Beat-to-beat ATP synthesis is coupled to intracellular Ca ²⁺ release through distinct high- and low-gain metabolic transfer functions in SA node myocytes. (A) Representative confocal image of an isolated SA node myocyte expressing the cytosolic ATP sensor (cyto-iATP). Scale bar, 5 µm. (B and C) Simultaneous confocal line-scan recordings of intracellular Ca²⁺ (red, top) and cyto-iATP (green, bottom). The dashed line indicates baseline fluorescence (F/F₀ = 1). (B) Example of a myocyte classified as high-gain, showing temporally aligned Ca²⁺ transients and cyto-iATP signals. Traces extracted from local ROIs (colored traces) illustrate spatially resolved Ca²⁺ and ATP dynamics. (C) Example of a myocyte classified as low-gain, showing Ca²⁺ transients with minimal corresponding cyto-iATP signals. (D) Event-level relationship between Ca²⁺ signal mass (input) and cyto-iATP signal mass (output). Data are separated into two populations: high-gain events (blue circles) and low-gain events (orange circles). Solid lines indicate linear fits for each population. (E) Cell-averaged relationship between Ca²⁺ and cyto-iATP signal mass, preserving bimodal separation between high-gain (blue) and low-gain (orange) groups. (F and G) Summary quantification of Ca²⁺ signal mass rate (F) and cyto-iATP signal mass rate (G) for high- and low-gain populations. P values are shown above comparisons. Large circles denote per-animal means; small circles indicate individual biological replicates.