Multi-parametric imaging of spatio-temporal cAMP signaling, transmembrane potential, and intracellular calcium in the Langendorff-perfused heart

Summary The spatiotemporal dynamics of intracellular second messengers and signaling molecules, including cyclic adenosine monophosphate (cAMP), have been studied extensively in isolated cardiomyocytes using Förster resonance energy transfer (FRET)-based reporters. However, little is known about how molecular signaling events affect tissue-level function. Optical mapping of transmembrane potential (V m ) and intracellular calcium (Ca 2+ i ) is frequently performed in isolated intact hearts to reveal tissue-level electrophysiological function but cannot reveal molecular underpinnings. Here, we developed a novel multi-parametric optical imaging system that enables multi-parametric recording of four wavelengths to visualize cAMP and real-time electrophysiological responses (V m and Ca 2+ i ) by utilizing concurrent FRE T imaging and d ual o ptical m apping (FRE-DOM) in the intact heart from a cardiac-specific FRET-based cAMP reporter mouse. We showed that cAMP is strongly and heterogeneously activated throughout the heart (atria and ventricles) in response to β-AR stimulation, the time course of which matches heart rate, action potential, and Ca 2+ responses. This novel imaging system will provide insight into the relationship between cAMP signaling, electrophysiology, and arrhythmogenesis at high spatio-temporal resolution.