bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.07.07.663572

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

Rhana, P.·Matsumoto, C.·Santana, L. F.
View original source ↗Export metadata ↓

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

Explore related subjects

physiologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Rhana, P., Matsumoto, C., Santana, L. F.. 2025-07-10. Demonstration of Beat-to-Beat, On-Demand ATP Synthesis in Ventricular Myocytes Reveals Sex-Specific Mitochondrial and Cytosolic Dynamics.. https://doi.org/10.1101/2025.07.07.663572

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Hypothalamic Farnesoid X Receptor deficiency alters energy balance by modulating hepatic glucose production and adipose tissue metabolism through central insulin signaling.

Objectives: The bile acid nuclear receptor Farnesoid X Receptor (FXR, NR1H4) is a major regulator of metabolism and energy homeostasis in peripheral organs. It modulates bile acid, glucose, and lipid metabolism, as well as fat mass and body weight. However, FXR is also expressed in the brain, particularly in the hypothalamus, a key center for the regulation of energy homeostasis. Although one study has demonstrated a role for brain FXR activation in energy balance, its specific hypothalamic role is still unknown. Here, we examined the role of FXR in the mediobasal hypothalamus in the regulation of energy balance. Methods: We used a genetic approach combined with metabolic phenotyping to determine the effect of FXR invalidation in the mediobasal hypothalamus on metabolic parameters involved in the central regulation of energy homeostasis. Results: Our results demonstrate that hypothalamic FXR deficiency induces a positive energy balance, resulting in a reduction in energy expenditure due to alterations in glucose metabolism accompanied by structural changes in white adipose tissues. Conclusion: This study uncovers a previously unrecognized role for hypothalamic FXR in the central homeostatic control of energy balance, providing new insights into its contribution to peripheral glucose metabolism and adipose tissue structural remodeling.

physiology↗
bioRxiv2026

Rad and Phospholamban are Key Drivers of the Ventricular Adrenergic Response and Stress-Induced Arrhythmia

The adrenergic response is a fundamental mechanism that regulates heart rate (chronotropy), cardiac contractility (inotropy) and relaxation (lusitropy). Adrenergic stress is also a recognized trigger of arrhythmia in disease. Yet, our understanding of the underlying molecular basis remains incomplete. Protein kinase A (PKA) and the calcium/calmodulin-dependent kinase II (CaMKII) phosphorylate multiple targets proposed to participate in the adrenergic response, including the GTP-binding protein Rad, phospholamban (PLB) and ryanodine receptor 2 (RyR2). Here we demonstrate that phosphorylation of both Rad and PLB is necessary for inotropy and lusitropy. We show that changes in cardiac contractility and relaxation are primarily dependent on intracellular calcium handling. Finally, we report that Rad and PLB control stress-induced arrhythmogenesis, despite the phosphorylation of other pro-arrhythmic targets. We have identified the essential molecular components of the adrenergic response, resolving a long-standing debate in cardiac excitation-contraction coupling and refining current models of sympathetic regulation in health and disease.

physiology↗
bioRxiv2026

Light-cycle time-restricted feeding remodels a hidden layer of the cardiac transcriptome through sex-specific transcript switching

Light-cycle time-restricted feeding disrupts daily cardiovascular and thermoregulatory rhythms, but the molecular effects of light-cycle time-restricted feeding on the heart have been measured only at the level of total gene expression. We used Oxford Nanopore long-read RNA sequencing to resolve the full-length ventricular transcriptome from male and female mice under ad libitum feeding or light-cycle time-restricted feeding across the 24-hour cycle. Greater than 20% of cardiac transcripts represent unannotated variants of known genes absent from the current GENCODE reference annotation. Light-cycle time-restricted feeding reorganizes transcript usage across hundreds of genes, including genes encoding splicing regulators, largely without changing total gene expression. The genes affected are sex-specific, with fewer than 2% of changes shared at the gene, transcript, and transcript-usage levels. We show that transcript-level regulation is a previously underrecognized component of the cardiac response to altered feeding behavior, undetected by conventional short-read approaches.

physiology↗