2D+T Imaging of Calcium Signaling Microdomains in Cardiac Myocytes

Abstract

Myocyte contraction is coupled with electrical depolarization via the process of excitation contraction coupling (ECC). During ECC, CM depolarization results in opening of voltage-gated Ca channels and calcium influx, which in turn triggers a larger calcium release via RyR calcium release channels on the sarcoplasmic reticulum (SR) store. Central to this process is the close apposition of the SR localized RyRs and plasma membrane voltage-gated calcium channels. This close coupling relies on membranous invaginations of the sarcolemma termed t-tubules, which ensure homogenous calcium elevations throughout the cytosol. In large mammals and in disease, t-tubules are not regularly distributed. Consequently, not all calcium release units (RyR clusters) are directly engaged by calcium influx, leading to potential inhomogeneities in cytosolic calcium. Here we optimized a multi-beam array confocal microscope to image signaling microdomains at high spatial (215 nm pixel size) and temporal resolution (125 fps) in intact pig myocytes. We fitted pixel-by-pixel the time evolution of calcium transients, extracted local key functional parameters and registered these pixel-based calcium changes with WGA stained cell membranes using a descriptor-based algorithm. When correlating functional parameters to the distance to closest membrane, we observed gradients in local calcium handling. Such differences were smaller during prolonged stimulation, suggesting a potential role for functional modulation, possibly through phosphorylation. We complemented live cells analysis with immunofluorescence staining of cells that were fixed after varying the duration of stimulation. We examined location and distribution of RyR clusters, their phosphorylation state and their distance to the closest membrane. We observed a site-specific RyR cluster density as well as the setting of local phosphorylation gradients that evolve with stimulation parameters and over time. Together, these data suggest that spatio-temporal patterns of phosphorylation contribute to the synchrony of calcium signals throughout the cell volume.