Local recovery of cardiac calcium‐induced calcium release interrogated by ultra‐effective, two‐photon uncaging of calcium

Abstract

In cardiac myocytes Ca²⁺ -induced Ca²⁺ release (CICR) from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs) governs activation of contraction. Ca²⁺ release occurs via subcellular Ca²⁺ signalling events, Ca²⁺ sparks. Local recovery of Ca²⁺ release depends on both SR refilling and restoration of Ca²⁺ sensitivity of the RyRs. We used two-photon (2P) photolysis of the ultra-effective caged Ca²⁺ compound BIST-2EGTA and laser-scanning confocal Ca²⁺ imaging to probe refractoriness of local Ca²⁺ release in control conditions and in the presence of cAMP or low-dose caffeine (to stimulate CICR) or cyclopiazonic acid (CPA; to slow SR refilling). Permeabilized cardiomyocytes were loaded with BIST-2EGTA and rhod-2. Pairs of short 2P photolytic pulses (1 ms, 810 nm) were applied with different intervals to test Ca²⁺ release amplitude recovery and trigger probability for the second spark in a pair. Photolytic and biological events were distinguished by classification with a self-learning support vector machine (SVM) algorithm. In permeabilized myocytes data recorded in the presence of CPA showed a lower probability of triggering a second spark compared to control or cAMP conditions. Cardiomyocytes from a mouse model harbouring the arrhythmogenic RyR^R420Q mutation were used for further validation and revealed a higher Ca²⁺ sensitivity of CICR. This new 2P approach provides composite information of Ca²⁺ release amplitude and trigger probability recovery reflecting both SR refilling and restoration of CICR and RyR Ca²⁺ sensitivity. It can be used to measure the kinetics of local CICR recovery, alterations of which may be related to premature heart beats and arrhythmias.