Synchronized Cardiac Impulses Emerge From Heterogeneous Local Calcium Signals Within and Among Cells of Pacemaker Tissue

Objectives: This study sought to identify subcellular Ca²⁺ signals within and among cells comprising the sinoatrial node (SAN) tissue. Background: The current paradigm of SAN impulse generation: 1) is that full-scale action potentials (APs) of a common frequency are initiated at 1 site and are conducted within the SAN along smooth isochrones; and 2) does not feature fine details of Ca²⁺ signaling present in isolated SAN cells, in which small subcellular, subthreshold local Ca²⁺ releases (LCRs) self-organize to generate cell-wide APs. Methods: Immunolabeling was combined with a novel technique to detect the occurrence of LCRs and AP-induced Ca²⁺ transients (APCTs) in individual pixels (chronopix) 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 signaling exhibited several distinguishable patterns of LCR/APCT interactions within and among cells. Rhythmic APCTs that were apparently conducted within the meshwork were transferred to a truly conducting HCN4⁻/CX43⁺ network of striated cells via narrow functional interfaces where different cell types intertwine, that is, the SAN anatomic/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 and asynchronous. Conclusions: The study has discovered a novel, microscopic Ca²⁺ signaling paradigm of SAN operation that has escaped detection using low-resolution, macroscopic tissue isochrones employed in prior studies: synchronized APs emerge from heterogeneous subcellular subthreshold Ca²⁺ signals, resembling multiscale complex processes of impulse generation within clusters of neurons in neuronal networks.