Beat-locked ATP microdomains in the sinoatrial node map a Ca²⁺-timed energetic hierarchy and regional pacemaker roles

Pacemaker myocytes of the sinoatrial (SA) node initiate each heartbeat through coupled voltage and Ca²⁺ 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 Ca²⁺ transient and segregated into high- and low-gain phenotypes defined by the Ca²⁺–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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ clock times oxidative phosphorylation under a local O₂ ceiling, unifying vascular architecture, mitochondrial organization, and Ca²⁺ 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.