Modulation of ryanodine receptor open probability has no maintained effect on the amplitude of systolic calcium release in sheep or dog ventricular myocytes

Greensmith, David; Trafford, Andrew; Dibb, Katherine; Eisner, David

Abstract

Caffeine is frequently used as an agent to increase the open probability of the Ryanodine receptor (RyR). However, our group has previously demonstrated that in rat, application of 500 µM caffeine has no sustained effect on systolic Ca. Rather, following an initial increase, the intracellular calcium ([Ca2+]i) transient amplitude returns to that of control steady state in around 20 s (Trafford et al., 2000). This temporary potentiation was due to a concurrent decrease in SR Ca, which was measured at steady state but calculated on a beat by beat basis. Our aim was to determine if the above phenomenon could also be observed in larger mammals. In addition, we measured beat by beat SR Ca in real time using the low affinity Ca indicator Mag-Fura-2. Young (~18 months) Sheep were killed in accordance with The Home Office Animal (Scientific Procedures) Act 1986 for enzymatic isolation of left ventricular mid myocardial myocytes. Dog ventricular myocytes were kindly provided by Michael Morton (Astra Zeneca). Myocytes were either loaded with Fura-2 for cytoplasmic Ca measurement or co-loaded with Fluo-3 and Mag-Fura-2 for cytoplasmic and SR Ca measurement respectively. Myocytes were current clamped via perforated patch and paced at 0.5 Hz. Sustained application of 500 µM caffeine in sheep myocytes initially increased the [Ca2+]i transient amplitude which subsequently returned to control levels. The rate at which the [Ca2+]i transient amplitude decreased following potentiation was faster when compared to rat, typically occurring within 5 beats. In dog myocytes loaded with Fluo-3 and Mag-Fura-2, sustained application of 500 µM caffeine also resulted in a temporary potentiation of the [Ca2+]i transient amplitude, which returned to that of control at around the same rate as those in sheep. We also observed an initial increase in the amplitude of SR Ca loss in the presence of 500 µM caffeine, which returned to that of control. The decrease of the [Ca2+]i transient amplitude and the amplitude of SR Ca loss occurred at the same rate. In the presence of caffeine, SR Ca content was decreased to a lower steady state, agreeing qualitatively with the calculated SR Ca reduction observed in rat. These data demonstrate that modulation of RyR open probability has no maintained effect on the [Ca2+]i transient amplitude, even in larger mammals. The direct measurement of SR Ca during these experiments also confirms that SR Ca loss underlies the post potentiation reduction of systolic Ca, compensating for a sustained increase in RyR open probability and allowing the myocyte to maintain a constant inotropy. The rapid rate at which the [Ca2+]i transient returns to control values in these larger mammals when compared to rat is presumably due to a greater dependence on sodium calcium exchange.

• Presentation Conference Type: Conference Abstract
• Conference Name: New insights into the molecular basis of cardiac arrhythmias: from animal models to computations
• Start Date: Sep 4, 2012
• End Date: Sep 6, 2012
• Acceptance Date: Sep 4, 2012
• Online Publication Date: Sep 4, 2012
• Publication Date: Sep 4, 2012
• Deposit Date: Jun 24, 2025
• Peer Reviewed: Peer Reviewed
• Article Number: PC28
• Series Number: 28
• Book Title: Proceedings of The Physiological Society
• Publisher URL: https://www.physoc.org/abstracts/modulation-of-ryanodine-receptor-open-probability-has-no-maintained-effect-on-the-amplitude-of-systolic-calcium-release-in-sheep-or-dog-ventricular-myocytes/