Analysis of Local Calcium Fluctuations in Cardiac Myocytes

Calcium sparks are key to muscle function; activation of ∼104 of these local calcium release events within each cardiac myocyte enables contraction with every heartbeat. Each calcium spark results from the concerted opening of a cluster of ryanodine receptors (RyRs) located on the sarcoplasmic reticulum. Though RyRs are activated by a positive-feedback mechanism (calcium-induced calcium release), calcium sparks show robust termination (a stereotypic calcium spark lasts ∼40 ms) in most cases. Much longer calcium sparks (up to seconds) have been observed in ventricular myocytes from animal models of heart failure and when RyRs have been pharmacologically inhibited. While the underlying mechanism for release prolongation remains unclear, calcium (dye signal) fluctuations present during the plateau of such long-lasting events hints at the underlying RyR gating activity. Using the intracellular fluorescent calcium indicator, fluo-4, we examined calcium sparks in cells treated with a low dose of tetracaine to partially inhibit RyRs and promote long-lasting calcium release events. Image analysis of release origin was unable to detect a change in location during the long-lasting event, while noise analysis was used to deduce the ensemble RyR gating rates. Finally, a computer model that incorporates calcium binding, diffusion and RyR gating was able to reproduce long-lasting events when the number of stochastically gating RyRs available was reduced and SR calcium content was increased. Large alterations in RyR cluster geometry were not required, but sub-clustering can alter fluctuation properties.