Beat–locked ATP microdomains in the sinoatrial node map a calcium–timed energetic hierarchy and regional pacemaker roles

Pacemaker myocytes of the sinoatrial (SA) node initiate each heartbeat through coupled voltage and Ca 2+ oscillators, but whether ATP supply is regulated on a beat–by–beat schedule in these cells has been unclear. Using genetically encoded sensors targeted to the cytosol and mitochondria, we tracked beat–resolved ATP dynamics in intact mouse SA node and isolated myocytes. Cytosolic ATP rose transiently with each Ca 2+ transient and segregated into high– and low–gain phenotypes defined by the Ca 2+ –ATP coupling slope. Mitochondrial ATP flux adopted two stereotyped waveforms—Mode–1 ″gains″ and Mode–2 ″dips″. Within Mode–1 cells, ATP gains mirrored the cytosolic high/low–gain dichotomy; Mode–2 dips scaled linearly with Ca 2+ load and predominated in slower-firing cells. In the intact node, high–gain/Mode–1 phenotypes localized to superior regions and low–gain/Mode–2 to inferior regions, paralleling gradients in rate, mitochondrial volume, and capillary density. Pharmacology placed the Ca 2+ clock upstream of ATP production: the HCN channel blocker ivabradine slowed the ATP cycle without changing amplitude, whereas the SERCA pump inhibitor thapsigargin or the mitochondrial uncoupler FCCP abolished transients. Mode–2 recovery kinetics indicate slower ATP replenishment that would favor low–frequency, fluctuation–rich firing in a subset of cells. Together, these findings reveal beat-locked metabolic microdomains in which the Ca 2+ clock times oxidative phosphorylation under a local O 2 ceiling, unifying vascular architecture, mitochondrial organization, and Ca 2+ signaling to coordinate energy supply with excitability. This energetic hierarchy helps explain why some SA node myocytes are more likely to set rate whereas others may widen bandwidth.