Abstract Body: Background: Energetic demands from ion transport and cross-bridge cycling are known, yet spatiotemporal ATP supply/demand dynamics during excitation-contraction coupling are poorly understood and often modeled as a stable ATP pool. We tested the hypothesis that ATP is produced and distributed in beat-synchronized microdomains, and tuned by sex-specific mitochondrial architecture. Methods: Adult mouse ventricular myocytes (♀,♂) were imaged by confocal microscopy using genetically encoded ATP sensors targeted to mitochondrial matrix and cytosol. Beat-locked ATP dynamics were quantified during field stimulation (1-2 Hz). Mitochondrial Ca²⁺ uptake and nucleotide exchange were inhibited with Ru360 and bongkrekic acid, respectively. 3D confocal segmentation quantified mitochondrial volume; SR-mitochondrial proximity and mitochondrial enrichment of mitofusin-2 (Mfn2) and ATP synthase α-subunit (ATP5A) were assessed. Results: Mitochondrial ATP fluctuated with every beat in spatially confined Mode 1 (M1) “gain” and Mode 2 (M2) “dip” microdomains (M1: ♀1.53±0.27, ♂1.46±0.20 F/F₀; M2: ♀0.71±0.08 /♂0.70±0.09 F/F₀). Ru360 markedly suppressed beat-locked [ATP]_mito oscillations (M1: ♀1.22±0.18, ♂1.24±0.11 F/F₀; M2: ♀0.79±0.05, ♂0.78±0.05 F/F₀, P<0.05), and ANT inhibition similarly reduced transients. Females exhibited lower mitochondrial volume (6391±1700 vs 8192±1264 μm³; P<0.05) but tighter SR-mitochondrial apposition (59±7% vs 47±9%; P<0.05) and higher mitochondrial Mfn2 and ATP5A density. With increased pacing, diastolic [ATP]_i rose to a similar plateau (♀1.22±0.11; ♂1.25±0.07 F/F₀), but females reached steady-state faster (3.62±0.77 vs 4.72±0.94 min; P<0.05). Beat-locked ATP gain amplitude increased in both sexes yet scaled more in males (e.g., M1: ♂587±58→811±111 μM vs ♀548±31→722±57 μM; P<0.05). Conclusions: Ventricular myocytes operate “paycheck-to-paycheck”, generating ATP on-demand in beat-synchronized microdomains that require MCU-driven Ca²⁺ entry and ANT-mediated nucleotide exchange. Females appear to optimize energetic delivery via tighter SR-mitochondrial coupling and higher per-mitochondrion catalytic capacity, whereas males scale beat-linked ATP swings via greater mitochondrial mass.