Paradoxical SR Ca2+ release in guinea-pig cardiac myocytes after β-adrenergic stimulation revealed by two-photon photolysis of caged Ca2+

In heart muscle the amplification and shaping of Ca²⁺ signals governing contraction are orchestrated by recruiting a variable number of Ca²⁺ sparks. Sparks reflect Ca²⁺ release from the sarcoplasmic reticulum (SR) via Ca²⁺ release channels (ryanodine receptors, RyRs). RyRs are activated by Ca²⁺ influx via L-type Ca²⁺ channels with a specific probability that may depend on regulatory mechanisms (e.g. β-adrenergic stimulation) or diseased states (e.g. heart failure). Changes of RyR phosphorylation may be critical for both regulation and impaired function in disease. Using UV flash photolysis of caged Ca²⁺ and short applications of caffeine in guinea-pig ventricular myocytes, we found that Ca²⁺ release signals on the cellular level were largely governed by global SR content. During β-adrenergic stimulation resting myocytes exhibited smaller SR Ca²⁺ release signals when activated by photolysis (62.3% of control), resulting from reduced SR Ca²⁺ content under these conditions (58.6% of control). In contrast, local signals triggered with diffraction limited two-photon photolysis displayed the opposite behaviour, exhibiting a larger Ca²⁺ release (164% of control) despite reduced global and local SR Ca²⁺ content. This apparent paradox implies changes of RyR open probabilities after β-adrenergic stimulation, enhancing local regenerativity and reliability of Ca²⁺ signalling. Thus, our results underscore the importance of phosphorylation of RyRs (or of a related protein), as a regulatory physiological mechanism that may also provide new therapeutic avenues to recover impaired Ca²⁺ signalling during cardiac disease.