Demonstration of Beat-to-Beat, On-Demand ATP Synthesis in Ventricular Myocytes Reveals Sex-Specific Mitochondrial and Cytosolic Dynamics.

Abstract

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 -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 summaryO_LIIt 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. C_LIO_LIUsing 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. C_LIO_LIMitochondrial ATP levels rose or fell with each beat in spatially confined regions, forming ATP "gain" or "dip" microdomains that were synchronized with Ca2+ transients. C_LIO_LIAt 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. C_LIO_LIVentricular 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. C_LI O_FIG O_LINKSMALLFIG WIDTH=192 HEIGHT=200 SRC="FIGDIR/small/663572v2_ufig1.gif" ALT="Figure 1"> View larger version (108K): org.highwire.dtl.DTLVardef@12722org.highwire.dtl.DTLVardef@1b041cdorg.highwire.dtl.DTLVardef@b007eeorg.highwire.dtl.DTLVardef@d8c3a4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstract figureC_FLOATNO Beat-locked mitochondrial ATP transients reveals modular, sex-specific bioenergetic control during excitation-contraction coupling. (A) Each action potential activates L-type CaV1.2 channels, producing a Ca2+ influx that triggers Ryanodine receptors (RyR2) and elicits SR Ca2+ release. (B) The cytosolic Ca2+ signal is decoded by mitochondria into spatially distinct "high-gain" and "low-gain" regions, shaped by the extent of SR-mitochondrial tethering via mitofusin 2 (Mfn2), yielding heterogeneous mitochondrial activation rather than a uniform, cell-wide metabolic response. (C) Mitochondria generate rhythmic, phase-locked ATP transients within discrete microdomains, such that ATP increases and ATP dips can coexist within the same cell. Ca2+ entry through the outer membrane (via VDAC) and into the matrix (via MCU) stimulates oxidative phosphorylation, increasing ATP production; ATP is exported by ANT to create local cytosolic "supply bursts" aligned with beat-to-beat demand ("paycheck-to-paycheck" energetics). Female myocytes show a higher prevalence of tightly coupled, high-gain ATP-producing microdomains, whereas male myocytes display a shifted balance toward lower-gain regions, consistent with sex-dependent SR-mitochondrial coupling and ATP microdomain patterning. C_FIG