The role of L-type calcium current in the generation of repolarization-induced contraction in cardiac myocytes.

OBJECTIVE: Early experiments into the arrhythmogenic transient inward current frequently showed apparent coupling of this current to repolarization from a depolarizing voltage clamp step. Calcium transients have subsequently been shown to couple to such repolarization and are the result of calcium release from the sarcoplasmic reticulum. We have investigated whether this phenomenon is due to calcium entry via non-inactivated calcium channels or to voltage-activated SR release. METHODS: Voltage clamp steps were imposed on isolated guinea pig and rabbit cardiac myocytes. Calcium release was monitored by tracking cell contraction. L-type calcium current at the moment of repolarization was manipulated by the rapid application of 2 mM cadmium or 10 mM calcium. RESULTS: Repolarization-induced contraction was abolished by the rapid application of 2 mM cadmium immediately prior to repolarization, and was augmented by the rapid change of extracellular calcium concentration from 2 mM to 10 mM immediately prior to repolarization. There is no evidence of coupling of drive train-induced aftercontractions to repolarization from the final action potential of the drive train and 2 mM cadmium does not alter the appearance or timing of these aftercontractions. Simulation of phase 1 repolarization in the mammalian cardiac action potential decreases rather than increases twitch amplitude. CONCLUSION: Repolarization-induced contraction results from calcium entry through non-inactivated calcium channels, not from voltage-activated release. It plays no physiological role in contributing to the stimulated twitch and no pathological role in generating drive train-induced aftercontractions.

Relative importance of SR load and cytoplasmic calcium concentration in the genesis of aftercontractions in cardiac myocytes.

OBJECTIVE: To determine whether calcium overload of the sarcoplasmic reticulum underlies drive train-induced aftercontractions in cardiac myocytes. METHODS: Sarcoplasmic reticulum calcium contents were measured immediately prior to drive train-induced aftercontractions in isolated guinea pig cardiac myocytes, using caffeine application under voltage clamp conditions. Cell shortening during caffeine exposure and cell shortening during the final stimulated beat of the drive train and the delay between caffeine exposure and the onset of inward current were also used as indirect measures of sarcoplasmic reticulum load. RESULTS: At the threshold for aftercontractions, all four measures of sarcoplasmic reticulum load showed interruption of the positive relationship between stimulation frequency and sarcoplasmic reticulum content, the sarcoplasmic reticulum being no more loaded prior to an aftercontraction than following subthreshold drive trains. Intracellular calcium concentration, estimated with the calcium-sensitive dye indo-1, was higher in cells showing aftercontractions than those not. CONCLUSIONS: We conclude that calcium overload of the sarcoplasmic reticulum does not underlie spontaneous calcium release in this situation and the primary trigger for spontaneous release may instead be raised cytoplasmic calcium concentration.

Calcium extrusion during aftercontractions in cardiac myocytes: the role of the sodium-calcium exchanger in the generation of the transient inward current.

Spontaneous release of calcium from the sarcoplasmic reticulum leads to delayed afterdepolarizations which may represent an arrhythmogenic mechanism in the intact heart. The current underlying delayed afterdepolarizations is the transient inward current, but how this is triggered by a spontaneous rise in cytoplasmic calcium concentration is a matter of debate. We have investigated this by rapid application of caffeine to isolated guinea-pig cardiac myocytes, before and after drive train-induced aftercontractions. Mean (+/- s.e.m.) sarcoplasmic reticulum content reduced from 85 +/- 11 micromol/l accessible cell volume to 53 +/- 9 micromol/l accessible cell volume (n=11) during the course of the aftercontraction. The charge movement expected to result from extrusion of this calcium via the sodium-calcium exchanger was 70.1 +/- 5.4 pC, compared with charge measured during the transient inward current of 70.1 +/- 10.8 pC in the same cells (P=0.9969). Rapid inhibition of the sodium-calcium exchanger, by replacement of the superfusate with a sodium and calcium free solution between the end of the drive train and the aftercontraction, completely abolished the transient inward current (from 90.4 +/- 10.2 pA inward current to 23.8 +/- 14.9 pA outward current, P<0.001). We conclude that the transient inward current in this species is explained entirely by sodium-calcium exchange current without the need to invoke other calcium-activated conductances.

The release of soluble adhesion molecules ICAM-1 and E-selectin after acute myocardial infarction and following coronary angioplasty.

Endothelial cells express surface adhesion molecules for leukocytes in response to myocardial ischaemia. These molecules may be released into plasma by activated cells and be detectable in soluble form. Samples were collected from the peripheral vein of 14 consecutive patients with acute myocardial infarction (AMI) at the time of admission, 6 h, and 1 and 5 days post-admission. Additionally, samples were drawn from the coronary sinus ostium and peripheral artery of seven patients undergoing coronary angioplasty (PTCA) before and after the first balloon inflation. We measured the plasma levels of soluble intercellular adhesion molecule-1 (sICAM-1) and soluble E-selectin (sELAM-1). In patients with AMI plasma levels of sICAM-1 exceeded those observed in age and sex-matched healthy subjects, (mean+/-SEM; 220.6+/-18 ng/ml) at all the time intervals assessed (358.9+/-24.5; 330.9+/-24.4; 379.4+/-39.7 and 366.8+/-47.5 ng/ml, respectively, p<0.01). sELAM-1 levels, however, were normal on admission, increased at 6 h to 52.7+/-3.8 ng/ml, p<0.05, and at day 1 (56.0+/-4.6 ng/ml) before decreasing to normal levels on the fifth day. After brief myocardial ischaemia occurring during PTCA, an increased level of sICAM-1 was observed following balloon deflation in the coronary sinus (329.2+/-20 ng/ml; p<0.05) as compared to the subjects undergoing coronary angiography, but not in the peripheral artery. sELAM-1 levels remained unchanged during angioplasty. Thus, soluble adhesion molecules expressed by activated endothelial cells are released into peripheral blood during both AMI and brief myocardial ischaemia and measurement of such molecules may prove useful for monitoring vascular endothelium activation following myocardial ischaemia/necrosis.

Plasma-mediated neutrophil activation during acute myocardial infarction: role of platelet-activating factor.

1. Polymorphonuclear neutrophils are involved in the development of myocardial injury during ischaemia through the release of free oxygen radicals and by adhesion of activated polymorphonuclear neutrophils to endothelium, resulting in plugging of coronary capillaries. Polymorphonuclear neutrophil activation may be a result of contact with ligands expressed by endothelial cells and/or a response to soluble stimuli released from ischaemic tissue to the plasma. 2. To investigate this we studied plasma-mediated polymorphonuclear neutrophil activation in vitro using plasma samples collected from 14 patients with acute myocardial infarction at time of admission and 6 h and 1, 2, 5 and 7 days later. Plasma samples were incubated with washed polymorphonuclear neutrophils isolated from healthy donors. Expression of adhesion molecules CD18/CD11b integrin and L-selectin (Leu-8) were measured by flow cytometry and superoxide anion production in polymorphonuclear neutrophils was measured by chemiluminescence. 3. Plasma samples obtained 6 h and 1 day after admission were capable of inducing CD18/CD11b antigen expression, superoxide anion production and L-selectin shedding in the washed polymorphonuclear neutrophils, and this effect was significant when compared with plasma taken at 5 and 7 days after admission. 4. The plasma-mediated polymorphonuclear neutrophil stimulation was prevented when the PMN were pretreated with platelet-activating factor receptor antagonists BN52021 or BN50739. The platelet-activating factor concentrations detected in the plasma samples were not higher than those detected in plasma from healthy subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of neutrophil activity by nitric oxide during acute myocardial ischaemia and reperfusion.

Nitric oxide (NO) exerts an inhibitory effect on polymorphonuclear neutrophil (PMN) function, via a cyclic GMP-mediated mechanism, while PMNs are known to play an important role in myocardial ischaemia-reperfusion injury (MI-R). Since the major source of NO, vascular endothelium, becomes functionally impaired during MI-R, it is attractive to hypothesize that it is this loss of endothelial nitric oxide production that allows PMN adherence and activation. The studies reviewed here add substance to this hypothesis. Authentic NO, administered during MI-R both reduces myocardial necrosis and PMN accumulation, while basal NO release, as estimated by coronary artery ring responses to L-NAME, an NO synthase inhibitor, declines during reperfusion with a time-course mirrored by PMN adherence in the same preparation. Reduction in infarct size and decreased PMN accumulation can also be demonstrated with L-arginine and NO donors. Since endothelial dysfunction leads to PMN adherence and PMNs have been shown to contribute to endothelial dysfunction, it seems probable that a positive feedback loop is generated during MI-R, leading to the amplification of PMN activity and subsequent myocardial damage.