Rhana, P. | bioRxiv Science

bioRxiv2025

Beat-locked ATP microdomains in the sinoatrial node map a calcium-timed energetic hierarchy and regional pacemaker roles

Pacemaker myocytes of the sinoatrial (SA) node initiate each heartbeat through coupled voltage and Ca2+ oscillators, but whether ATP supply is regulated beat-by-beat in these cells remains unclear. Using genetically encoded sensors targeted to the cytosol and mitochondria, we tracked beat-resolved ATP dynamics in intact mouse SA node and isolated myocytes. Cytosolic ATP rose transiently with each Ca2+ transient and segregated into high- and low-gain phenotypes defined by the Ca2+-ATP coupling slope. Mitochondrial ATP flux adopted two stereotyped waveforms--Mode 1 "gains" and Mode 2 "dips." Within Mode 1 cells, ATP gains mirrored the cytosolic high/low-gain dichotomy; Mode 2 dips scaled linearly with Ca2+ load and predominated in slower-firing cells. High-gain/Mode 1 phenotypes localized to superior regions and low-gain/Mode 2 to inferior regions, paralleling gradients in rate, mitochondrial volume, and capillary density. Mechanistic dissection placed sarcoplasmic reticulum (SR) Ca2+ release upstream of ATP production, showing that Ca2+ triggers metabolic transients while membrane voltage primarily modulates their frequency. Inhibiting mitochondrial Ca2+ uptake and adenine nucleotide exchange eliminated beat-locked mito- and cyto-ATP signals, indicating that the mitochondrial Ca2+ uniporter (MCU)-adenine nucleotide translocase (ANT) machinery couples Ca2+ release to ATP fluctuations. Mode 2 recovery kinetics indicate slower ATP replenishment, which would favor low-frequency, fluctuation-rich firing in a subset of cells. Together, these findings reveal beat-locked metabolic microdomains in which Ca2+ transients time oxidative phosphorylation under a local O2 ceiling, unifying vascular architecture, mitochondrial organization, and Ca2+ signaling to match energy supply to excitability. This energetic hierarchy helps explain why some pacemaking myocytes are more likely to set the rate, whereas others may widen the bandwidth. SummaryBeat-locked cytosolic and mitochondrial ATP transients in SA-node myocytes sort into high-gain, low-gain, or consumption-dominant modes aligned with superior-inferior vascular-mitochondrial gradients. This energetic hierarchy lets high-gain cells set fast rates while low-gain/dip cells stabilize slow rhythms, broadening operating range but capping maximal bandwidth.

physiology↗

bioRxiv2025

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 -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

physiology↗

bioRxiv2023

The formation of KV2.1 macro-clusters is required for sex-specific differences in L-type CaV1.2 clustering and function in arterial myocytes

In arterial myocytes, the canonical function of voltage-gated CaV1.2 and KV2.1 channels is to induce myocyte contraction and relaxation through their responses to membrane depolarization, respectively. Paradoxically, KV2.1 also plays a sex-specific role by promoting the clustering and activity of CaV1.2 channels. However, the impact of KV2.1 protein organization on CaV1.2 function remains poorly understood. We discovered that KV2.1 forms micro-clusters, which can transform into large macro-clusters when a critical clustering site (S590) in the channel is phosphorylated in arterial myocytes. Notably, female myocytes exhibit greater phosphorylation of S590, and macro-cluster formation compared to males. Contrary to current models, the activity of KV2.1 channels seems unrelated to density or macro-clustering in arterial myocytes. Disrupting the KV2.1 clustering site (KV2.1S590A) eliminated KV2.1 macro-clustering and sex-specific differences in CaV1.2 cluster size and activity. We propose that the degree of KV2.1 clustering tunes CaV1.2 channel function in a sex-specific manner in arterial myocytes.

physiology↗