Maximal acceleration of Ca²⁺ release refractoriness by β-adrenergic stimulation requires dual activation of kinases PKA and CaMKII in mouse ventricular myocytes

Key points: Refractoriness of calcium release in heart cells is altered in several disease states, but the physiological mechanisms that regulate this process are incompletely understood. We examined refractoriness of calcium release in mouse ventricular myocytes and investigated how activation of different intracellular signalling pathways influenced this process. We found that refractoriness of calcium release is abbreviated by stimulation of the 'fight-or-flight' response, and that simultaneous activation of multiple intracellular signalling pathways contributes to this response. Data obtained under several conditions at the subcellular, microscopic level were consistent with results obtained at the cellular level. The results provide insight into regulation of cardiac calcium release and how alterations to this process may increase arrhythmia risk under different conditions. Time-dependent refractoriness of calcium (Ca²⁺) release in cardiac myocytes is an important factor in determining whether pro-arrhythmic release patterns develop. At the subcellular level of the Ca²⁺ spark, recent studies have suggested that recovery of spark amplitude is controlled by local sarcoplasmic reticulum (SR) refilling whereas refractoriness of spark triggering depends on both refilling and the sensitivity of the ryanodine receptor (RyR) release channels that produce sparks. Here we studied regulation of Ca²⁺ spark refractoriness in mouse ventricular myocytes by examining how β-adrenergic stimulation influenced sequences of Ca²⁺ sparks originating from individual RyR clusters. Our protocol allowed us to separately measure recovery of spark amplitude and delays between successive sparks, and data were interpreted quantitatively through simulations with a stochastic mathematical model. We found that, compared with spark sequences measured under control conditions: (1) β-adrenergic stimulation with isoproterenol (isoprenaline) accelerated spark amplitude recovery and decreased spark-to-spark delays; (2) activating protein kinase A (PKA) with forskolin accelerated amplitude recovery but did not affect spark-to-spark delays; (3) inhibiting PKA with H89 retarded amplitude recovery and increased spark-to-spark delays; (4) preventing phosphorylation of the RyR at serine 2808 with a knock-in mouse prevented the decrease in spark-to-spark delays seen with β-adrenergic stimulation; (5) inhibiting either PKA or Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) during β-adrenergic stimulation prevented the decrease in spark-to-spark delays seen without inhibition. The results suggest that activation of either PKA or CaMKII is sufficient to speed SR refilling, but activation of both kinases appears necessary to observe increased RyR sensitivity. The data provide novel insight into β-adrenergic regulation of Ca²⁺ release refractoriness in mouse myocytes.