Accurate measurements of the time course of Ca²⁺ transients (CaTs) during cardiac action potentials are essential for our understanding of the underlying physiology.  This is particularly important for evaluation of Ca²⁺ flux balance across the surface and intracellular membranes.  Fluorescent probes for Ca²⁺ have been crucial for the measurement of CaTs.  However, all experimental methods have their limitations and the kinetics of Ca²⁺ binding to the probe will inevitably influence the time course of the measured CaT.  Greater Ca²⁺ binding would be expected to slow the signal. The dissociation equilibrium constant (K_d) for Ca²⁺ binding to the probe gives some indication of the extent of this disturbance (although the magnitude of forward and backward rate constants for binding will be important as well as their ratio), and stronger binding of Ca²⁺ is expected to cause greater slowing of CaT time course.  The commonly used Fura-2 shows strong Ca²⁺ binding with a K_d of approximately 0.14 μM.  CaTs measured with Fura-2 show very little decline during the action potential plateau (e.g. Banyasz et al, 2012).  Fluo-3 (Kd = 0.39 μM) gives a slighter faster time course though still slow, while Fluo-5F (Kd 2.3 μM) gave CaTs with a more rapid rapid upstroke and decay (e.g. Capel et al, 2015).  Rhod-2 (Kd = 0.57 μM) gave rapid upstroke and decay both in isolated myocytes (Fenandez-Tenorio & Niggli, 2016) and whole hearts (Nemec et al 2010).

The experiments presented here were in whole hearts and isolated myocytes.  CaTs were measured in ventricular muscle in Langendorff-perfused guinea-pig hearts under conditions similar to those previously published (Nemec et al 2010; Lee et al 2012).  Rhod-2 gave a CaT with a peak at around 25 ms (24.6 ± 4.1 ms, n=3) at body temperature, recorded from the left ventricle.  This is similar to that measured in previous experiments in Langendorff hearts (Lee et al, 2012, Nemec et al 2010), and also in isolated ventricular myocytes (Fenandez-Tenorio & Niggli, 2016).  It might be thought that Rhod-FF with a Kd of 19 μM would give a negligibly small signal, but this was not the case and CaTs were measurable with a peak at approximately 10 ms (10.0 ± 0.9 ms, n=4, significantly different from Rhod-2, P<0.05, two sample t-test).  CaTs and Ca²⁺ sparks were also recorded using Rhod-FF in mouse isolated ventricular myocytes.

The above observations underline the importance of the kinetics of Ca²⁺ binding to the fluorescent probe for the measurement of the time course of CaTs.  The necessity of Ca²⁺ binding to any Ca²⁺ probe will inevitably slow CaTs, but the measurements with Rhod-FF show that the time to peak can be as little as 10 ms, and the time course of CaTs is expected to be even faster in the absence of a Ca²⁺-binding fluorescent probe.