Hyperkalemic normothermic hypoperfusion of the isolated heart

It is established that both normothermic hypoperfusion of the heart by cardioplegic solution and pharmacocardioplegia with cold protect myocardium from the ischemic damage.     

Flow heterogeneity following global no-flow ischemia in isolated rabbit heart

The purpose of this study was to evaluate flow heterogeneity and impaired reflow during reperfusion after 60-min global no-flow ischemia in the isolated rabbit heart. Radiolabeled microspheres were used to measure relative flow in small left ventricular (LV) segments in five ischemia + reperfused hearts and in five nonischemic controls. Relative flow heterogeneity was expressed as relative dispersion (RD) and computed as standard deviation/mean. In postischemic vs. preischemic hearts, RD was increased for the whole LV (0.92 +/- 0.41 vs. 0.37 +/- 0.07, P < 0.05) as well as the subendocardium (Endo) and subepicardium considered separately (1.28 +/- 0.74 vs. 0.30 +/- 0.09 and 0.69 +/- 0.22 vs. 0.38 +/- 0.08; P < 0.05 for both comparisons, respectively) during early reperfusion. During late reperfusion, the increased RD for the whole LV and Endo remained significant (0.70 +/- 0.22 vs. 0.37 +/- 0.07 and 1.06 +/- 0.55 vs. 0.30 +/- 0.09; P < 0.05 for both comparisons, respectively). In addition to the increase in postischemic flow heterogeneity, there were some regions demonstrating severely impaired reflow, indicating that regional ischemia can persist despite restoration of normal global flow. Also, the relationship between regional and global flow was altered by the increased postischemic flow heterogeneity, substantially reducing the significance of measured global LV reflow. These observations emphasize the need to quantify regional flow during reperfusion after sustained no-flow ischemia in the isolated rabbit heart.     

Advantages of perfluorochemical perfusion in the isolated working rabbit heart preparation using 31P-NMR

Quantitative 31P-NMR and enzymatic analysis of high-energy phosphates were used to characterize an isolated perfused working rabbit heart preparation. In this model, the left side of the heart works against a physiological after-load. Two perfusates, Krebs-Henseleit saline and the perfluorocarbon emulsion FC-43 (perfluorotributylamine), were evaluated in their ability to maintain cardiac function and high-energy phosphate metabolites over a period of 2-3 h. Adenine nucleotides ATP, ADP, phosphocreatine and inorganic phosphate (Pi) were measured by 31P-NMR while monitoring cardiac output and coronary flow. Intracellular pH was determined using the chemical shift of Pi. At the end of each experiment, hearts were freeze clamped and enzymatically assayed for adenine nucleotides, phosphocreatine and Pi. In every experiment, hearts perfused with FC-43 emulsion maintained the same rate of cardiac output as hearts perfused with Krebs-Henseleit saline, but with half the coronary flow rate: FC-43, 22 +/- 2.5 (n = 5), Krebs-Henseleit saline 42 +/- 2.7 (n = 6) ml/min, P less than 0.001. Hearts perfused with FC-43 emulsion showed higher [phosphocreatine] and [ATP] measured by 31P-NMR. For [phosphocreatine]: FC-43 3.2 +/- 0.7 (n = 5), Krebs-Henseleit saline 1.7 +/- 0.2 (n = 6) mumol/g wet wt., P less than 0.01. For [ATP]: FC-43 1.8 +/- 0.7 (n = 5), Krebs-Henseleit saline 0.9 +/- 0.2 (n = 6) mumol/g wet wt., P less than 0.02. [phosphocreatine] and [ATP] determined by 31P-NMR values were identical within experimental error to those values obtained by enzymatic analysis. Comparing [Pi] determined by both methods, 36% of Pi in FC-43-perfused hearts, and only 24% of Pi in Krebs-Henseleit saline-perfused hearts were visible by NMR, indicating that a large proportion of Pi is bound in the intact functioning heart. Similar results were obtained for [ADP]. Using the combined techniques of 31P-NMR and enzymatic assay, we have shown in this model of the isolated working rabbit heart preparation, that FC-43 emulsion maintains significantly better function and high-energy phosphate levels than Krebs-Henseleit saline.     

Desferrioxamine reduces susceptibility to lipid peroxidation in rabbit kidneys subjected to warm ischaemia and reperfusion

Rabbit kidneys were clamped and subjected to warm ischaemia for 60 or 120 min then reperfused with blood for 60 min or for 24 hr. Treated rabbits received desferrioxamine at 15 or 50 mg/kg i.v. 15 min before reperfusion. Their kidneys were then removed and assayed for phospholipid Schiff base fluorescence (ex. 360 nm, em. 435 nm), diene and triene conjugates by UV spectrophotometry (240 nm and 268 nm respectively), for superoxide dismutase and for reduced and oxidised glutathione to provide an index of glutathione redox activity. All indices of lipid peroxidation were significantly elevated in untreated rabbits and glutathione redox activity was reduced. Treatment with desferrioxamine however effectively prevented these deviations and in many cases maintained them at the levels in fresh rabbit kidneys. These data provide further evidence that lipid peroxidation occurring during the reperfusion period is superimposed on the damage set up during warm ischaemia and may be preventable by administration of suitable therapeutic agents.     

An improved isolated working rabbit heart preparation using red cell enhanced perfusate

The performance of isolated working rabbit hearts perfused with Krebs-Henseleit (KH) buffer was compared with those in which the buffer was supplemented with washed human red blood cells (KH + RBC) at a hematocrit of 15 percent. When perfused with KH alone at 70 cm H2O afterload and paced at 240 beats/minute, coronary flow was more than double, whereas aortic flow was 40-60 percent of that in hearts perfused with KH + RBC, regardless of left atrial filling pressures (LAFP). Peak systolic pressure reached a plateau at 120 mm Hg in KH + RBC, but at 95 mm Hg in the KH group. Stroke work, however, was similar in the two groups. Despite the high coronary flow, oxygen uptake by hearts perfused with KH was substantially less and did not respond to increases in LAFP as in those perfused with KH + RBC. There was a 20 percent drop in ATP and glycogen content after 90 minutes' perfusion. In contrast, isolated hearts perfused with RBC-enriched buffer remained stable for at least 150 minutes. Irrespective of the perfusate, triacylglycerol content of the muscle remained at similar levels throughout the course of study. Increasing RBC in the perfusate from 15 percent to 25 percent had no additional effect on cardiac performance or oxygen consumption. Our findings demonstrate that in the isolated working rabbit heart inclusion of RBC in the perfusate improves mechanical and metabolic stability by providing an adequate oxygen supply.     

Glutamine cycling in isolated working rat heart

To what extent does glutamine turnover keep pace with oxidative metabolism in the rat heart? To address this question, the following groups of substrates were presented to the isolated, working rat heart: 1) glucose (5 mM), insulin (40 microU/ml), and [2-13C]acetate (5 mM; high workload, n = 5); 2) pyruvate (2.5 mM) and [2-13C]acetate (5 mM; normal workload, n = 5); or 3) propionate (1 mM) and [2-13C]acetate (2.5 mM; normal workload, n = 3). In a subset of these experiments, the exchange of glutamate and glutamine was quantified by separation with ion exchange chromatography and analysis by GC-MS. There was an apparent equilibration of mass isotopomers of glutamate and glutamine after 50 min of perfusion, although the extent of equilibration was not determined. The fractional enrichment in glutamine was 31% of the enrichment of glutamate with the three different perfusates. From high-resolution nuclear magnetic resonance spectra, we found a ratio of glutamine to glutamate content of 94.1, 53.4, and 96.9%, respectively, for each experimental group. In experiments for which l-[1-13C]glutamine (5 mM) was included in the perfusate of group 2, [1-13C]glutamine was detected in the heart, but transfer of 13C from glutamine to glutamate was not detected (n = 4). We conclude that, in the perfused working heart, production of glutamine by amidation of glutamate takes place and can be detected, whereas the reverse process, generation of glutamate from glutamine, remains undetected.     

Duration of ischaemia determines matrix metalloproteinase-2 activation in the reperfused rabbit heart

It has been hypothesised that activation of matrix metalloproteinase-2 (MMP-2) contributes to reversible myocardial dysfunction (stunning) following short-term ischaemia and reperfusion. Gelatin zymography was used to measure release of both pro-MMP-2 (72 kDa) and MMP-2 (62 kDa), into the coronary effluent from isolated, perfused rabbit hearts during 90 min aerobic perfusion (control), or low-flow ischaemia (15 or 60 min at 1 mL/min), followed by 60 min reperfusion. In controls, pro-MMP-2 was detected in the coronary effluent throughout the first 30 min of aerobic perfusion, but MMP-2 was not detected. In contrast, MMP-2 was detected in the coronary effluent during reperfusion after both 15 and 60 min ischaemia. However, while left ventricular systolic function was impaired after both 15 min and 60 min ischaemia, a significant increase in the release of MMP-2 was only detected in hearts following 60 min ischaemia. The dissociation between mechanical function and MMP-2 levels suggest that MMP-2 does not contribute to myocardial stunning in this model, but may contribute to myocardial dysfunction following prolonged ischaemia.     

Selective effects of adenosine receptor agonists upon coronary resistance and heart rate in isolated working rabbit hearts

Dose dependent changes in heart rate (HR) and coronary resistance (CR) were determined for adenosine, 5'-N-ethylcarboxamide adenosine (NECA) and l-N6-phenylisopropyladenosine (l-PIA) over a dose range of 1 X 10(-9) to 1 x 10(-5) M. Changes in CR were determined under controlled metabolic demand conditions (constant mean aortic pressure, constant mean left atrial pressure, and constant HR). Decreases in HR were determined by allowing the paced hearts to beat spontaneously between doses for 15 seconds. Adenosine significantly decreased CR and HR at greater than or equal to 1 X 10(-5) M. NECA significantly decreased both CR and HR at greater than or equal to 3 X 10(-8) M. l-PIA significantly decreased HR at greater than or equal to 3 X 10(-8) M; however a dose at greater than or equal to 3 X 10(-6) M was required to significantly decrease CR. These results provide evidence that the coronary vasodilator action of adenosine may primarily be mediated by A2 receptors. Furthermore, the data are in support of previous observations that the bradycardic action of adenosine is principally mediated via A1 receptors.     

Hormone selective lipase activation in the isolated rabbit heart

The synthesis and release of PGs by the isolated perfused rabbit heart upon bradykinin stimulation results from lipase stimulation which liberates arachidonic acid for PG biosynthesis. The [¹⁴C]-labelled fatty acids, arachidonate, linoleate, and oleate, when infused into the heart preparation, were efficiently incorporated into the phospholipid pool in the heart, mostly in the 2-position of phosphatidylcholine. On the other hand, [¹⁴C]-palmitate was esterified into both the 1- and the 2-position. Bradykinin released bioassayable PG when injected into the rabbit hearts regardless of which fatty acid label was incorporated into the phospholipid pool. However, only [¹⁴C]-arachidonic acid (but not [¹⁴C]-linoleate, oleate or palmitate) was liberated from the variously labelled hearts upon hormone stimulation. This selective bradykinin effect on fatty acid release suggests that hormone stimulation either activates a specific lipase that distinguishes different fatty acids in the 2-position or activates lipase which is selectively compartmented with arachidonate-containing phospholipids. Ischemia, on the other hand, appeared to non-specifically stimulate tissue lipases, resulting in a non-selective release of oleic as well as arachidonic acid. A disproportionally large release of arachidonic acid was observed accompanying a relatively small PG (10:1 arachidonate: PG ratio) production during ischemia, as compared to bradykinin (3:1 ratio), suggesting distinct mechanisms for PG biosynthesis induced by bradykinin and ischemia.This work was supported by NIH grants: SCOR-HL-17646, HE-14397, HL-20787, and Experimental Pathology training grant (WH) 5 TO1 GM00897-16. Address correspondence to Dr. Philip Needleman, Department of Pharmacology, Washington University Medical School, St. Louis, Missouri 63110.     

Atriopeptin release from the isolated perfused rabbit heart

Mammalian atrial extracts have been shown to contain bioactive peptides which exert natruiretic, diuretic, and smooth muscle relaxant effects. These extracts include several low molecular weight (< 5,000 Mr) atrial peptides (atriopeptins) which exhibit identical sequences over a central core region which are derived from the high molecular weight peptide (atriopeptigen) precursor which has been purified and sequenced. In the current study we found that extracts of rabbit atria possess both high and low molecular weight bioactive atrial peptides, however, the coronary venous effluent obtained from the isolated perfused rabbit heart only contained the low molecular weight peptide. This trypsin labile activity causes a dose-dependent relaxation of rabbit aorta and chicken rectum assay strips. Separation of the bioactivity with gel filtration chromatography and reversed phase HPLC indicates the heart releases a single substance similar to atriopeptin III. There was no evidence that atriopeptigen was released from the isolated perfused rabbit heart. We suggest that atriopeptigen is proteolytically processed in the atria to an atriopeptin which is subsequently the released form of the atrial peptide.     

Temperature sensitivity of afferent receptors in the isolated rabbit heart

Changes in the positive chronotropic effects induced by epicardial irrigation with heated Krebs-Henseleit solution were studied in the isolated rabbit heart before and after intracoronary infusion of a ganglionic blocking agent, Arfonad (10 mg/ml). 2-3 minutes after Arfonad infusion the positive chronotropic effects decreased to 37.9% and 5-10 minutes later they returned to control levels. It is concluded that epicardial surface warming causes an increase in afferent receptor activity. It is suggested that neurogenic component of the positive chronotropic effect may be produced through the activation of intracardiac reflectory pathways.     

Utilization of energy-providing substrates in the isolated working rat heart.

1. An improved perfusion system for the isolated rat heart is described. It is based on the isolated working heart of Neely, Liebermeister, Battersby & Morgan (1967) (Am. J. Physiol. 212, 804-814) and allows the measurement of metabolic rates and cardiac performance at a near-physiological workload. The main improvements concern better oxygenation of the perfusion medium and greater versatility of the apparatus. Near-physiological performance (cardiac output and aortic pressure) was maintained for nearly 2 h as compared with 30 min or less in the preparations of earlier work. 2. The rates of energy release (O2 uptake and substrate utilization) were 40-100% higher than those obtained by previous investigators, who used hearts at subphysiological workloads. 3. Values are given for the rates of utilization of glucose, lactate, oleate, acetate and ketone bodies, for O2 consumption and for the relative contributions of various fuels to the energy supply of the heart. Glucose can be replaced to a large extent by lactate, oleate or acetate, but not by ketone bodies. 4. Apart from quantitative differences there were also major qualitative differences between the present and previous preparations. Thus insulin was not required for maximal rates of glucose consumption at near-physiological, in contrast with subphysiological, workloads when glucose was the sole added substrate. When glucose oxidation was suppressed by the addition of other oxidizable substrates (lactate, acetate or acetoacetate), insulin increased the contribution of glucose as fuel for cardiac energy production at high workload. 5. In view of the major effects of workload on cardiac metabolism, experimentation on hearts performing subphysiologically or unphysiologically is of limited value to the situation in vivo.     

Pharmacological evidence for catecholamine-independent contractile effects of X-537A on the isolated working rat heart preparation.

The ionophore X-537A increased heart rate and contractility of the isolated, working rat heart preparation. The increased heart rate appeared to be caused solely by release of catecholamines as the response was completely eliminated by reserpine pretreatment or addition of propranolol to the perfusate. The inotropic response, however, had an apparent catecholamine-independent component as neither propranolol, nor propranol in combination with phentolamine, completely eliminated the inotropic response to X-537A. On the other hand, reserpine pretreatment did abolish the inotropic effect of the ionophore but this action appeared to be a nonspecific one as the responses to norepinephrine and to CaCl2 were substantially diminished.