Beat-locked ATP microdomains in the sinoatrial node map a Ca ²⁺ -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 beat-by-beat in these cells remains 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. 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. Mechanistic dissection placed sarcoplasmic reticulum (SR) Ca ²⁺ release upstream of ATP production, showing that Ca ²⁺ triggers metabolic transients while membrane voltage primarily modulates their frequency. Inhibiting mitochondrial Ca ²⁺ uptake and adenine nucleotide exchange eliminated beat-locked mito- and cyto-ATP signals, indicating that the mitochondrial Ca ²⁺ uniporter (MCU)–adenine nucleotide translocase (ANT) machinery couples Ca ²⁺ release to ATP fluctuations. Mode 2 recovery kinetics indicate slower ATP replenishment, which would favor low-frequency, fluctuation-rich firing in a subset of cells. Together, these findings reveal beat-locked metabolic microdomains in which Ca ²⁺ transients time oxidative phosphorylation under a local O ₂ ceiling, unifying vascular architecture, mitochondrial organization, and Ca ²⁺ signaling to match energy supply to excitability. This energetic hierarchy helps explain why some pacemaking myocytes are more likely to set the rate, whereas others may widen the bandwidth.