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

Pacemaker cells of the sinoatrial (SA) node fire spontaneously and continuously under normal conditions, sustaining a high energetic cost with every heartbeat. How they meet this demand has remained poorly understood. Using genetically encoded fluorescent sensors targeted to the cytosol and mitochondria, we tracked ATP in real time within myocytes of the mouse SA node. Rather than maintaining a steady energy reserve, these cells produce and consume ATP in precise, beat-by-beat bursts in both compartments, synchronized to each Ca2+ transient that triggers a heartbeat-a just-in-time energetic strategy. Not all pacemaker cells operate equally. Cells in the superior SA node, better supplied by blood vessels and rich in mitochondria, produce ATP more efficiently with each beat-a high-gain phenotype. Cells in the inferior node, more sparsely vascularized, operate in a lower gain or energy-deficit mode. These distinct energetic profiles set limits on the firing frequencies each cell can sustain, determining which cells drive fast rates and which support stable rhythm across a broader frequency range. Blocking Ca2+ transfer into mitochondria or impairing ATP export abolished beat-locked energy signals, consistent with mitochondrial Ca2+ uniporter-ANT machinery coupling Ca2+ release to ATP fluctuations. Strikingly, disrupting mitochondrial energy production rendered pacemaker cells electrically silent, suggesting that mitochondrial ATP synthesis is essential for excitability. Together, these findings suggest that beat-locked, just-in-time ATP generation is integral to cardiac pacemaking itself, with local blood supply, mitochondrial capacity, and Ca2+ signaling shaping which cells preferentially set heart rate and which support stable firing across a broader frequency range.