Cardiac dysfunction in isolated perfused hearts from spontaneously diabetic BB rats

The purpose of this investigation was to examine cardiac function and biochemistry in spontaneously diabetic BB rats, a strain in which diabetes occurs spontaneously and closely resembles insulin-dependent diabetes in humans. The study involved two groups: nondiabetic littermates of BB rats and BB diabetic rats treated daily with a very low insulin dose such that the rats were severely hyperglycemic and hyperlipidemic. The hearts from these two groups were isolated and heart function (using isolated perfused working hearts) and biochemistry were examined 6 weeks after the onset of diabetes. BB diabetic rats exhibited a lower calcium-stimulated myosin ATPase activity and depressed left ventricular developed pressure, cardiac contractility, and ventricular relaxation rates compared with BB nondiabetic littermates. These results suggest that the chronically diabetic state in the BB rat produces cardiac changes similar to those demonstrable after chemical diabetes induced by alloxan or STZ, or that seen during human diabetes mellitus.     

Cardiac anaphylaxis in isolated guinea pig hearts perfused at constant flow or constant pressure

Acute responses to antigen-antibody interactions (anaphylactic reactions) in isolated guinea pig hearts are reported to include decreases in coronary flow, increases in heart rate, prolongation of impulse propagation, development of arrhythmias, and transient increases followed by substantial decreases in ventricular contractile force. It is not clear from these studies, however, whether all of the changes are direct effects of the mediators released by the antigen-antibody reaction or whether some of them are indirect results of the severe reduction in flow evoked by coronary vasoconstriction. Therefore, the present study was designed to assess cardiac anaphylactic events in isolated hearts of guinea pigs passively sensitized with IgG antibody to ovalbumin under conditions in which coronary perfusion pressure was maintained constant and to compare the responses to those of hearts in which coronary flow was maintained at a constant rate. Our data indicate that when coronary flow decreased during anaphylaxis (constant pressure perfusion), hearts responded to antigen challenge with greater prolongation of the PR interval, duration of arrhythmias, suppression of left ventricular systolic pressure, and release of histamine and adenosine plus inosine into the venous effluent than when coronary flow was maintained during anaphylaxis (constant flow perfusion). The data suggest that maintenance of coronary flow during cardiac anaphylaxis may attenuate the severity of the functional derangement.     

Differential electrophysiologic effects of global and regional ischemia and reperfusion in perfused rat hearts. Effects of Mg2+ concentration

The effects of regional and global ischemia on cellular electrical activity and on arrhythmias induced by reperfusion were studied at different Mg²⁺ concentrations (Mg²⁺ _o, 0, 1.2, and 4.8 mM) in perfused rat hearts. Surface electrograms and transmembrane potentials were recorded during control, 10 min of ischemia (perfusion arrest or coronary ligation), and reperfusion. Increasing Mg²⁺ _o from 0-4.8 mM decreased heart rate, did not alter action potential morphology, and had a strong antiarrhythmic action on reperfusion following coronary ligation. At low and normal Mg²⁺ _o, the incidence of tachyarrhythmias was between 70 and 80%. Global ischemia led to progressive atrioventricular block and the final ventricular beating rate was similar at all Mg²⁺ _o despite unequal initial values. The severity of arrhythmias was similar to that found after regional ischemia in Mg²⁺ _o = 0, but much lower at normal and high Mg²⁺ _o. The resting depolarization induced by coronary ligation decreased as Mg²⁺ _o was raised, but such a relation was not seen during global ischemia where the depolarization was less marked. The action potential duration did not vary with the ventricular rate between 160 and 380 beats per min but increased considerably when sinus rate was markedly slowed (40 to 80 bpm) by raising Mg²⁺ _o to 9.6 mM. Our data show that a high Mg²⁺ _o exerts a strong protection against reperfusion arrhythmias regardless of the type of ischemia. Modulation of the sinus rhythm by Mg²⁺ may contribute to its protective effect by decreasing K⁺ _o accumulation and Na⁺ _i loading during ischemia.     

Influence of extracellular potassium on the antiarrhythmic effect of global preconditioning in isolated perfused rat hearts

The objective of this study was to investigate if a variation in extracellular-K+ concentrations alters the effects of global preconditioning on ischemia-induced arrhythmias. Rat hearts were Langendorff-perfused with Krebs-Henseleit solution and randomised in 8 groups (n = 12/group): four control groups (K⁺: 2, 4, 6, or 8 mmol/L) which underwent 30-min coronary artery occlusion and four preconditioned groups (K⁺: 2, 4, 6, or 8 mmol/L) in which the 30-min regional ischemia was preceded by 2 cycles of 3 min global ischemia. In the presence of low K⁺ (2 mmol/L), there were no differences between control and preconditioning groups in the number of ventricular premature beats (VPBs): 194 ± 64 vs. 217 ± 81, the incidence of ventricular tachycardia (VT): 100% vs. 100% and of ventricular fibrillation (VF): 100% vs. 100%. In the presence of normal K⁺ concentration (4 mmol/L), ischemic preconditioning reduced the number of VPBs from 88 ± 26 to 25 ± 10, (p < 0.05), the incidence of VT from 100 to 50% (p < 0.05), and of VF from 67 to 16% (p < 0.05). In the condition of higher K⁺ concentration (6 mmol/L), VPBs (34 ± 8 vs. 11 ± 4), the incidence of VT (100% vs. 25%; p < 0.05 ) and VF (25% vs. 8%) were further reduced in preconditioned hearts. In the condition of K⁺ concentration (8 mmol/L), there were no differences in VPBs (11 ± 3 vs. 7 ± 2), the incidence of VT (8% vs. 0%) and VF (8% vs. 0%) between control and preconditioned hearts. Our data show that ischemic preconditioning affords protection against arrhythmias during coronary artery occlusion in the isolated rat heart and that hypokalemia abolishes the antiarrhythmic effects of global preconditioning.     

Myocardial triglyceride turnover during reperfusion of isolated rat hearts subjected to a transient period of global ischemia.

Triglyceride turnover in reperfused/ischemic rat hearts was investigated. Hearts were initially perfused under aerobic conditions for a 1-h "pulse" perfusion with 1.2 mM [1-14C]palmitate to label the endogenous lipid pools, followed by a 30-min period of no-flow ischemia or a 10-min period of retrograde perfusion (control). Hearts were then reperfused under aerobic conditions with buffer containing 1.2 mM [9,10-3H]palmitate. All buffers contained 11 mM glucose and 500 microunits/ml insulin. Rates of endogenous triglyceride lipolysis and synthesis were measured during reperfusion, whereas rates of exogenous palmitate oxidation were measured both prior to ischemia and during reperfusion following ischemia. During reperfusion of ischemic hearts, a 20% increase in exogenous fatty acid oxidation rates was seen compared with pre-ischemic rates. Despite an initial burst of endogenous fatty acid oxidation, no acceleration of steady state endogenous triglyceride lipolysis was seen compared with their nonischemic hearts. In contrast, a significant increase in triglyceride synthesis was observed. Triglyceride turnover was also measured in a series of hearts reperfused following ischemia in the absence of exogenous fatty acids. A significant enhancement of functional recovery was seen compared with hearts reperfused with 1.2 mM palmitate. In addition, a significant increase in fatty acid oxidation from endogenous triglyceride lipolysis was observed. We conclude that the heart quickly recovers its ability to oxidize exogenous fatty acids during reperfusion and that although triglyceride lipolysis is not accelerated during reperfusion of ischemic hearts in the presence of 1.2 mM palmitate, a significant increase in triglyceride synthesis does occur.     

4-Aminopyridine: Effects on electrical activity during ischemia and reperfusion in perfused rat hearts

We investigated the effects of 2 and 4 mM 4-aminopyridine (4-AP, – blocker of the transient outward current I_to) on the electrophysiological response to regional ischemia and reperfusion. Spontaneously beating rat hearts were subjected to coronary occlusion (10 min) followed by reperfusion. The surface electrogram and the membrane potential from subepicardial left ventricular cells were recorded throughout. The basal effect of 4-AP was a dose dependent increase in the action potential duration (APD₉₀) without changes in the resting potential or the heart rate. During early ischemia resting depolarization (from 87.4 ± 1.9–70.1 ± 2.5 mV in the controls) was enhanced by 4 mM, 4-AP (84.3 ± 1.4 mV vs. 61.7 ± 1.3 mV) whereas APD₉₀ increased by 73.5%. These effects resulted in a marked reduction in the duration of diastolic intervals that led to conduction failure and aborted responses. A partial recovery was found by the end of ischemia concomitant with APD₉₀ shortening in both, control and 4-AP treated hearts. On reperfusion, 4-AP did not influence the initial incidence of ventricular tachyarrhythmias but decreased their duration from 531.5 ± 56.3–260.7 ± 100 sec (2 mM) and to 75.6 ± 10.5 sec (4 mM). These data confirm others obtained by Henry et al. [11] in isolated cells indicating that ischemia induces sequential changes in several K⁺ conductances. In addition, they show that changes in action potential characteristics may exert beneficial effects on reperfusion arrhythmias by acting on the arrhythmic substrate without suppressing the trigger mechanism.     

Electrophysiological effects of ethmozine in perfused rabbit hearts

His-bundle electrocardiography was used to evaluate the effects of ethmozine on cardiac conduction in isolated perfused rabbit hearts electrically driven at cycle lengths of 320 and 250 ms. There was no significant change in conduction until high concentrations of ethmozine were reached. His-Purkinje and atrioventricular (AV) nodal conduction were slowed significantly at 0.1 microgram/mL and atrial conduction at 1.0 microgram/mL. Conduction block occurred at 10.0 micrograms/mL in all the hearts treated. Effects of the drug (0.1 and 0.01 microgram/mL) on conduction of extrasystoles were also studied in hearts driven at a basic cycle length of 270 ms. No significant change was observed in atrial conduction of extrasystoles throughout the coupling intervals tested at both concentrations. Ethmozine (0.01 and 0.1 microgram/mL) caused slowing of His-Purkinje conduction of extrasystoles but the effect of the drug did not change as a function of the coupling interval. An interval-dependent increase in AV-nodal conduction time was observed, with the maximum slowing of conduction occurring at coupling intervals close to the effective refractory period of the AV node. AV-nodal functional refractory period was increased significantly by ethmozine (0.01 and 0.1 microgram/mL). The effective refractory period was significantly increased only at the higher concentration.     

Synthesis of stress-induced protein in isolated and perfused rat hearts

Isolated and perfused rat hearts were examined by two-dimensional gel electrophoresis and liquid scintillation counting for alterations in protein synthesis following incubation with L-[3H]leucine at 0.5-2.5, 2.5-4.5, or 4.5-6.5 h of perfusion. When 35-mL volumes of three different buffers were recycled for a 2-h period from 0.5 to 2.5 h, by fluorography little effect was seen on the normal patterns of protein synthesis and there was a moderate synthesis of a stress-induced protein (heat-shock protein) with a molecular mass of 71 X 10(3) daltons (SP71). However, hearts perfused with Krebs-improved Ringer 1 bicarbonate had the highest incorporation of L-[3H]leucine. When buffers were recycled for 30-min periods from 0.5 to 2.5 h, SP71 was synthesized in hearts perfused with Krebs-Henseleit original Ringer bicarbonate. Hearts perfused in a similar fashion with Krebs-improved Ringer 1 bicarbonate had the lowest incorporation of label into SP71 and in fact SP71 was undetectable on fluorograms. Overall protein synthesis was decreased and the ratio of SP71 to the total synthesis was increased at 4.5-6.5 h of perfusion when 35-mL volumes of Krebs-improved Ringer 1 bicarbonate was recycled for 2-h periods. A similar result was observed at 2.5-4.5 h of perfusion when this buffer was recycled for either the duration of the experiment or 30-min periods.     

Modification of basal release of prostaglandins from rabbit isolated hearts

Rabbit isolated hearts, perfused through the coronary circulation with Krebs' solution continuously release a prostaglandin-like substance (PLS) into the effluent. The basal release was decreased by fibrillation, increased by mechanical disturbance, anoxia or arterial emboli and abolished by indomethacin. We conclude that mechanical distortion and trauma are important stimuli for prostaglandin release in the heart.     

Myocardial actions of prostaglandins in isolated cat cardiac tissue.

The inotropic responses to prostaglandins (PG) A₁, E₁, E₂ and F_2α were studied in isolated cat myocardial tissue. PGA₁ and F_2α exhibited no significant inotropic effects, whereas, PGE₂ and PGE₁ produced negative inotropic effects at concentrations of 2.8 × 10⁻⁷ and 2.8 × 10⁻⁶ M in isolated cat papillary muscles.In isolated perfused cat hearts, PGE₁ (2.8 × 10⁻⁶M) produced a negative inotropic effect along with a significant increase in coronary flow. As flow declined, the negative inotropic effect became more severe. PGE₁ at 2.8 × 10⁻⁹ M produced a sustained increase in coronary flow and oxygen consumption with no inotropic effect. PGE₂ and F_2α did not exert significant changes in coronary flow or contractile force.Thus prostaglandins do not appear to exert significant positive inotropic effects at physiologic or at generally accepted pharmacologic concentrations in isolated cat heart preparations. At extremely high concentrations, prostaglandins E₁ and E₂ exert a negative inotropic effect; however, this would not explain the protective effect of these prostaglandins in circulatory shock.     

Pharmacologic protection of perfused rat heart against global ischemia

Using a Langendorff rat heart model, studies were performed on the effects of three drugs in protecting the heart against global ischemia. The drugs used were: (a) MR-256, a prostaglandin oligomeric derivative, which is a calcium chelating agent and at the same time, is an inhibitor of phospholipase A2 activity, (b) chlorpromazine which is not a calcium chelator, but is a calmodulin antagonist and is an inhibitor of phospholipase A2 activity, and (c) BAPTA/AM, a calcium chelating agent, but which is not an inhibitor of phospholipase A2 activity. The perfused heart was exposed to 15 minutes of global ischemia. In control experiments (no drug), the ventricular pressure recovered to 26.4 +/- 6.7% (n = 22) of the original level. With pretreatment of (a) MR-256 (b) chlorpromazine, and (c) BAPTA/AM, maximum recoveries were 0.5 +/- 6.7% (n = 5), 88.7 +/- 8.5% (n = 5), 45.3 +/- 26.6% (n = 5), respectively. MR-256 and chlorpromazine were found to react with free radicals. The modes of action of these three different types of drugs are discussed.     

Isolated perfused rat hearts release secondary free radicals during ischemia reperfusion injury: Cardiovascular effects of the spin trap α-phenyl N-tert-butylnitrone

Free radicals produced during myocardial post-ischemic reperfusion are aggravating factors for functional disturbances and cellular injury. The aim of our work was to investigate the significance of the secondary free radical release during non ischemic perfusion and post-ischemic reperfusion and to evaluate the cardiovascular effects of the spin trap used. For that purpose, isolated perfused rat hearts underwent 0, 20, 30 or 60 min of a total ischemia, followed by 30 min of reperfusion. The spin trap: α-phenyl N-tert-butylnitrone (PBN) was used (3 mM). Functional parameters were recorded and samples of coronary effluents were collected and analyzed using Electron Paramagnetic Resonance (EPR) to identify and quantify the amount of spin adducts produced. During non ischemic perfusion, almost undetectable levels of free radical release were observed. Conversely, a large and long-lasting (30 min) release of spin adducts was detected from the onset of reperfusion. The free radical species were identified as alkyl and alkoxyl radicals with amounts reaching 40 times the pre-ischemic values. On the other hand, PBN showed a cardioprotective effect, allowing a significant reduction of rhythm disturbances and a better post-ischemic recovery for the hearts which were submitted to 20 min of ischemia. When the duration of ischemia increased, the protective effects of PBN disappeared and toxic effects became more important. Our results have therefore confirmed the antioxidant and protective properties of a spin trap agent such as PBN. Moreover, we demonstrated that the persistent post-ischemic dysfunction was associated with a sustained production and release of free radical species.     

Isolated perfused rat hearts release secondary free radicals during ischemia reperfusion injury: cardiovascular effects of the spin trap alpha-phenyl N-tert-butylnitrone.

Free radicals produced during myocardial post-ischemic reperfusion are aggravating factors for functional disturbances and cellular injury. The aim of our work was to investigate the significance of the secondary free radical release during non ischemic perfusion and post-ischemic reperfusion and to evaluate the cardiovascular effects of the spin trap used. For that purpose, isolated perfused rat hearts underwent 0, 20, 30 or 60 min of a total ischemia, followed by 30 min of reperfusion. The spin trap: alpha-phenyl N-tert-butylnitrone (PBN) was used (3 mM). Functional parameters were recorded and samples of coronary effluents were collected and analyzed using Electron Paramagnetic Resonance (EPR) to identify and quantify the amount of spin adducts produced. During non ischemic perfusion, almost undetectable levels of free radical release were observed. Conversely, a large and long-lasting (30 min) release of spin adducts was detected from the onset of reperfusion. The free radical species were identified as alkyl and alkoxyl radicals with amounts reaching 40 times the pre-ischemic values. On the other hand, PBN showed a cardioprotective effect, allowing a significant reduction of rhythm disturbances and a better post-ischemic recovery for the hearts which were submitted to 20 min of ischemia. When the duration of ischemia increased, the protective effects of PBN disappeared and toxic effects became more important. Our results have therefore confirmed the antioxidant and protective properties of a spin trap agent such as PBN. Moreover, we demonstrated that the persistent post-ischemic dysfunction was associated with a sustained production and release of free radical species.