ESTIMATION OF EC-COUPLING EFFICACY.

Nicolas Lindegger, Ernst Niggli
University of Bern, Institute of Physiology.

In cardiac muscle, Ca2+ entering the cell via L-type Ca2+ channels triggers Ca2+ release from the sarcoplasmic reticulum (SR) through ryanodine receptors (RyRs) by Ca2+-induced Ca2+ release (CICR). Changes in efficacy, amplification and positive feedback of the CICR may result from regulatory mechanisms (e.g. b-adrenergic stimulation, change in SR Ca2+ load). In addition, several cardiac diseases may impair CICR efficacy (e.g. heart failure).
Two photon photolysis (TPP) of caged Ca2+ (DM-Nitrophen) was used as a diffraction-limited trigger for CICR in guinea pig ventricular cells. The Ca2+ signals were simultaneously recorded with Fluo-3 using laser-scanning confocal microscopy. To determine the efficacy of CICR, we rapidly elicited Ca2+ signals with trains of increasing photolytic power (power-response curve). Droplet test showed a nearly quadratic relation between Ca2+ signals and TPP power while experiments in cells showed a sigmoidal curve with saturation at high photolytic power (90 mW, Hill coefficient: 1.8±0.9). Under high SR Ca2+ load conditions Ca2+ release signals had larger amplitudes and the dose-response curve of TPP was shifted to the left, suggesting an increase in the Ca2+ sensitivity of CICR. Similarly, after ß-adrenergic stimulation, TPP elicited larger Ca2+ signals. Thus, dose-response curves of Ca2+ signals triggered by TPP provide a rapid measurement of CICR sensitivity.
In conclusion, changes of SR Ca2+ load and/or enhanced Ca2+ sensitivity after RyR phosphorylation could account for the observed changes of EC-coupling efficacy.