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OBJECTIVES: The relative contribution of oxygen free radicals, disturbances in calcium homeostasis and Na+/H+ exchange in the development of injury in the ischemic/reperfused heart was investigated. The study was designed to assess whether these factors initiate independent mechanisms of injury or, alternatively, they share a common mechanism of toxicity. METHODS: Isolated working rat hearts were subjected to different periods (30-55 min) of global ischemia and then were reperfused for 30 min. We compared the effects of oxygen radical scavengers (10 mM dimethylthiourea, DMTU and 0.6 mM desferrioxamine), inhibitors of Na+/H+ exchange (0.15 mM amiloride and 15 microM dimethylamiloride, DMA) and of 0.1 microM diltiazem, which was used to limit calcium overload, given alone or in combination, on the rate of myocardial injury development (recovery of hemodynamic function, LDH release, incidence of severe arrhythmias and structural integrity of cardiomyocytes were estimated at reperfusion following different periods of ischemia). RESULTS: All interventions studied, when given alone, provided nearly equivalent cardioprotection. DMTU or desferrioxamine when applied in combination with diltiazem provided additive cardioprotection, relatively limited, however, as compared to the remarkable cardioprotection achieved by DMTU or desferrioxamine in combination with amiloride. CONCLUSIONS: All mechanisms studied may contribute in an equal manner to the rate of injury development in the ischemic/reperfused heart. The oxygen free radicals-induced myocardial injury may be partially attributed to some disturbance in intracellular calcium homeostasis, possibly calcium overload, whereas the damaging effect of the Na+/H+ exchange activated upon reperfusion is probably largely related to some other mechanism.

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(1) Electrical stimulation (2 Hz) of guinea-pig hearts, perfused with medium containing 11 mM D-glucose plus 0.1 mM octanoate as substrate, resulted in an increase in the percentage of pyruvate dehydrogenase in the active form (PDHa) from 16 to 68%. (2) Rapid isolation of mitochondria by a technique designed to minimize net loss or gain of Ca2+ revealed an increase in mitochondrial Ca2+ content of the stimulated hearts, as measured with 45Ca (2.74 +/- 0.27 versus 1.37 +/- 0.11 nmol/mg protein; stimulated versus rested). (3) Perfusion of rested hearts with a medium containing a reduced Na+ concentration (20 mM, with the remainder replaced with Li+) also gave increased values of PDHa content (30.9% versus 16% for the normal, physiological medium). This procedure is known to raise cytosol Ca2+ concentrations and would be expected to give mitochondrial Ca2+ loading. (4) These results are consistent with a role of mitochondrial Ca2+ in activating pyruvate dehydrogenase in the intact heart.

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The inhibitory action of caffeine on calcium (Ca2+) release from the sarcoplasmic reticulum (SR) and interference with mitochondrial (Ca2+) fluxes by a mitochondrial uncoupler protonfore CCCP were utilized to define a calcium pool responsible for potentiation of post-rest twitch tension in guinea-pig atria. The Ca2+ fluxes were assessed by means of 45Ca2+. Caffeine and CCCP when applied separately did not affect post-rat 45Ca2+ content. Yet, when they were applied together it was markedly reduced to the resting level. It is concluded that a possible source of contractile Ca2+ may be located in mitochondria and an eventual shift of Ca2+ between mitochondria and the SR seems to be a plausible assumption.

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Examination of influence of divalent cations (Co2+, Ni2+, Mn2+) and organic blockers (verapamil and D600) on calcium efflux from resting mammalian myocardium shows that they either inhibit or increase transiently 45Ca2+ efflux, depending on the site of action. It seems that those agents whose sites of action are limited to the sarcolemma inhibit Ca2+ efflux. Co2+, Ni2+, verapamil and D600 belong to this group. Mn2+ ions which act also apparently on intracellular structures increase transiently Ca2+ efflux. Such a finding illustrates a diversity in mechanisms of action in a group of agents generally classified as calcium channel blockers.

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Calcium shifts accompanying rest and post-rest phenomena in isolated left atrial appendages of guinea-pig heart were investigated by means of isotope 45Ca2+. Experiments were performed under conditions of full equilibration of preparations with isotope-containing solution (at least 45 min) in order to investigate the changes in content of exchangeable Ca, or at short exposure to isotope (2-5 min) in order to measure the excitation-dependent Ca2+ influx. Atria stimulated at the rate of 60/min for 55 min in radioactive solution contained 3.79 +/- 0.21 mM of 45Ca2+/kg of wet weight (w.w.). The rested preparations contained 2.61 +/- 0.23 mM/kg w.w. When the previously stimulated for 45 min atria were allowed to rest for 10 min, their content of 45Ca2+ dropped to 3.01 +/- 0.17 mM/kg w.w. despite continued exposure to this isotope. The first post-rest contraction was by 50% +/- 25% stronger than the control steady-state beats. Contractile force (CF) decreased during the two following beats to 25% of control. Calcium-45 content dropped during these beats to 2.50 +/- 0.14 mM/kg w.w. whereas influx of 45Ca2+ amounted to 0.24 mM/kg w.w. CF recovered to control values during 10 min of the following stimulation. Calcium-45 content reached at this time 4.09 +/- 0.12 mM/kg w.w. It is proposed that there are two intracellular store compartments in guinea-pig atrial muscle. The capacity of one of them is rate-dependent and its Ca2+ is lost at rest. This Ca2+ is partially trapped by the other compartment (presumably sarcoplasmic reticulum) and is released to activate the strong post-rest contraction.

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We studied the pattern of post-rest activation and shifts of 45Ca in the isolated mammalian atrial muscle. The first contraction evoked in the rabbit and guinea-pig atrial muscle after 10 min rest was several times stronger than the steady-state beats at a rate of 60/min. Contractile force (CF) declined to 20-50% of control during the next 1-3 beats and recovered to the pre-rest level upon subsequent stimulation. The post-rest beats were negligibly affected by noradrenaline (NA), isoproterenol (IS) acetylcholine (ACh), carbachol (CCh) and Ni, whereas the steady-state beats were readily affected by all these drugs or ions. Post-rest potentiation was completely inhibited by caffeine in a concentration of 10 mM. The guinea-pig atria, equilibrated for 60 min in 45Ca containing solution and stimulated at a rate of 60/min, contained 4.47 +/- 0.16 mmol 45Ca/kg wet weight (w.w.). Ten min of rest resulted in a drop of this content to 3.52 +/- 0.13 mmol/kg w.w. despite the continued presence of 45Ca in the superfusing solution. Three initial post-rest beats resulted in further drop of the content of 45Ca to 2.62 +/- 0.14 mmol/kg w.w. Continued post-rest stimulation resulted in a recovery of the pre-rest 45Ca content. This recovery was inhibited by ACh and CCh. Both drugs inhibited 45Ca loss during the initial 3 beats. Neither this loss nor recovery were affected by IS. It is concluded that calcium (Ca) fraction described in the previous papers [11, 15] as Ca2 in the guinea-pig ventricular muscle plays an important role in the force-frequency relations also in the atrial muscle. However, unlike in ventricular muscle only about half of it is released from the cells upon rest whereas the remaining Ca is taken up by the release compartment and used to activate the strong post-rest contraction. It is thereafter extruded from the cells which results in severe depletion of intracellular Ca stores. Fraction Ca2 is re-accumulated during the post-rest stimulation resulting in recovery of the contractile force.

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The rest- and excitation-dependent shifts of Ca and 45Ca in the isolated, perfused ventricles of guinea-pig hearts were investigated. As much as 50% of the total Ca content (2.2 mmol/kg ww) found in the ventricular muscle stimulated at a steady rate of 60/min, was released into perfusate during 4 min of rest. In the preparations perfused with 45Ca containing solution during the 4 min of rest or during the last 20 s of rest only, a single beat resulted in extra uptake of 0.359 and 0.287 mmol of labelled calcium (45Ca) per kg ww, respectively. Single post-rest excitation evoked in the ventricles which were previously perfused with radioactive solution for 64 min, resulted in increase in tissue 45Ca content by 0.229 mmol/kg ww. In these preparations, the gain in 45Ca is equivalent to the net Ca uptake. Continued post-rest stimulation at the rate of 60/min resulted in recovery of pre-rest content of 45Ca and of total Ca. Gain of 45Ca was paralleled by recovery of contractile force. Uptake of 45Ca in the preparations stimulated at the steady rate of 60/min was 0.137 mmol/kg ww and its value did not depend on the number of beats during exposure to the isotope. Thus 45Ca uptake over a number of steady-state beats may be regarded as equal to the uptake in a single beat. This uptake is by orders of magnitude larger than reported previously by other authors. It is proposed that contraction is triggered by Ca influx into the excited cells (Ca1), and that the response of contractile proteins to this trigger is controlled by a large intracellular Ca2 fraction whose volume is rate-dependent.

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Isolated perfused guinea pig heart ventricles were rendered quiescent by means of atrioventricular block and elevation of perfusate potassium concentration to 7.4 mM. Muscles were exposed to 45Ca2+ during 3 min of rest followed by a single or multiple excitations. Tissue analysis after a half minute of nonradioactive perfusion showed that the 45Ca content had increased after a single excitation by 0.183 mmol above control unstimulated muscles. The difference in calcium uptake between stimulated and unstimulated muscle was not decreased by exposure to 2 mM lanthanum during washout of 45Ca. No difference in uptake of radioactivity between the stimulated and control muscles was found if 63Ni2+ in a concentration 0.001 mM was used instead of 45Ca2+. The 45Ca uptake was strongly inhibited by 2 mM Ni2+ applied during exposure to this isotope. When the experimental muscles were subjected to rhythmic postrest stimulation, most of the 45Ca was taken up during the initial three beats, mostly during the first. A single excitation applied during washout of radioactivity resulted in rapid decrease in 45Ca content in the experimental muscles. It is concluded that about 0.18 mmol of Ca diffuses into myocardial cells at a single postrest excitation. Excitation-dependent extrusion of Ca2+ is postuated.

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The effect of acetylcholine (ACh) on the efflux of 45Ca from atrial myocardial tissue was studied on left guinea-pig auricles. ACh at concentrations of 10(-7)-10(-6) g/ml abolished the twitch responses and caused development of week transient contracture in non-stimulated preparations. The extracellular space (ECS) was not affected. ACh at concentrations of 10(-8)-10(-6) g/ml increased 45Ca efflux from quiescent preparations in a dose-dependent manner. This effect was antagonized by atropine. The contracture developed in Ca2+, Na+-free medium was further enhanced by ACh at concentrations of 10(-7)-10(-6) g/ml. These results suggest that ACh causes the release of calcium from atrial tissue. This effect may contribute to the mechanism of the negative inotropic action of ACh.

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Effect of Ni, Co and Mn ions and of D-600 on rested state contractions (RSCs) of cat and rabbit papillary muscles and on post rest contractions (PRCs) of cat and rabbit atrial muscle was investigated. Ni and Co in concentration 2 mmol/l reduced the force of RSCs in papillary muscles by 78%, increased the dip between the phase 1 and 2 of respective action potentials (APs), lowered the level of plateau and decreased the total duration of AP. Strong PRCs in atrial muscle were reduced by Ni only by 25%, while the subsequent beats were strongly inhibited. Ni completely inhibited RSCs in papillary muscles increased by caffeine (10 mmol/1) or by reduction in Na concentration by 50%. Mn ions in concentration 1--2 mmol/l inhibited strongly both RSCs in papillary muscles and strong PRCs in atrial muscle, increased the dip between phase 1 and 2 and increased total duration of AP. Mn ions stimulated 45Ca efflux from papillary muscles and atrial strips. D-600 did not affect RSCs nor the shape of respective APs. It is concluded that RSCs in papillary muscles are directly activated by Ca inflow during the respective excitation while strong PRCs in atrial muscle are activated mostly by Ca released from intracellular stores.

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