Simultaneous measurement of cytoplasmic and SR calcium during modulation of ryanodine receptor open probability in canine ventricular myocytes

Greensmith, David; Galli, Gina; Morton, Michael; Pollard, Christoper; Trafford, Andrew; Eisner, David

Abstract

We have previously shown in rat ventricular myocytes that modulation of ryanodine receptor (RyR) open probability using low concentrations of caffeine has no maintained effect on the amplitude of systolic Ca (Trafford et al, 2000). We suggested that the increased RyR open probability was compensated for as the initial increase in systolic Ca enhanced sarcolemmal Ca efflux thus reducing SR Ca content. Here we present data that address two outstanding issues. (1) Are similar results seen in a large animal model? (2) We wished to measure SR Ca content directly rather than, as in our previous work, inferring it from sarcolemmal Ca fluxes. Canine ventricular myocytes were loaded with Fluo-3 for cytoplasmic Ca measurement or co-loaded with Fluo-3 and the low-affinity Ca indicator, Mag-Fura-2 for cytoplasmic and intra SR Ca measurement respectively and paced at 0.5 Hz under voltage or current clamp. Under voltage clamp, application of 0.5 mM caffeine initially increased the amplitude of systolic Ca by 115 %. This was associated with a 19 % decrease in Ca influx on the L-type Ca current and a 145 % increase in sarcolemmal Ca efflux. However, within 3 or 4 beats in caffeine, all these parameters recovered to control levels. Upon caffeine washout, we observed an under shoot in the amplitude of systolic Ca which subsequently recovered to control levels. In the steady state, 0.5 mM caffeine reduced SR Ca content (quantified by application of 10 mM caffeine) by 47 % which subsequently recovered during washout. To directly measure beat by beat SR Ca, myocytes were co-loaded with Fluo-3 and Mag-Fura-2 and paced under current clamp. Upon caffeine application, a 154 % potentiation of systolic Ca was associated with a 755 % increase in the magnitude of systolic SR Ca depletion. Both these parameters rapidly recovered to control levels during caffeine application, coinciding with a beat by beat reduction of SR Ca to 84 % of control. Upon washout, an initial 41 % decrease in systolic Ca was associated with a 55 % decrease in the magnitude of systolic SR Ca depletion. Both these parameters and SR Ca content subsequently recovered to control levels. These data demonstrate that in a large animal model, modulation of RyR open probability has no maintained effect on the amplitude of systolic Ca. Increasing RyR open probability initially increases systolic Ca leading to reduced Ca influx and enhanced Ca efflux and so a net loss of Ca from the cell. This results in a beat by beat loss of SR Ca to a level that compensates for the increased RyR open probability, at which point the amplitude of systolic Ca is restored. The converse is true during the relative decrease of RyR open probability during wash.

• Presentation Conference Type: Conference Abstract
• Conference Name: 37th Congress of the International Union of Physiological Sciences (IUPS)
• Start Date: Jul 21, 2013
• End Date: Jul 26, 2013
• Acceptance Date: Jul 21, 2013
• Online Publication Date: Jul 21, 2013
• Publication Date: Jul 21, 2013
• Deposit Date: Jun 24, 2025
• Peer Reviewed: Peer Reviewed
• Article Number: PCA053
• Publisher URL: https://www.physoc.org/abstracts/simultaneous-measurement-of-cytoplasmic-and-sr-calcium-during-modulation-of-ryanodine-receptor-open-probability-in-canine-ventricular-myocytes/