Resumption Of Stimulation Research Articles

Discontinued stimulation of cardiomyocytes provides protection against hypothermia-rewarming-induced disruption of excitation-contraction coupling.

What is the central question of this study? Will discontinued stimulation of isolated cardiomyocytes (asystole) during hypothermia mitigate hypothermia-rewarming-induced cytosolic Ca2+ overload? What is the main finding and its importance? Mimicking asystole or hypothermic cardiac arrest by discontinued stimulation of cardiomyocytes during hypothermia resulted in normal contractile function after rewarming. This result suggests that asystole during severe hypothermia provides protection from hypothermia-rewarming-induced contractile dysfunction in cardiomyocytes. After exposure of spontaneously beating hearts or electrically stimulated isolated cardiomyocytes to hypothermia-rewarming (H/R), cardiac dysfunction or alteration in excitation-contraction coupling, respectively, is a consequence. In contrast, hypothermic cardiac arrest, as routinely applied during cardiac surgery, will not impose any hazard to cardiac function after rewarming. We hypothesize that by maintaining asystole during H/R, cardiomyocytes will avoid Ca2+ overload attributable to the transient stimulation-evoked elevation of [Ca2+ ]i and thus, H/R-induced elevation of phosphorylated cardiac troponin I and reduced Ca2+ sensitivity after rewarming. To test this hypothesis, the aim of the study was to determine whether discontinued electrical stimulation (to imitate hypothermic cardiac arrest) versus stimulation during 3h of H/R prevents disruption of excitation-contraction coupling in our established cardiomyocyte H/R model. Cytosolic Ca2+ and the contractile response (sarcomere length shortening) were measured using an IonOptix system, and the dynamic assessment of Ca2+ sensitivity of contraction was conducted using a phase-loop plot. Cardiomyocytes were divided into three groups. Group 1 (time-matched control) was continuously stimulated at 0.5Hz for 3h at 35°C. Group 2 was continuously stimulated during H/R at 0.5Hz, whereas in group 3 stimulation was discontinued during H/R and thus the cells remained quiescent until the resumption of stimulation after rewarming. The results demonstrate that discontinued stimulation of cardiomyocytes during H/R, imitating hypothermic cardiac arrest during cardiac surgery, provides protection against H/R-induced disruption of excitation-contraction coupling. We suggest that protective effects are caused by preventing the protein kinaseA-induced elevation of phosphorylated cardiac troponin I, which is a key mechanism to reduce myofilament Ca2+ sensitivity of contraction.

Cytosolic calcium staircase in ventricular myocytes isolated from guinea pigs and rats

The aim was to study intracellular calcium dynamics underlying positive or negative tension staircase of mammalian hearts. Changes in cytosolic calcium concentration [( Ca2+]i) in single ventricular myocytes were investigated using a Ca2+ indicator, fura-2. Beat to beat alterations in fura-2 fluorescence and cell edge movement on resumption of stimulation were recorded on video tape, and analysed by a computer based image processing system. Single ventricular myocytes were enzymatically isolated from the hearts of 30 adult guinea pigs and 25 adult rats. In guinea pig ventricular myocytes, the positive contractile staircase was associated with ascending staircases of both peak systolic and end diastolic [Ca2+]i because of a cumulative increase in diastolic [Ca2+]i. In rat ventricular myocytes, the negative contractile staircase was accompanied by a descending staircase of peak systolic [Ca2+]i, while end diastolic [Ca2+]i level was unchanged due to the rapid decay of [Ca2+]i transients. Ryanodine (10 microM) reversed the mode of [Ca2+]i and contractile staircases from negative to positive in rat myocytes, whereas it caused minimal alteration in guinea pig myocytes. Tension staircase of mammalian hearts depends on diastolic Ca2+ level as well as Ca2+ handling by the sarcoplasmic reticulum. The positive staircase may require progressive increase in diastolic [Ca2+]i, while the negative staircase may be mediated by depletion of activator Ca2+ in the sarcoplasmic reticulum.

Rest decay of calcium transients and contractility in feline ventricular myocytes

To define the respective contributions of sarcoplasmic reticulum Ca2+ release and action potential-dependent Ca2+ influx to the activator Ca2+, rest decay of the cytosolic free Ca2+ (Ca2+i) transient and contractility was measured in feline ventricular myocytes loaded with the Ca2(+)-sensitive fluorescent dye indo-1. Myocytes were stimulated to a steady state and then rested for periods ranging from 2 to 120 s. Rest decay was assayed with both resumption of stimulation and rapid extracellular applications of caffeine. The protocol was then repeated after exposure to ryanodine. Ryanodine changed the Ca2+i transients during steady-state stimulation from large, rapidly rising transients starting near the resting Ca2+i level to small, slowly rising Ca2+i transients superimposed on an elevated diastolic Ca2+i. Under control conditions the stimulation- and caffeine-induced Ca2+i transients exhibited slow monotonic rest decay with similar time constants of decay (180 and 202 s, respectively, from a representative cell). After ryanodine, the rest decay of stimulation-induced Ca2+i transients was seemingly no different. However, no Ca2+i transient could be elicited by caffeine after 15 s of rest. These results suggest that 1) under normal conditions the primary source of activator Ca2+ is the sarcoplasmic reticulum, and 2) the rest decay of contractility seen in feline ventricular myocytes results from time-dependent loss of Ca2+ from this organelle.

Compartmentation of cellular calcium in rabbit ventricle dependent upon its transsarcolemmal route

Augmentation of calcium influx may be buffered in the myocardium by slowly exchangeable compartments. Myocardial calcium influx was enhanced in rabbit interventricular septa by interventions which affect either calcium channel current (BAY k 8644) or Na/Ca exchange (ouabain, low Na0). The effect of these interventions on slowly exchangeable calcium was measured by performing 45Ca washouts. In addition, beating was resumed following a period of prolonged quiescence and the effect on slowly exchangeable calcium measured. Previous work had shown that at a Ca0 of 1.0 mM, low sodium perfusion induced a significant increment in slowly exchangeable calcium. The present investigation demonstrates that ouabain and resumption of stimulation following quiescence also augment this calcium. At 1.0 mM Ca0, as few as 15 beats administered in a 10 min interval were found to induce a significant increment in calcium content, which could be inhibited by the mitochondrial inhibitor CCCP but not by ryanodine. In contrast, BAY k 8644 did not augment slowly exchangeable calcium, although it produced at 0.5 mM Ca0 the same inotropic response as ouabain and 75 mM Na0. In conclusion, slowly exchangeable calcium is not affected by augmentation of calcium channel current, but is enhanced by ouabain, resumption of stimulation, and low Na0. A common characteristic shared by those interventions which enhance slowly exchangeable calcium is an increased Nai/Na0 ratio, which results in an augmented calcium uptake via on Na/Ca exchange.

Cumulative depletions of extracellular calcium in rabbit ventricular muscle monitored with calcium-selective microelectrodes.

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 quiescence. These experiments provide insight into transsarcolemmal calcium movements and certain aspects of cellular calcium regulation.

Calcium channel block and recovery from block in mammalian ventricular muscle treated with organic channel inhibitors.

The effects of a bradycardic agent, AQA 39, on the action potential, tension and membrane current of cat ventricular muscle have been compared with the effects of 1. AQA 39 and D600 both lengthened the action potential duration; neither drug affected the resting potential (−83 to −85 mV). Both drugs depressed the action potential plateau amplitude and tension when the stimulation rate was 120 min−1; ED50's for the negative inotropic effect were 7×10−8 M for D600 and 3×10−5 M for AQA 39. At 20 min−1, D600 was only slightly less depressant (ED50 for negative inotropy 4×10−7 M) but 5×10−5 M AQA 39 had no effect, neither on tension nor on the action potential plateau amplitude. 2. In voltage clamp experiments (holding potential −50 mV), 2×10−5 M AQA 39 and 2×10−6 M D600 were equipotent. With depolarizing pulses (250 ms steps to −10 mV at 20 min−1), they depressed slow inward calcium current (Isi) by 70% and peak tension by 80% in 30 min. 3. At a membrane potential of −50 mV, Isi in muscle exposed to either drug for 30 min without stimulation was not depressed during the first post-rest clamp but the block of calcium channels was rapid upon resumption of stimulation (i.e. a “use-dependent” block). 4. Following steady-state block, Isi could be restored to control amplitude with hyperpolarizing pre-pulses, whereas depolarizing pre-pulses enhanced the block of Isi. 5. It seems that both drugs block calcium channels only after the channels have been activated (opened) and that the degree of activation and the degree of block are linked. The drugs dissociate from a blocking site at a rate dependent upon the diastolic membrane potential; the more negative the membrane potential inbetween subsequent depolarizations, the quicker the dissociation. While the dissociation of AQA 39 at −50 mV is only about twice as fast as that of D600, it is nearly 100 times faster at −80 mV and more than 150 times faster at potentials more negative than −90 mV. These differences might be related to the different lipophilic properties of the two drugs.

The decay of the potentiated state in sheep and calf ventricular myocardial fibers. Influence of agents acting on transmembrane Ca2+ flux.

Paired pulse stimulation increases the contractile strength of mammalian myocardium. If stimulation is discontinued the "potentiated state" takes several minutes to decay, as shown by the first contraction following resumption of stimulation. From earlier experiments it has been inferred that contractile force depends on Ca2+ released from intracellular stores rather than on an influx of Ca2+ associated with a given action potential. This view now receives support from the following findings: (1) Ni2+ and Co2+, known to inhibit Ca2+ influx during the action potential, when applied during a 2-minute rest period, are practically without effect on the strength of the first beat but strongly depress steady state contractions. (2) Caffeine, known to enhance Ca2+ efflux from and inhibit Ca2+ uptake into intracellular stores, greatly accelerates the decay of the potentiated state during a rest period. (3) Na+ -poor solution, known to inhibit Ca2+ efflux, has a strong positive inotropic effect. Paired pulse stimulation fails to increase contractile strength in Na+ -poor solution, and a rest period of many minutes is practically without effect on the amplitude of the first beat after resumption of stimulation. The results indicate that contraction is due to Ca2+ released from internal stores whose degree of filling can be altered.

Contraction dependency of the positive inotropic action of cardiac glycosides.

The concept that the positive inotropic effect of cardiac glycosides is dependent on the contractile state of the myocardium was tested. Isolated left atria of rabbits, when driven electrically at 15, 30, 60 and 120/min (at 30, 33, or 37°C), responded to ouabain (I µ/ml) in proportion to the number of contractions and not to the time of exposure or frequency of drive. Contraction-related effects were proportional to concentration of ouabain, acetylstrophanthidin, and digoxin. A contraction-related effect of ouabain was observed in anesthetized dogs with surgical A-V block, ventricular electrical drive and right ventricular contractile force recording. Quiescent rabbit atria exposed to ouabain for 30 min at 30°C and then washed in ouabain-free medium, showed minimal positive inotropic effect when stimulation was resumed. Toxic concentration of ouabain (5 µg/ml) in contact with quiescent atria for 30 min and then washed out, on resumption of stimulation had an immediate positive intropic (but no toxic) effect which diminished progressively, in contrast to the rapid development of toxic effects (ectopic contractions and contracture) in atria exposed to this dose of auabain during repetitive contractions. It is concluded that a major part of the positive intropic effect of cardiac glycosides is contraction dependent.