Improved functional recovery of ischemic myocardium by suppression of adenosine catabolism.

Isolated working rat heart preparations were used to ascertain whether the addition of adenosine and prevention of its catabolism could aid in the functional recovery of hearts following global ischemia. Hearts were infused with either 80 micro M EHNA (an adenosine deaminase inhibitor) or 20 micro M adenosine and EHNA in either normal (2.4 mM) or low (0.05 mM) calcium-containing buffer prior to clamping of the aorta for 30 minutes. In one series of hearts, postischemic concentrations (mumoles/gram wet weight) of adenosine triphosphate (ATP), diphosphate (ADP), and monophosphate (AMP), adenosine, inosine, and hypoxanthine were measured; in another series, the recovery of aortic flow rate was used as a measure of functional recovery of ventricular muscle. With normal electrolyte balance, EHNA was unable to protect hearts against ATP loss and ventricular failure. Hearts with EHNA + adenosine recovered 14% of preischemic aortic output and ATP levels were slightly elevated at 0.93 mumole/gm. Those treated with either EHNA or EHNA + adenosine in low-calcium buffer recoverd 100% of their original aortic output. However, EHNA + adenosine maintained considerably higher ATP levels (1.57 mumoles/gm) than did EHNA alone (1.14 mumoles/gm) and was associated with faster initial recovery of aortic output. Thus the prevention of adenosine catabolism was insufficient for adequate ventricular recovery unless the tissue ATP was maintained above about 1.0 mumole/gm. EHNA + adenosine in a 0.05 mM Ca++ infusion solution conserved ATP, markedly improved the functional recovery of hearts, and thus may have a role to play in myocardial preservation during elective cardiac arrest.

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

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 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.

The electron microscopic localization of nephrotoxic antibodies in isolated glomeruli.

Scanning electron microscopy showed that most glomeruli isolated by sieving from normal and nephrotoxic rats were cleanly decapsulated and undisrupted. An anti-IgG antibody-horesradish peroxidase conjugate was applied to such isolated glomeruli and also to slices of renal cortex which were sebsequently embedded in epoxy resin. Linear staining along the glomerular basement membranes of nephrotoxic rats was evident and subendothelial electron-dense deposits were shown to contain anti-glomerular basement membrane antibody. Whereas the linear reaction was faint and limited to superficial parts of glomeruli in tissue slices, it was intense and present in most regions of all of the isolated glomeruli. Thus, the fine details of the distribution of intraglomerular antibody are more clearly and consistently demonstrated in isolated glomeruli than in kidney slices.

An association between the onset of rigor and loss of vascular competence in early myocardial infarcts.

Experimental myocardial ischaemia was induced in 12 anaesthetized mongrel dogs by ligation of the circumflex branch of the left coronary artery. Twenty minutes after ligation 1% sodium fluorescein injected into the artery distal to the ligature evenly perfused the left ventricular wall in the vicinity of the posterior papillary muscle (PPM) but, when injected 90 minutes after ligation, the PPM and adjacent subendocardial myocardium was not perfused by this tracer. Measurements with a linear variable transducer and standard load demonstrated that after 20 minutes ischaemia, the PPM had a similar percentage compressibility to the corresponding unaffected anterior papillary muscle but, after 90 minutes, the compressibility of the PPM was significantly reduced. Scanning electron microscopy of the marginal zone between the perfused and unperfused parts of the myocardium revealed many collapsed vessels which contained small groups of tightly packed erythrocytes indicating that the loss of vascular competence was probably due to the plugging of small vessels by erythrocytes.

The effect of rigor mortis on the passage of erythrocytes and fluid through the myocardium of isolated dog hearts.

The effect of normal and artificially induced rigor mortis on the vascular passage of erythrocytes and fluid through isolated dog hearts was studied. Increased rigidity of 6-mm thick transmural sections through the centre of the posterior papillary muscle was used as an indication of rigor. The perfusibility of the myocardium was tested by injecting 10 ml of 1% sodium fluorescein in Hanks solution into the circumflex branch of the left coronary artery. In prerigor hearts (20 minute incubation) fluorescein perfused the myocardium evenly whether or not it was preceded by an injection of 10 ml of heparinized dog blood. Rigor mortis developed in all hearts after 90 minutes incubation or within 20 minutes of perfusing the heart with 50 ml of 5 mM iodoacetate in Hanks solution. Fluorescein injected into hearts in rigor did not enter the posterior papillary muscle and adjacent subendocardium whether or not it was preceded by heparinized blood. Thus the vascular occlusion caused by rigor in the dog heart appears to be so effective that it prevents flow into the subendocardium of small soluble ions such as fluorescein.

The effect of ischaemia on the function and fine structure of the microvasculature of myocardium.

Seventeen mongrel dogs were subjected to ligation of the circumflex branch of the left coronary artery. At intervals from 10 to 300 minutes after ligation, 10 ml of a sodium fluorescein and colloidal thorium hydroxide solution were injected via an intra-arterial catheter placed distal to the ligature. Up to 20 minutes after ligation this tracer solution evenly perfused the left ventricular wall including the posterior papillary muscle (PPM). During 25 to 50 minutes there was a progressive patchy loss of perfusion into the PPM although the sub-epicardial region was evenly perfused. After 60 or more minutes of ischaemia it was not possible to inject this tracer solution into the subendocardial region. Electron microscopic examination indicated that this loss of vascular competence was possibly due to vasospasm of some vessels in the marginal zone between the perfused and non-perfused areas and was unlikely to be due to intravascular thrombosis, endothelial cell swelling, or to compression of vessels due to cell or tissue oedema.

The potassium/sodium ratio in the demonstration of sudden ischemic cardiac death: a critical appraisal.

The practical value of measuring the ratio of potassium ion (K+) to sodium ion (Na+) in myocardium as an indicator of early inapparent infarction in sudden cardiac death was assessed using a series of 29 human hearts from selected coroner's autopsies together with experimental material from dogs, including infarcts of 5 min to 4 h duration. Samples for electrolyte analysis were derived from a transverse slice of each heart, taken through both ventricles midway between base and apex, all slices being completely subdivided into a numbered sequence of blocks. Ratios were mapped and compared with macroscopic enzyme staining and histological stains for injured muscle. Detailed examination of coronary arteries was performed on all human cases. Measurement of the K+/Na+ ratio did not detect all human cases of proven acute coronary occlusion and did not unequivocally demonstrate experimental infarcts less than 2 h old. Moreover, all ratios fell with increasing duration of autolysis, emphasizing the need for multiple sampling so that each heart may serve as its own control. As a routine test, therefore, the method is both impracticable and unreliable and as previously used has been subject to misinterpretation.

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.

Myocardial adenine pool depletion and recovery of mechanical function following ischemia.

The loss of nucleotide pool precursors from the heart during ischemia and reperfusion may affect resynthesis of ATP and consequently mechanical recovery. Isolated working rat hearts were made globally ischemic for from 15 to 25 min, and the tissue content of adenine pool metabolites, creatine, creatine phosphate (CP), and inorganic phosphate (Pi), were measured after 20 min of reperfusion. In addition, the coronary effluent was assayed for nucleotides, nucleosides, and oxypurines. Hearts that recovered 75% or more of their preischemic hemodynamic function had significantly lower ATP and NAD but greater CP and Pi than controls. Complete failure of hearts was associated with severely depleted ATP but not CP. All hearts released 25% or more of their preischemic adenine pool during the 20-min reperfusion. This loss correlated more closely with a reduction in recovery from 100 to 75% than with complete failure. Thus extensive loss of adenine pool precursors is not critically related to the failure of heart muscle to recover function but may be an important limiting factor in determining the extent and time course of mechanical recovery.

Regional variations in the "adenine/oxypurine" pool of the heart in normoxia and oxygen deficiency.

The regional distribution of energy-related metabolites was determined for the normal dog heart by using high performance liquid chromatography to analyse the levels of ATP, ADP, AMP, IMP, adenosine, inosine, hypoxanthine/xanthine, uric acid and NAD in tissue samples from 7 defined areas of the myocardium and 2 of the conduction system, and calculating the overall concentration of these metabolites in each area. From 8 of the 20 hearts used, additional samples were taken and allowed to undergo autolysis in vitro at 37 degrees C for up to 2 h before being analysed in the same manner. The total concentration of the metabolites assayed ("adenine/oxypurine" pool) did differ from one area to another, most notably between ventricles (left = 6.08, right = 5.93, apex = 6.08 microM/g wet tissue weight) and atria (left wall = 4.44, left appendage = 4.12, right wall = 4.34, right appendage = 3.57 microM/g wet tissue weight), but for each area remained essentially constant during the period of autolysis studied.

Freeze-drying from tertiary butanol in the preparation of endocardium for scanning electron microscopy.

The scanning electron microscope appearances and shrinkage of blocks of canine endocardium prepared by freeze-drying directly, by freeze-drying after replacing tissue water with tertiary butanol (2-methyl propan-2-ol) and by critical point drying were compared. All three methods demonstrated endothelial cells which showed nuclear prominences, microvilli and intercellular boundaries. The microvilli varied in size and number from dog to dog but were generally less well defined in specimens freeze-dried from water. Shrinkage due to t-butanol dehydration was significantly less than that which occurred in ethanol in the critical point drying method. Overall the reduction in surface area was significantly less in specimens freeze-dried directly at -65 C (6.8%) than in those dried from t-butanol at -20 C (15.4%) and those prepared bly critical point drying (22.1%). However the amount of shrinkage observed in t-butanol treated tissue was not significantly different from that which was critical point dried. It was not possible to distinguish between comparable samples prepared by these two methods on the basis of their scanning electron microscopic appearances. Thus the relative simplicity and convenience of the t-butanol method, together with its saving of time, its use of standard freeze-drying equipment and the avoidance of ice-crystal artefact justify its consideration as an alternative method of preparing wet biological tissue for scanning electron microscopy.

Comparative biochemistry and fine structure of atrial and ventricular myocardium during autolysis in vitro.

In a previous study we found that the development of fine structural alteration in atrial myocardium made ischaemic in vivo was slower than has been observed for ventricular myocardium. To explore possible reasons for this, parallel samples of atrial (A) and ventricular (V) myocardium undergoing autolysis (ischaemic necrosis) in vitro at 37 degrees C were studied for up to 2 hours. At 15-minute intervals tissue was snap-frozen for measurement of pH, lactate, and adenine metabolites by HPLC. In half the experiments comparable specimens were taken for electron microscopic examination as well. Fine structural alteration developed less uniformly and more slowly in A than in V. The most striking metabolic differences between A and V were: A had a consistently higher tissue pH and lower lactate level The sum of the adenine + hypoxanthine metabolites was essentially constant but significantly different for each (A = 5.04 +/- 0.12 (s.e.m.), V = 7.71 +/- 0.15 (s.e.m.) mumol/g wet tissue weight) Initial ATP levels were lower (40% less) in A The maximum accumulation of AMP was higher in A, despite its smaller pool of adenine metabolites Both adenosine and inosine showed slower rates of change in A. These results suggest that during early, severe ischaemic injury A and V show differing activities of 5'-nucleotidase.