Sarcoplasmic reticulum (SR) Ca²⁺ cycling is key to normal excitation–contraction coupling but may also contribute to pathological cardiac alternans and arrhythmia.

To measure intra-SR free [Ca²⁺] ([Ca²⁺]_SR) changes in intact hearts during alternans and ventricular fibrillation (VF).

Simultaneous optical mapping of V_m (with RH237) and [Ca²⁺]_SR (with Fluo-5N AM) was performed in Langendorff-perfused rabbit hearts. Alternans and VF were induced by rapid pacing. SR Ca²⁺ and action potential duration (APD) alternans occurred in-phase, but SR Ca²⁺ alternans emerged first as cycle length was progressively reduced (217±10 versus 190±13 ms; P<0.05). Ryanodine receptor (RyR) refractoriness played a key role in the onset of SR Ca²⁺ alternans, with SR Ca²⁺ release alternans routinely occurring without changes in diastolic [Ca²⁺]_SR. Sensitizing RyR with caffeine (200 μmol/L) significantly reduced the pacing threshold for both SR Ca²⁺ and APD alternans (188±15 and 173±12 ms; P<0.05 versus baseline). Caffeine also reduced the magnitude of spatially discordant SR Ca²⁺ alternans, but not APD alternans, the pacing threshold for discordance, or threshold for VF. During VF, [Ca²⁺]_SR was high, but RyR remained nearly continuously refractory, resulting in minimal SR Ca²⁺ release throughout VF.

In intact hearts, RyR refractoriness initiates SR Ca²⁺ release alternans that can be amplified by diastolic [Ca²⁺]_SR alternans and lead to APD alternans. Sensitizing RyR suppresses spatially concordant but not discordant SR Ca²⁺ and APD alternans. Despite increased [Ca²⁺]_SR during VF, SR Ca²⁺ release was nearly continuously refractory. This novel method provides insight into SR Ca²⁺ handling during cardiac alternans and arrhythmia.