thapsigargin and Cardiac-Output--Low

Defects in myocyte contraction and relaxation are key features of human heart failure. Sodium/calcium exchanger-mediated contribution to contraction and relaxation were separated from other mechanisms [L-type calcium current, sarco(endo)plasmic reticulum (SR) Ca(2+)-ATPase] based on voltage, temperature, and selective blockers. Rod-shaped left ventricular myocytes were isolated from failed human explants (n = 29) via perfusion with collagenase-containing Krebs solution. Action potentials using perforated patch and contractions using an edge detector were recorded at 0.5-1.5 Hz in Tyrode solution at 25 degrees C and 37 degrees C. Contraction duration was dependent on action potential (AP) duration at 37 degrees C but not at 25 degrees C, suggesting the role of the exchanger in relaxation and linking myocyte relaxation to the repolarization phase of the AP. Voltage-clamp experiments from -50 to +10 mV for 1,500 ms in Tyrode or Na(+)- and K(+)-free solutions after conditioning pulses triggered biphasic contractions that included a rapid SR-mediated component and a slower voltage-dependent exchanger-mediated component. We used thapsigargin to block the SR, which eliminated the rapid component, and we used an exchanger blocker, Kanebo 7943, which eliminated the slow component. The exchanger was shown to contribute to contraction through reverse-mode exchange, as well as to play a key role in relaxation of human ventricular myocytes.

Topics: Action Potentials; Cardiac Output, Low; Enzyme Inhibitors; Humans; Myocardial Contraction; Myocardium; Patch-Clamp Techniques; Sodium-Calcium Exchanger; Temperature; Thapsigargin; Thiourea; Ventricular Function

We have previously shown that ANG II increases microvascular permeability in normal dog lungs but not after pacing-induced heart failure. This study investigated how ANG II induces permeability in isolated blood-perfused canine lung lobes and what alterations occur during heart failure. In normal lobes, the protein kinase C (PKC) inhibitors staurosporine (500 nM) or chelerythrine (10 microM) did not modify ANG II-induced increases in the capillary filtration coefficient (Kf,c, ml . min-1 . cmH2O-1 . 100 g-1; an index of microvascular permeability), suggesting that PKC is not involved. Thapsigargin (150 nM) was used to stimulate capacitative Ca2+ entry in lobes from control dogs and dogs paced at 245 beats/min for 4 wk to induce heart failure. In control lobes, Kf,c rose after thapsigargin, from 0.06 +/- 0.01 to 0.17 +/- 0.03 ml . min-1 . cmH2O-1 . 100 g-1 (mean +/- SE, P < 0.05) but did not change in the paced group. A Ca2+ ionophore, A-23187, increased Kf,c in both control (10 microM; 0.05 +/- 0.01 to 0.17 +/- 0.05 ml . min-1 . cmH2O-1 . 100 g-1, P < 0.05) and pace (5 microM; 0.06 +/- 0.01 to 0. 21 +/- 0.07 ml . min-1 . cmH2O-1 . 100 g-1, P < 0.05) lobes, indicating that increasing intracellular Ca2+ is sufficient to induce pulmonary microvascular permeability after pacing. We conclude that during heart failure, Ca2+ signaling within the pulmonary microvascular endothelium is altered.

Topics: Alkaloids; Angiotensin II; Animals; Benzophenanthridines; Calcimycin; Calcium; Capillary Permeability; Cardiac Output, Low; Cardiac Pacing, Artificial; Dogs; Endothelium, Vascular; Enzyme Inhibitors; Ionophores; Lung; Phenanthridines; Protein Kinase C; Signal Transduction; Staurosporine; Thapsigargin; Vascular Resistance

It has been reported that the balance between the two main Ca2+ removal systems in the cardiac cells, the sarcoplasmic reticulum (SR) and Na+/Ca2+ exchanger, is altered in failing human heart. We have studied post-rest contraction behaviour as a non-invasive probe of the amount of Ca2+ stored in the SR in myocytes from failing and non-failing human ventricle. The first beat following a rest interval, as a percentage of the preceding steady state (B1/SS), was larger and more variable in cells from failing heart, indicating some accumulation of Ca2+ in the SR during rest. This could be mimicked by treatment of myocytes with digoxigenin, a compound which increases intracellular Na+, suggesting that alterations in the Na+ balance of the cell might contribute to the effect. Isoprenaline, which stimulates Ca2+ uptake by the SR while the myocyte is beating, prevented SR Ca2+ accumulation during rest in susceptible myocytes. We hypothesize that loss of SR function in the failing heart is partially compensated for by increased Ca2+ extrusion via the Na+/Ca2+ exchange in the contracting myocyte, leading to increased intracellular Na+ during activity. This Na+ is lost at rest, predisposing the cells to accumulate Ca2+ in the SR. Experiments to test this hypothesis are proposed.

Topics: Analysis of Variance; Calcium; Cardiac Output, Low; Cardiotonic Agents; Colforsin; Digoxigenin; Drug Evaluation, Preclinical; Heart Ventricles; Humans; In Vitro Techniques; Isoproterenol; Linear Models; Myocardial Contraction; Rest; Thapsigargin