteries.

The influence of adenosine on the no-reflow phenomenon in anoxic and ischaemic hearts

Summary Adenosine and the adenosine deaminase inhibitor erythro‐9‐(2‐hydroxy‐3‐nonyl) adenine (EHNA), separately and in combination, were added to the perfusate of isolated rat hearts which were then subjected to ischaemia or anoxia. The effect of these infusates on the vascular competence of the myocardium subjected to oxygen deprivation of from 15–90 min was compared to control hearts. Vascular competence at selected time intervals was assessed from the distribution of injected 1% sodium fluorescein in the cut surface of the ventricles. A region of non‐perfusion surrounding the left ventricular lumen and involving 25% of the ventricular myocardium developed within 15 min of anoxic perfusion, with little change thereafter. Adenosine had no significant effect on this. The no‐reflow phenomenon following ischaemia had a similar distribution, but developed more slowly and eventually involved twice as much of the myocardium (59% after 90 min). Surprisingly, pre‐treatment with adenosine greatly increased (from 14–46%) the extent of no‐reflow after 30 min of ischaemia. Pre‐treatment with Ehna caused a slight reduction (59–43%) in its extent but only after 60 min. Thus the no‐reflow phenomenon which developed in ischaemic myocardium is unlikely to be due to the reduced vasodilatory action of adenosine.

Changes in the Contractile state, fine structure and metabolism of cardiac muscle cells during the development of rigor mortis

SummaryTransmural slices from the left anterior papillary muscle of dog hearts were maintained for 120 min in a moist atmosphere at 37° C. At 15-min intervals tissue samples were taken for estimation of adenosine triphosphate (ATP) and glucose-6-phosphate (G6P) and for electron microscopic examination. At the same times the deformability under standard load of comparable regions of an adjacent slice of tissue was measured. ATP levels fell rapidly during the first 45 min to a relative plateau which was maintained from 45 to 75 min after excision of the heart. During a subsequent further decline in ATP, the mean deformability of myocardium fell from 30 to 12% indicating the development of rigor mortis. Conversely, G6P levels increased during the first decline in adenosine triphosphate but remained relatively steady thereafter. Whereas many of the myocardial cells fixed after 5 min contracted on contact with glutaraldehyde, all cells examined after 15 to 40 min were relaxed. A progressive increase in the proportion of contracted cells was observed during the rapid increase in myocardial rigidity. During this late contraction the cells showed morphological evidence of irreversible injury. These findings suggest that ischaemic myocytes contract just before actin and myosin become strongly linked to maintain the state of rigor mortis.

Factors affecting the development of contraction band necrosis during reperfusion of the isolated isovolumic rat heart

The effects of time, oxygen availability and transmural fibre location on the post-ischaemic development of contraction-band necrosis was investigated in isolated isovolumic rat hearts. Anoxic reperfusion after 60 min of total ischaemia was associated with the slow development of contraction bands (8% of myocytes affected after 20 min), particularly in the circumferentially oriented mid-myocardial muscle fibres. This could be completely prevented by the presence of 3 mM amytal. Oxygenated reperfusion caused a rapid development of contraction bands (6% of myocytes affected after 5 mins) which was significantly (P less than 0.05) increased to 18% of cells affected by 20 mins reoxygenation. The mid-myocardium always contained the greatest number of contraction bands with up to 46% of cells involved after 20 mins of oxygenated reperfusion. In all groups many cells were observed to have disrupted sarcolemmae irrespective of the presence or absence of contraction bands. The predominance of contraction bands in the constrictor muscles encircling the heart suggests that their formation may be influenced by the tension or strain imposed upon myocardial fibres during the ischaemic episodes.