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

ABSTRACTBackground 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 Ca2+ signalling present in isolated SAN cells, in which small subcellular, subthreshold local Ca2+ releases (LCRs) self-organize to generate cell-wide APs.Objectives To study subcellular Ca2+ 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 Ca2+ transients (APCTs) in individual pixels (chonopix) across the entire mouse SAN images.Results At high magnification, Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+signals within and among cell clusters of an HCN4+/CX43- meshwork in mouse sinoatrial node. These Ca2+ 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 Ca2+ 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.Competing Interest StatementThe authors have declared no competing interest.Abbreviations listAPaction potentialAPCTAP-induced Ca2+ transientChronopixchrono-pixelCX43Connexin 43 (Gap junction alpha-1 protein)FFTFast Fourier TransformHCN4Hyperpolarization-Activated Cyclic Nucleotide-Gated Channel 4ICaLL-type Ca2+ currentICaTT-type Ca2+ currentIVCInferior Vena CavaLCRLocal Ca2+ ReleaseNCXNa+/Ca2+ exchangerPBSPhosphate-Buffered SalinepreBötCpreBötzinger ComplexROIRegion of InterestSANSinoatrial NodeSVCSuperior Vena CavaView Full Text