Abstract
In cardiac myocytes, calcium sparks exhibit time-dependent refractoriness such that triggering of a second spark soon after an initial spark has terminated is improbable. Recent studies in rat ventricular myocytes suggested that spark amplitude recovery is controlled by local sarcoplasmic reticulum (SR) refilling whereas refractoriness of spark triggering depends on both refilling and ryanodine receptor (RyR) sensitivity.Here we examined spark refractoriness in mouse ventricular myocytes by exposing Fluo-3 loaded quiescent cells to 50 nM ryanodine, recording sparks with a confocal microscope, and analyzing the repeated sparks that were produced at singular RyR clusters.Beta-adrenergic stimulation accelerated spark amplitude recovery and decreased median spark-to-spark delay compared to control. Spark amplitude recovery was also accelerated/decelerated, respectively, by either activating or inhibiting PKA with forskolin or H89. Spark-to-spark delays were not affected by forskolin but increased in H89. Experiments performed on S2808A mice with a mutated PKA phosphorylation site corroborate these results.Forskolin results suggest that during beta-adrenergic stimulation pathways other than PKA can be activated. Beta-adrenergic stimulation in the presence of PKA or CaMKII blockers (H89 and KN93, respectively) were studied. In both conditions, time constant of amplitude recovery was increased and spark-to-spark delay was slightly higher than control. Employing a mathematical model allowed us to gain further insight into experimental results. Under control conditions SR refilling is enhanced due to activation of SERCA via endogenous PKA activity. Additional activation of either PKA or CaMKII is sufficient to accelerate spark amplitude restitution through faster refilling. However, inhibition of either pathway prevents RyR sensitivity to be increased. Thus, activity of both kinases is necessary to explain the changes in RyR gating observed during beta-adrenergic stimulation.
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