Oxidative phosphorylation drives distinct modes of beat-to-beat ATP dynamics in SA node mitochondria. (A) Representative confocal image of an isolated SA node myocyte co-expressing mito-iATP (green) and labeled with MitoTracker (red), illustrating mitochondrial localization of the mito-ATP sensor. Scale bar, 5 µm. (B and C) Representative confocal line-scan images and corresponding normalized fluorescence traces (F/F₀) from superior (B) and inferior (C) SA node regions. The dashed line indicates baseline fluorescence (F/F₀ = 1). Two classes of mito-iATP signals are resolved: Mode 1 transients, characterized by positive deflections, and Mode 2 transients, characterized by negative deflections. Traces acquired after application of the mitochondrial uncoupler FCCP (1 µM) are shown for comparison. (D–F) Summary quantification of Mode 1 mito-iATP dynamics, including signal mass rate (D), peak amplitude (E), and transient frequency (F). (G–I) Summary quantification of Mode 2 mito-iATP dynamics, including signal mass rate (G), peak amplitude (H), and transient frequency (I). P values are shown above comparisons. Data are presented as means ± SEM (N = 6 mice per group). Large circles denote per-animal means; small circles indicate individual biological replicates. N represents the number of independent mice.