Sarcoplasmic reticulum (SR) Ca²⁺ cycling is tightly regulated by ryanodine receptor (RyR) Ca²⁺ release and sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) Ca²⁺ uptake during each excitation-contraction coupling cycle. We previously showed that RyR refractoriness plays a key role in the onset of SR Ca²⁺ alternans in the intact rabbit heart, which contributes to arrhythmogenic action potential duration (APD) alternans. Recent studies have also implicated impaired SERCA function, a key feature of heart failure, in cardiac alternans and arrhythmias. However, the relationship between reduced SERCA function and SR Ca²⁺ alternans is not well understood. Simultaneous optical mapping of transmembrane potential (V_m) and SR Ca²⁺ was performed in isolated rabbit hearts (n = 10) using the voltage-sensitive dye RH237 and the low-affinity Ca²⁺ indicator Fluo-5N-AM. Alternans was induced by rapid ventricular pacing. SERCA was inhibited with cyclopiazonic acid (CPA; 1-10 μM). SERCA inhibition (1, 5, and 10 μM of CPA) resulted in dose-dependent slowing of SR Ca²⁺ reuptake, with the time constant (tau) increasing from 70.8 ± 3.5 ms at baseline to 85.5 ± 6.6, 129.9 ± 20.7, and 271.3 ± 37.6 ms, respectively (p < 0.05 vs. baseline for all doses). At fast pacing frequencies, CPA significantly increased the magnitude of SR Ca²⁺ and APD alternans, most strongly at 10 μM (pacing cycle length = 220 ms: SR Ca²⁺ alternans magnitude: 57.1 ± 4.7 vs. 13.4 ± 8.9 AU; APD alternans magnitude 3.8 ± 1.9 vs. 0.2 ± 0.19 AU; p < 0.05 10 μM of CPA vs. baseline for both). SERCA inhibition also promoted the emergence of spatially discordant alternans. Notably, at all CPA doses, alternation of SR Ca²⁺ release occurred prior to alternation of diastolic SR Ca²⁺ load as pacing frequency increased. Simultaneous optical mapping of SR Ca²⁺ and V_m in the intact rabbit heart revealed that SERCA inhibition exacerbates pacing-induced SR Ca²⁺ and APD alternans magnitude, particularly at fast pacing frequencies. Importantly, SR Ca²⁺ release alternans always occurred before the onset of SR Ca²⁺ load alternans. These findings suggest that even in settings of diminished SERCA function, relative refractoriness of RyR Ca²⁺ release governs the onset of intracellular Ca²⁺ alternans.

Cyclic adenosine 3',5'-monophosphate (cAMP) is a key second messenger in cardiomyocytes responsible for transducing autonomic signals into downstream electrophysiological responses. Previous studies have shown intracellular heterogeneity and compartmentalization of cAMP signaling. However, whether cAMP signaling occurs heterogeneously throughout the intact heart and how this drives sex-dependent functional responses are unknown. Here, we developed and validated a novel cardiac-specific fluorescence resonance energy transfer-based cAMP reporter mouse and a combined voltage-cAMP whole-heart imaging system. We showed that in male hearts, cAMP was uniformly activated in response to pharmacological β-adrenergic stimulation. In contrast, female hearts showed that cAMP levels decayed faster in apical versus basal regions, which was associated with nonuniform action potential changes and notable changes in the direction of repolarization. Apical phosphodiesterase (PDE) activity was higher in female versus male hearts, and PDE inhibition prevented repolarization changes in female hearts. Thus, our imaging approach revealed sex-dependent regional breakdown of cAMP and associated electrophysiological differences.