Synchronized cardiac impulses emerge from multi-scale, heterogeneous local calcium signals within and among cells of heart pacemaker tissue

ABSTRACT Background The current paradigm of Sinoatrial Node (SAN) impulse generation: (i) is that full-scale action potentials (APs) of a common frequency are initiated at one site and are conducted within the SAN along smooth isochrones; and (ii) does not feature fine details of Ca 2+ signalling present in isolated SAN cells, in which small subcellular, subthreshold local Ca 2+ releases (LCRs) self-organize to generate cell-wide APs. Objectives To study subcellular Ca 2+ signals within and among cells comprising the SAN tissue. Methods We combined immunolabeling with a novel technique to detect the occurrence of LCRs and AP-induced Ca 2+ transients (APCTs) in individual pixels (chonopix) across the entire mouse SAN images. Results At high magnification, Ca 2+ signals appeared markedly heterogeneous in space, amplitude, frequency, and phase among cells comprising an HCN4 + /CX43 - cell meshwork. The signalling exhibited several distinguishable patterns of LCR/APCT interactions within and among cells. Apparently conducting rhythmic APCTs of the meshwork were transferred to a truly conducting HCN4 - /CX43 + network of straited cells via narrow functional interfaces where different cell types intertwine, i.e. the SAN anatomical/functional unit. At low magnification, the earliest APCT of each cycle occurred within a small area of the HCN4 meshwork and subsequent APCT appearance throughout SAN pixels was discontinuous. Conclusions We have discovered a novel, microscopic Ca 2+ signalling paradigm of SAN operation that has escaped detection using low-resolution, macroscopic tissue isochrones employed in prior studies: APs emerge from heterogeneous subcellular subthreshold Ca 2+ signals, resembling multiscale complex processes of impulse generation within clusters of neurons in neuronal networks. Condensed abstract By combining immunolabeling with a novel optical technique we detected markedly heterogenous Ca 2+ signals within and among cell clusters of an HCN4 + /CX43 - meshwork in mouse sinoatrial node. These Ca 2+ signals self-organized and transferred, throughout the node, to projections from an HCN4 - /CX43 + network connected to a highly organized, rapidly conducting part of the CX43 + network. Thus, APs emerge from heterogeneous, subthreshold Ca 2+ signaling not detected in low-resolution macroscopic isochrones. Our discovery requires a fundamental paradigm shift from concentric impulse propagation initiated within a leading site, to a multiscale/complex process, resembling the emergence of organized signals from heterogeneous local signals within neuronal networks.