Positive Inotropic Effects of ATP Released via the Maxi-Anion Channel in Langendorff-Perfused Mouse Hearts Subjected to Ischemia-Reperfusion

The organic anion transporter SLCO2A1 constitutes an essential core component of the ATP-conductive large-conductance anion (Maxi-Cl) channel. Our previous experiments using Langendorff-perfused mouse hearts showed that the Maxi-Cl channel contributes largely to the release of ATP into the coronary effluent observed during 10-min reperfusion following a short period (6 min) of oxygen-glucose deprivation. The present study examined the effect of endogenous ATP released via Maxi-Cl channels on the left ventricular contractile function of Langendorff-perfused mouse hearts, using a fluid-filled balloon connected to a pressure transducer. After the initial 30-min stabilization period, the heart was then perfused with oxygen-glucose-deprived Tyrode solution for 6 min, which was followed by a 10-min perfusion with oxygenated normal Tyrode solution in the absence and presence of an ATP-hydrolyzing enzyme, apyrase, and/or an adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). In the absence of apyrase and DPCPX, the left ventricular developed pressure (LVDP) decreased from a baseline value of 72.3 ± 7.1 to 57.5 ± 5.5 mmHg (n = 4) at the end of 6-min perfusion with oxygen-glucose-deprived Tyrode solution, which was followed by a transient increase to 108.5 ± 16.5 mmHg during subsequent perfusion with oxygenated normal Tyrode solution. However, in the presence of apyrase and DPCPX, the LVDP decreased to the same degree during 6-min perfusion with oxygen-glucose-deprived Tyrode solution, but failed to exhibit a transient increase during a subsequent perfusion with oxygenated normal Tyrode solution. These results strongly suggest that endogenous ATP released through Maxi-Cl channels contributes to the development of transient positive inotropy observed during reperfusion after short-period hypoxia/ischemia in the heart.

O2 consumption of mechanically unloaded contractions of mouse left ventricular myocardial slices

Left ventricular (LV) myocardial slices were isolated from murine hearts (300 μm thick) and were stimulated at 1 Hz without external load. Mean myocardial slice O2 consumption (MVo2) per minute (mMVo2) without stimulation was 0.97 ± 0.14 ml O2·min−1·100 g LV−1 and mean mMVo2 with stimulation increased to 1.80 ± 0.17 ml O2·min−1·100 g LV−1 in normal Tyrode solution. Mean ΔmVo2 (the mMVo2 with stimulation − the mMVo2 without stimulation) was 0.83 ± 0.12 ml O2·min−1·100 g LV−1. There were no differences between mean mMVo2 with and without stimulation in Ca2+-free solution. The increases in extracellular Ca2+ concentrations up to 14.4 mM did not affect the mMVo2 without stimulation but significantly increased the mMVo2 with stimulation up to 140% of control. The ΔmMVo2 significantly increased up to 190% of the control in a dose-dependent manner. In contrast, the shortening did not increase in a dose-dependent manner. Cyclopiazonic acid (CPA; 30 μM) significantly reduced the ΔmMVo2 to 0.27 ± 0.06 ml O2·min−1·100 g LV−1 (35% of control). The combination of 5 mM 2,3-butanedione monoxime (BDM) and 30 μM CPA did not further decrease ΔmMVo2. Although BDM (3–5 mM) decreased the ΔmMVo2 by 28–30% of control in a dose-independent manner, 3–5 mM BDM decreased shortening in a dose-dependent manner. Our results indicate that the ΔmMVo2 of mouse LV slices during shortening under mechanically unloaded conditions consists of energy expenditure for total Ca2+ handling during excitation-contraction coupling, basal metabolism, but no residual cross-bridge cycling.

Reloading of Ca(2+)-depleted sarcoplasmic reticulum during rest in guinea pig ventricular myocytes

The effects of rest on a Ca(2+)-depleted sarcoplasmic reticulum (SR) in guinea pig ventricular myocytes were investigated. Cell shortening was measured using a video edge-detection system, and cytoplasmic Ca2+ was monitored using the fluorescent indicator indo-1. Rapid cooling and rewarming in the presence of 10 mM caffeine were used to deplete the SR of Ca2+. The resting cell was then superfused for variable time intervals with a normal Tyrode solution containing 2 mM Ca2+. Another rapid cooling in caffeine was performed to assess the SR Ca2+ load at the end of rest. Rapid cooling after 1- and 2-min rest elicited an increase of indo-1 fluorescence of 51.9 +/- 7.7 (n = 17) and 72.7 +/- 6.7% of control (n = 9), respectively. This increase was not detectable when Ca2+ was absent from the superfusing solution. In contrast, the increase was larger when external Ca2+ was elevated to 4 mM. Nickel (5 mM) and nifedipine (20 microM) added to the superfusing solution during the rest interval did not alter the increase in indo-1 fluorescence. We conclude that Ca2+ is reaccumulated by a depleted SR during rest. Although this Ca2+ seems to originate from the extracellular space, its route from there to the SR is unclear.

Temperature and relative contributions of Ca transport systems in cardiac myocyte relaxation

The relative contributions of the different Ca transport systems involved in cardiac relaxation were evaluated at 25 and 35 degrees C in isolated rabbit, ferret, and cat ventricular myocytes during twitches, caffeine-induced contractures in normal Tyrode solution, and caffeine-induced contractures in Na- and Ca-free solution. The time course of intracellular [Ca] decline these contractions in rabbit ventricular myocytes allowed estimates of the relative contributions of the sarcoplasmic reticulum (SR) Ca pump, Na/Ca exchange, sarcolemmal Ca pump, and the mitochondrial calcium uniporter (with the latter two considered together as “slow mechanisms”).The percent contributions of the SR Ca pump, the Na/Ca exchange, and the slow mechanisms were 70, 27 and 3% at 25 degrees C and 74, 23, and 3% at 35 degrees C. Warming from 25 to 35 degrees C decreases twitch contractions in rabbit and ferret myocytes and caffeine-induced contractures in normal Tyrode solution and Na- and Ca-free solution in all species. In contrast, in cat myocytes warming increased twitches, possibly because of a stronger effect of temperature on Ca influx. We conclude that increased temperature accelerates all of the Ca transport systems involved in relaxation. Despite large changes in each Ca transport system with warming, the relative contributions during relaxation remain similar at physiological temperature.

Myocardial VO2 of mechanically unloaded contraction of rat ventricular slices measured by a new approach

We instituted a new approach of measuring mechanically unloaded myocardial oxygen consumption (VO2) by using rat left ventricular (LV) slices in an air-tight chamber filled with oxygenated Tyrode solution. Myocardial slices (300 microns in thickness) freely shortened without external load by electrical field stimulation (St). VO2 without St (n = 6) was 1.69 +/- 0.41 ml O2.min-1.100 g LV-1. VO2 with St (n = 6) increased to 2.28 +/- 0.36 ml O2.min-1.100 g LV-1. VO2 in Ca(2+)-free Tyrode solution irrespective of St was nearly equal to VO2 without St in normal Tyrode solution, indicating that all these VO2 correspond to basal metabolic VO2. The increment in VO2 by St (delta VO2) increased up to twice normal with the extracellular Ca2+ concentration up to 4 times normal. Inhibition of cross-bridge cycling by 2,3-butanedione monoxime (5 and 10 mM) did not decrease delta VO2. These results suggest that delta VO2 consists of VO2 primarily for excitation-contraction coupling but not for cross-bridge cycling.

Effects of allopurinol on reperfusion arrhythmias in isolated ventricles

Electrophysiological effects of allopurinol on arrhythmias were studied in isolated segments of guinea pig right ventricular free walls paced from endocardium. A high-gain electrocardiogram as well as transmembrane electrical activity from endo- and epicardium were recorded. Tissues were exposed to simulated ischemia for 15 min and then were reperfused with normal Tyrode solution. Sustained or nonsustained ventricular tachycardia, bigeminy, and trigeminy with characteristics of transmural reentry occurred in early reperfusion in 75% of 20 control preparations. Arrhythmias were associated with prolongation of transmural conduction time and abbreviation of endocardial effective refractory period (ERP). Allopurinol strongly reduced the incidence of reperfusion arrhythmias (20-33%) between 10 and 100 microM, whereas either lower or higher concentrations (5 or 500 microM) were less effective (43 and 50%). Antiarrhythmic efficacy correlated with significant attenuation of reperfusion-induced transmural conduction delay (P less than 0.05 or 0.01). Allopurinol did not affect endocardial conduction times nor did it significantly alter endocardial action potential duration or ERP. Our results indicate that allopurinol exerts antiarrhythmic efficacy during reperfusion by selectively attenuating defects related to anisotropic tissue properties.

Na+-Ca2+ exchange in regulation of contractility in canine cardiac Purkinje fibers

To study Na+-Ca2+ exchange, intracellular Na+ activity (aiNa), twitch tension, and transmembrane potential were simultaneously measured in canine cardiac Purkinje fibers driven at a constant rate (1 Hz) in the absence and presence of strophanthidin (5 X 10(-7) M) at normal, low, and high extracellular [Na+] ([Na+]o) or [Ca2+] ([Ca2+]o). Intracellular Ca2+ activity (aiCa) of the fibers was also measured in a normal Tyrode solution. Reductions of [Na+]o by 20, 40, and 60% decreased the ratio of extracellular Na+ activity (aoNa) and aiNa in the steady state but steeply increased twitch tension. This finding is consistent with the view that a decrease in aoNa/aiNa increases intracellular Ca2+ through Na+-Ca2+ exchange. In further agreement with this view, a Na+-free solution virtually depleted intracellular Na+ and increased the resting tension of the fibers. The slope of the relation of the logs of twitch tension and aiNa that was determined at normal [Na+]o and [Ca2+]o may reflect the properties of the Na+-Ca2+ exchange. Slope of log tension-aiNa relationship decreased when reducing [Na+]o or increasing [Ca2+]o had decreased the level of aiNa. On the other hand, the slope increased when a rise in [Na+]o or a reduction in [Ca2+]o had increased the level of aiNa. These results indicate that as the aiNa level increased, slope of tension-aiNa relation increased, which suggests that Na+-Ca2+ exchange may depend on level of aiNa.(ABSTRACT TRUNCATED AT 250 WORDS)

A Microelectrophysiologic Basis For The Fibrillation Retardant Effects Of Sulfinpyrazone And Salicylate

The antagonism of platelet function has been suggested as a mode of action for sudden death prophylaxis by sulfinpyrazone (S) and salicylates (A). To evaluate an antiarrhythmic means for such action, 5.0 to 20.0 mg/Kg A or S was administered i.v. to open-chest, intermittently cardiac paced canines in which arterial pressure and EKG were monitored. Fibrillation thresholds (FT) were measured before and after coronary arterial ligation and drug administration, by extrastimuli of increasing current strength during the vulnerable period of the T wave. Acutely both A and S increased FT 2 to 3 times that of post-infarcted values. To elucidate the mechanism for this response Purkinje fibers were isolated from the left ventricles of adult canines. These were pinned to the paraffin floor of a lucite isolation well, affixed with stimulating and surface recording electrodes, and partitioned with a plastic collar coated with vegetable fat to provide a fluid seal. Separate inflow and outflow ports allowed for individualized microenvironments for each chamber. After 60 min of tissue equilibration with well oxygenated, 36°C normal Tyrode Solution (TS), the fluid of the chamber distal to the stimulus source was altered to include 2-35 mg% A or 2-50 ugm/ml S, with or without CO2 substitution for O2 (pH 6.4) or HC1 (pH 6.4). Action potentials (AP) from each chamber were monitored during 60 min of altered TS and then 60 min of normal TS reperfusion. In a dose-dependent manner A depressed AP parameters to cause automaticity and inactivity, while S was without effect. Whereas S was able to prolong tissue survival and support electrical activity in a situation of local acidosis (if absolute hypoxia were not superimposed), A was not. Such information suggests that although both A and S are arrhythmia retardant in the postinfarction period, their modes of action may differ. Both might act as “membrane stabilizers”; however, it appears that electrogenic aberration is necessary before S manifests an effect.

Maximum Rate of Depolarization of Single Muscle Fiber in Normal and Low Sodium Solutions

The values of membrane action potentials and maximum depolarization rates of single muscle fibers in normal Tyrode solution and in low sodium solutions containing as little as 20 per cent of the sodium chloride were measured with intracellular microelectrodes. Under these conditions the membrane potential remains unchanged up to 36 per cent of [Na+]out concentration, whereas the overshoot of the action potential varies linearly with the logarithm of the external sodium concentration. The maximum depolarization rate is a linear function of the external sodium concentration. The results obtained support the ionic theory for sodium and the independence principle for sodium current related to the external sodium concentration.