Demonstration of beat‐to‐beat, on‐demand ATP synthesis in ventricular myocytes reveals sex‐specific mitochondrial and cytosolic dynamics

The energetic demands on ventricular myocytes imposed by the transport of ions and cross-bridge cycling are well known, yet the spatiotemporal dynamics of ATP supply and demand remain poorly understood. Here, using confocal microscopy and genetically encoded fluorescent sensors targeted to mitochondria and cytosol, we visualized beat-to-beat ATP dynamics in ventricular myocytes from male and female mice. These probes showed fluctuations in mitochondrial ATP levels with each contraction, revealing two distinct, spatially localized waveforms - ATP 'gain' and ATP 'dip' - representing transient increases or decreases in matrix ATP levels, respectively. These waveforms were tightly phase-locked to intracellular Ca²⁺ transients and organized into energetic microdomains. Inhibition of the mitochondrial Ca²⁺ uniporter or the adenine nucleotide translocase attenuated these ATP transients. Although female myocytes exhibited larger mitochondrial ATP transients than their male counterparts, their mitochondrial volume was lower. Female myocytes also exhibited tighter coupling between the sarcoplasmic reticulum and mitochondria and showed a higher density of mitofusin 2 and ATP synthase catalytic α-subunit per unit volume, suggesting more efficient ATP production. Cytosolic ATP transients mirrored mitochondrial waveforms and domain structure in both male and female myocytes. During faster pacing, diastolic cytosolic ATP rose more rapidly in female myocytes, whereas beat-locked ATP transients increased in both sexes but proportionally more in males than in females. These findings demonstrate that ATP is synthesized on a beat-to-beat basis in a modular, microdomain-specific manner. We propose that male myocytes rely on greater mitochondrial mass for energetic scaling, whereas female cells employ architectural precision to optimize ATP delivery. KEY POINTS: It is known that each heartbeat requires precise ATP delivery to fuel ion transport and cross-bridge cycling, but the timing and spatial organization of ATP production in heart cells has been unclear. Using advanced imaging and genetically encoded sensors, we visualized beat-to-beat ATP fluctuations in the mitochondria and cytosol of individual male and female mouse ventricular myocytes. Mitochondrial ATP levels rose or fell with each beat in spatially confined regions, forming ATP 'gain' or 'dip' microdomains that were synchronized with Ca²⁺ transients. At higher firing rates, beat-locked, diastolic ATP transients rose more quickly in female myocytes, but were larger in male myocytes, highlighting distinct sex-specific strategies for matching energy supply to contractile demand. Ventricular myocytes 'live paycheck-to-paycheck', producing just enough ATP on demand to fuel each beat. Male and female myocytes adopt distinct strategies to meet this demand: male myocytes scale output through greater mitochondrial mass, while female myocytes achieve energetic precision via enhanced sarcoplasmic reticulum-mitochondrial coupling.