ryanodine and acetylstrophanthidin

To elucidate the role of changes in [Ca2+]i in the induction of ventricular fibrillation (VF), Ca2+i signals, epicardial electrical potentials, and isovolumic left ventricular pressure were simultaneously recorded in isolated intact ferret hearts loaded with aequorin, a bioluminescent protein. When the preparations were perfused with 3 microM acetylstrophanthidin and 8 mM Ca2+, or with a low Na+ solution (18 mM Na+, 100 mM Li+), spontaneous transitions to the VF state were consistently observed within a short period of time. The initiation of spontaneous VF was preceded by development of a Ca2+i overload state, coincidental with the ascending phase of diastolic Ca2+i oscillations, and was followed by further elevation in Ca2+i levels, which were associated with augmented Ca2+i oscillations of a saw-toothed pattern. Pretreatment with 10 microM ryanodine, which blocked Ca2+i oscillations in the preparation, did not eliminate inducibility of VF by means of AC electrical stimulations; however, VF no longer occurred spontaneously, and the threshold for VF induction increased markedly. In the absence of a state of Ca2+i overload, spontaneous defibrillation occurred within a minute after the initiation of VF. We conclude that 1) VF can be induced in the absence of Ca2+i oscillations; however, 2) Ca2+i oscillations play a crucial role as a trigger for VF and therefore are an important determinant of the vulnerability to VF; and 3) the augmented Ca2+i oscillations after the transition to VF state may support the maintenance of this type of arrhythmia.

Topics: Aequorin; Animals; Calcium; Electric Stimulation; Electrophysiology; Ferrets; In Vitro Techniques; Male; Myocardial Contraction; Myocardium; Perfusion; Ryanodine; Sodium; Strophanthidin; Ventricular Fibrillation

Developed twitch tension and action potentials were recorded in rabbit ventricular muscle in physiological saline at 30 degrees C stimulated at 0.5 Hz. Addition of 5 microM nifedipine to block Ca entry via Ca channels almost abolished twitches (to 2.5 +/- 0.7%, S.E.M., n = 10 of control). This suggests that under normal conditions Ca entry via Na-Ca exchange is insufficient to activate contractions. However, when muscles are first exposed to 4 microM acetylstrophanthidin to elevate [Na]i the same exposure to nifedipine only partially suppresses twitches (to 59 +/- 12% of the original control). This suggests that when [Na]i is elevated, Ca entry via the Na-Ca exchange may be adequate to partially activate contraction. From this result it is not clear whether Ca entry via Na-Ca exchange is sufficient to activate contraction directly or whether sarcoplasmic reticulum (SR) Ca release is required. When these experiments were carried out in the presence of 5 to 10 mM caffeine or 100 nM ryanodine similar results were obtained. That is, nifedipine still abolished contractions in the presence of caffeine or ryanodine (to 3.8 +/- 0.3% and 1.3 +/- 0.4%, respectively), but only partially inhibited contractions in the presence of caffeine + acetylstrophanthidin (to 21 +/- 5%) or ryanodine + acetylstrophanthidin (10 +/- 2%). Thus, it appears that even in the absence of a functional SR and with Ca current blocked, Na-Ca exchange might bring sufficient Ca into the cell to activate appreciable contractions, but only when [Na]i is elevated. Action potential duration is decreased by nifedipine and acetylstrophanthidin and is further decreased when nifedipine is added on top of acetylstrophanthidin. If this Ca entry is by an electrogenic 3 Na: 1 Ca exchange, Ca entry will be favored at more positive membrane potentials. If the action potential were not so abbreviated with these drugs, Na-Ca exchange might bring in more Ca and activate additional tension.

Topics: Action Potentials; Animals; Caffeine; Calcium; Carrier Proteins; Heart Ventricles; In Vitro Techniques; Myocardial Contraction; Nifedipine; Rabbits; Ryanodine; Sodium-Calcium Exchanger; Strophanthidin; Ventricular Function

Reduction of the transsarcolemmal [Na] gradient in rabbit cardiac muscle leads to an increase in the force of contraction. This has frequently been attributed to alteration of Ca movements via the sarcolemmal Na/Ca exchange system. However, the specific mechanisms that mediate the increased force at individual contractions have not been clearly established. In the present study, the [Na] gradient was decreased by reduction of extracellular [Na] or inhibition of the Na pump by either the cardioactive steroid acetylstrophanthidin or by reduction of extracellular [K]. Contractile performance and changes in extracellular Ca (sensed by double-barreled Ca-selective microelectrodes) were studied in order to elucidate the underlying basis for the increase in force. In the presence of agents that inhibit sarcoplasmic reticulum (SR) function (10 mM caffeine, 100-500 nM ryanodine), reduction of the [Na] gradient produced increases in contractile force similar to that observed in the absence of caffeine or ryanodine. It is concluded that an intact, functioning SR is not required for the inotropic effect of [Na] gradient reduction (at least in rabbit ventricle). However, this does not exclude a possible contribution of enhanced SR Ca release in the inotropic response to [Na] gradient reduction in the absence of caffeine or ryanodine. Acetylstrophanthidin (3-5 microM) usually leads to an increase in the magnitude of extracellular Ca depletions associated with individual contractions. However, acetylstrophanthidin can also increase extracellular Ca accumulation during the contraction, especially at potentiated contractions. This extracellular Ca accumulation can be suppressed by ryanodine and it is suggested that this apparent enhancement of Ca efflux is secondary to an enhanced release of Ca from the SR. Under conditions where Ca efflux during contractions is minimized (after a rest interval in the presence of ryanodine), acetylstrophanthidin increased both the rate and the extent of extracellular Ca depletions. Thus, acetylstrophanthidin can increase both Ca influx and Ca efflux during the cardiac muscle contraction. These results can be explained by a simple model where the direction of net Ca flux via Na/Ca exchange during the action potential is determined by the changes in reversal potential of the Na/Ca exchange. Reduction of the [Na] gradient may well lead to net cellular Ca uptake (via Na/Ca exchange) and may also elevate the resting intracellular [Ca].(ABSTRAC

Topics: Alkaloids; Animals; Caffeine; Calcium; In Vitro Techniques; Myocardial Contraction; Papillary Muscles; Rabbits; Ryanodine; Sarcoplasmic Reticulum; Sodium; Strophanthidin

Transient changes of extracellular free calcium in rabbit ventricular muscle under nonsteady state conditions were measured with double-barreled calcium microelectrodes. Resumption of stimulation after a rest interval produces a cumulative decrease of extracellular free calcium often by more than 10% (with bulk extracellular free calcium = 0.2 mM). The extracellular free calcium returns to the bulk value as a new steady state is achieved. The changes of extracellular free calcium recorded presumably represent net calcium uptake and loss by cardiac muscle cells. These cumulative extracellular free calcium depletions are blocked by 0.5 mM cobalt and 1 microM nifedipine and are increased to 167 +/- 11% of control by the calcium agonist Bay k 8644 (1 microM) and to 620 +/- 150% of control by increasing stimulus frequency from 0.2-2 Hz. Caffeine (10 mM) inhibits the cumulative extracellular free calcium depletions, probably by rendering the sarcoplasmic reticulum unable to accumulate calcium. It is proposed that the extracellular free calcium depletions recorded represent, in large part, calcium which has entered the cells and has been taken up by the sarcoplasmic reticulum (which had become depleted of calcium during the rest interval). Nifedipine and cobalt inhibit these cumulative depletions presumably by preventing the calcium entry which could subsequently be accumulated by the sarcoplasmic reticulum. The net cellular calcium uptake produced by such a post-rest stimulation protocol can also be inhibited by 1-3 microM acetylstrophanthidin and reduction of extracellular sodium to 70 mM. Acetylstrophanthidin and low extracellular sodium would be expected to shift the sodium-calcium exchange in favor of increased calcium uptake, which may, in turn, prevent the loss of sarcoplasmic reticulum calcium during the rest interval. This would limit the amount of calcium which the sarcoplasmic reticulum could take up with subsequent activation. In contrast to the results with caffeine, ryanodine (1 microM) increases the magnitude and rate of calcium uptake after a rest interval, indicative of a fundamental difference in the actions of caffeine and ryanodine. When stimulation is stopped in the presence of ryanodine, extracellular free calcium increases much faster than in control. This suggests that ryanodine may enhance calcium uptake by the sarcoplasmic reticulum during repetitive stimulation and may enhance calcium efflux from the sarcoplasmic reticulum during quies

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Caffeine; Calcium; Cobalt; Electric Stimulation; Extracellular Space; Heart; In Vitro Techniques; Microelectrodes; Myocardium; Nifedipine; Rabbits; Ryanodine; Strophanthidin

To determine whether Na+-Ca2+ exchange is a physiologically significant Ca2+ efflux mechanism in rabbit ventricle, we investigated the effects exerted on postrest contractions by interventions that alter the transmembrane distribution of Na+ or Ca2+ so as to retard Ca2+ efflux via this system. Contractions elicited after rest periods of 0.25-10 min in duration were studied. The following interventions increased postrest contractions much more than those elicited by rhythmic stimulation: 1) Na+ pump inhibition by cardiac glycosides or by a reduction in extracellular K+, 2) reduction of extracellular Na+ (maintaining a constant [Ca2+]-to-[Na+]2 ratio), and 3) elevation of extracellular Ca2+. In contrast, isoproterenol, norepinephrine, and histamine produced comparable increases in both rhythmically stimulated and postrest contractions, suggesting that the postrest contractile potentiation was not just the result of a general increase in inotropic state. Ryanodine, which appears to antagonize sarcoplasmic reticulum (SR) Ca2+ release in cardiac muscle, markedly reduced the amplitude of the postrest contractions, but only modestly decreased rhythmically stimulated responses. Results suggest 1) that Ca2+ released from SR is involved in postrest response, 2) that Na+-Ca2+ exchange serves as a Ca2+ efflux pathway in normally polarized resting rabbit ventricle, and 3) that this activity in part determines the amount of Ca2+ available for release from SR.

Topics: Animals; Calcium; Cobalt; Heart; In Vitro Techniques; Male; Myocardial Contraction; Myocardium; Periodicity; Rabbits; Ryanodine; Sarcoplasmic Reticulum; Sodium; Strophanthidin