Inhibition of the sarcoplasmic reticulum Ca2+ pump with thapsigargin to estimate the contribution of Na+-Ca2+ exchange to ventricular myocyte relaxation

Abstract

Relaxation in the mammalian ventricle is initiated by Ca 2+ removal from the cytosol, which is performed by three main transport systems: sarcoplasmic reticulum Ca 2+ -ATPase (SR-A), Na + -Ca 2+ exchanger (NCX) and the so-called slow mechanisms (sarcolemmal Ca 2+ -ATPase and mitochondrial Ca 2+ uptake). To estimate the relative contribution of each system to twitch relaxation, SR Ca 2+ accumulation must be selectively inhibited, usually by the application of high caffeine concentrations. However, caffeine has been reported to often cause changes in membrane potential due to NCX-generated inward current, which compromises the reliability of its use. In the present study, we estimated integrated Ca 2+ fluxes carried by SR-A, NCX and slow mechanisms during twitch relaxation, and compared the results when using caffeine application (Cf-NT) and an electrically evoked twitch after inhibition of SR-A with thapsigargin (TG-TW). Ca 2+ transients were measured in 20 isolated adult rat ventricular myocytes with indo-1. For transients in which one or more transporters were inhibited, Ca 2+ fluxes were estimated from the measured free Ca 2+ concentration and myocardial Ca 2+ buffering characteristics. NCX-mediated integrated Ca 2+ flux was significantly higher with TG-TW than with Cf-NT (12 vs 7 µM), whereas SR-dependent flux was lower with TG-TW (77 vs 81 µM). The relative participations of NCX (12.5 vs 8% with TG-TW and Cf-NT, respectively) and SR-A (85 vs 89.5% with TG-TW and Cf-NT, respectively) in total relaxation-associated Ca 2+ flux were also significantly different. We thus propose TG-TW as a reliable alternative to estimate NCX contribution to twitch relaxation in this kind of analysis.

Length-Dependent Activation of Contractility and Ca-Transient Kinetics in Auxotonically Contracting Isolated Rat Ventricular Cardiomyocytes

“…Application of thapsigargin at 25°C had a similar negative inotropic effect but principally different lusitropic effect as well as effects on Ca-transient kinetics, compared to 35°C in normal saline. Thapsigargin, a non-competitive inhibitor of SERCA, prevents Ca 2+ repletion of SR and markedly attenuates and prolongs Ca-transient (Bassani and Bassani, 2003). These changes in intracellular Ca 2+ kinetics strongly modulate the contractile response of cardiac cell via EC-coupling and mechano-calcium feedback (Eisner et al, 2017).…”

“…An increase in temperature from 25 to 35°C was chosen to accelerate Ca 2+ uptake by SERCA2a, which is a main contributor to Ca 2+ extrusion from the interfilament space (Bassani et al, 1994;Mackiewicz and Lewartowski, 2006). Thapsigargin, in contrast, inhibits SERCA2a (Bassani et al, 1994;Treiman et al, 1998;Bassani and Bassani, 2003) and has an opposite effect on the kinetics of free cytosolic Ca 2+ . It is assumed that both conditions have a minor direct effect on the intrinsic properties of contractile machinery compared to Ca 2+ -induced modulation of contraction.…”

Особливості Na+/K+- ТА Н+-АТФазних активностей лімфоцитів периферичної крові жінок, хворих на рак яєчника

“…Визначення загальної ATФазної ензиматичної активності лімфоцитів проводили за +37 °С у середовищі інкубації (об'єм -1 мл) такого складу (мМ): 30 NaCl, 120 KCl, 5 МgС1 2 , 1,5 АТФ, 1 ЕГТА, 1 NaNО 3 (інгібітор мітохондріальної АТФази) [17], 20 Hepes-Tris-буфер 7,4), 0,1 мкМ тапсигаргін (селективний інгібітор Са 2+ ,Mg 2+ -ATФази ендоплазматичного ретикулуму) [18]. Наявність Са 2+ -хелатора ЕГТА в середовищі інкубації забезпечувало зв'язування в ньому ендогенних іонів Са 2+ .…”