In cardiac excitation-contraction coupling, L-type Ca2+ current (ICa) triggers Ca2+ release from the sarcoplasmic reticulum (SR) via ryanodine receptor (RyR) Ca2+ release channels. It is unclear why SR Ca2+ release cannot be elicited by premature stimuli, even though ICa is fully recovered. Here, we use calsequestrin null mice (Casq2 KO) and wild-type littermates (WT) to test the hypothesis that calsequestrin (Casq2) determines refractoriness of SR Ca2+ release. Ca2+ release refractoriness was measured in voltage-clamped myocytes dialyzed with Fluo-4 by applying premature extrastimuli (S2) at successively shorter S1-S2 coupling intervals following a 1 Hz train (S1 stimuli). To maintain constant trigger, Ca2+ release was activated with ICa tail currents that elicited maximal Ca2+ release during the S1 train. WT S2 Ca2+release was significantly depressed with short coupling interval whereas Casq2 KO cardiomyocytes exhibit no refractoriness of Ca2+ release (Figure, n=11 WT, 12 KO, p = 0.01). At the same time, ICa current density, SR Ca2+ content, and steady-state Ca2+ transients (S1) were not significantly different from WT-myocytes. We conclude that calsequestrin is a critical determinant of SR Ca2+ release refractoriness in cardiac muscle (Supported by NIH-R01HL71670, R01HL88635).View Large Image | View Hi-Res Image | Download PowerPoint Slide