Biology subjects

Santana, L. F.

Publications and source records attributed to Santana, L. F..

13 recordsLinked to original sources

bioRxiv2026

Endogenous subthreshold noise and PIP2 tune diastolic coherence across the intact sinoatrial node through stochastic resonance

The heartbeat originates in the sinoatrial (SA) node, where spontaneously firing pacemaker myocytes interact to generate a coherent rhythm. The classical entrainment account is deterministic: the fastest cells entrain the rest, and variability is an error term that coupling suppresses. We proposed previously that the node instead exploits its own noise through stochastic resonance, with rhythm quality peaking at intermediate fluctuation amplitude, and since mapped a metabolic gradient capable of supplying it. Two-photon imaging of the voltage sensor ASAP5 in the intact mouse node resolved the subthreshold regime that tissue-scale mapping averages away. Diastolic coherence followed an inverted-U against endogenous noise amplitude in both poles, with optima 3.5-fold apart, and each pole operated near its own. {beta}-adrenergic stimulation raised noise in both poles to a common level. The inferior pole, which began below its optimum, gained coherence, while the superior pole, already at its optimum, lost interval regularity. Blocking HCN-mediated If with ivabradine lowered superior coherence without changing superior noise, and lowered inferior noise without lowering inferior coherence, dissociating the two coordinates. Thus, coherence is a surface defined by noise and coupled-clock drive. Phosphoinositide 4,5-bisphosphate (PIP2), an ATP-dependent lipid cofactor for HCN channels, increased firing rate and noise in both poles but improved coherence only in the energy-poor inferior node. Prior If block abolished that rescue. Pacemaking is therefore graded by a bioenergetic supply chain that sets the noise, not by entrainment alone. Noise, long treated as a nuisance to be averaged away, is an active ingredient of the pacemaker.

physiology↗

bioRxiv2026

Intracellular Mechanosensation in Intestinal Smooth Muscle: Piezo1 Complexes Amplify Signaling Beyond the Surface

Mechanosensation is fundamentally viewed as a plasma membrane phenomenon. We challenge this paradigm by introducing intracellular mechanosensation in intestinal smooth muscle. We hypothesized that a distinct, organelle-based signaling axis exists to amplify mechanotransduction from the inside out. To test this, we investigated whether Piezo1, a canonical plasma membrane mechanosensor, also operates within the cell. Using tissue-level wire myography, high-resolution confocal microscopy, proximity ligation assays, and patch-clamp electrophysiology on freshly dissociated cells, we identified a functional intracellular signaling hub that starts at the sarcoplasmic reticulum (SR). Unlike surface transduction, this intracellular mechanism relies on a nanoscale multiprotein complex (<40 nm) comprising an SR sensor (intra-Piezo1) and an amplifier (Ryanodine Receptor, RyR), coupled with a PM effector (large-conductance, Ca2+-activated K+ channels, i.e., BKCa channels). Activating this intracellular complex generated massive BK-mediated outward currents independent of extracellular Ca{superscript 2} but strictly dependent on internal SR Ca{superscript 2} stores, confirming intrinsic organellar mechanotransduction. Within this complex, intra-Piezo1 and RyR are positioned to operate as a coupled SR Ca{superscript 2} release unit that activates BK channels at SR-PM junctions, driving potent membrane hyperpolarization that reduces smooth muscle contractility, revealing the intra-Piezo1 complex as a molecular brake on excitation. These findings demonstrate that mechanotransduction is not confined to the cell surface. Instead, a specialized Sensor-Amplifier-Effector complex originating at intracellular organelles amplifies cellular sensitivity to physical force, providing a critical gain-control system that restrains smooth muscle excitability and regulates GI motility. Key PointsO_LIIntracellular organelles contribute to mechanosensory signaling in GI smooth muscle cells, complementing plasma membrane mechanisms. C_LIO_LIIntra-Piezo1 form a nanoscale signaling complex (<40nm) on the sarcoplasmic reticulum (SR), linking the mechanosensor Piezo1 with RyR and large conductance, Ca2+-activated K+ channels. C_LIO_LIUnlike surface sensors, this intracellular complex functions via a "Sensor-Amplifier-Effector" mechanism in which intra-Piezo1 detects mechanical stress and triggers SR Ca2+ release, thereby activating a nearby RyR and large-conductance, Ca2+-activated K+ channel. C_LIO_LIEngaging this intracellular Piezo1-mediated axis significantly dampens smooth muscle contractility, identifying a critical gain-control system essential for regulating GI motility. C_LI

physiology↗

bioRxiv2025

Digital Twin for the Win: Personalized Cardiac Electrophysiology

Individual variability shapes how diseases manifest, how patients respond to therapy, and how rare phenotypes arise. Conventional experimental approaches obscure variation by averaging, which limits mechanistic insight and predictive accuracy. We present a computational framework that builds digital twins of human-induced pluripotent stem cell-derived cardiomyocytes from a single optimized voltage clamp experiment. The framework depends on massive synthetic datasets comprising simulated cells that span broad ionic and electrophysiological ranges. These synthetic data make it possible to control parameters precisely, explore biological variability comprehensively, and train models beyond the limits of experimental data. A neural network trained on synthetic data then inferred biophysical parameters from experimental recordings from live cells, reproducing distinct electrophysiological features. Our study unites computational modeling, data simulation, and learning to enable scalable, precise, individualized cardiac electrophysiology modeling and can be readily extended to any electrically active cell type.

physiology↗

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↗

bioRxiv2025

Parameter Optimization of Biochemical Models for Precision Medicine: A Case Study in PI(4,5)P2 Synthesis

Precision medicine requires models that can translate rich molecular measurements into individualized predictions of biological response. This challenge is particularly acute for phosphoinositide signaling disorders that often exhibit cell-type-specific responses to identical genetic or pharmacological perturbations. Here, we develop a New Approach Methodology (NAM) demonstrating that basal phosphoinositide pool composition, determined by the size of the PI(4)P reserve, determines the robustness of lipid signaling. The NAM comprises a core kinetic model of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PI(4)P), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), and inositol 1,4,5-trisphosphate (IP3) dynamics. The model also incorporates phospholipase C (PLC)-mediated hydrolysis and phosphatase-mediated turnover and explicitly accounts for IP3 biosensor binding during parameter optimization. Parameters were optimized using experimental measurements from superior cervical ganglion (SCG) neurons and validated against independent dose-dependent PI(4,5)P2 depletion data. Local and global sensitivity analyses were performed to identify the dominant parameter drivers of pathway behavior. These sensitivity relationships were then used to generate a population of model variants that captured phosphoinositide dynamics observed in tsA201 cells, human neuroblastoma cells, and hippocampal neurons. To infer cell-specific models, we developed two complementary inverse methods: sensitivity fingerprinting derived from mechanistic model sensitivities and a neural network trained on synthetic phosphoinositide time series. Both approaches reproduced experimental PI(4)P, PI(4,5)P2, and IP3 dynamics across cell types while preserving the baseline model structure. Importantly, the inferred models predicted experimentally observed differential vulnerability to kinase perturbation without additional fitting. Hippocampal neurons with large basal pools of PI(4)P maintained PI(4,5)P2 and IP3 signaling under phosphatidylinositol 4-kinase alpha (PI4KA) inhibition, whereas cells with small basal PI(4)P pools exhibited signaling failure. Simulations of PI4KA and phosphatidylinositol-4-phosphate 5-kinase type 1 gamma (PIP5K1C) loss-of-function mutations under repeated stimulation further revealed progressive signaling collapse in small-pool neurons but sustained function in large-pool neurons, demonstrating that basal lipid composition can determine genetic vulnerability. Together, this NAM provides a predictive, cell-specific framework for translating dynamic lipid measurements into mechanistic models that support precision medicine applications in phosphoinositide-related disorders.

biophysics↗

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↗

bioRxiv2023

A Computational Model Predicts Sex-specific Responses to Calcium Channel Blockers in Mesenteric Vascular Smooth Muscle

The function of the smooth muscle cells lining the walls of mammalian systemic arteries and arterioles is to regulate the diameter of the vessels to control blood flow and blood pressure. Here, we describe an in-silico model, which we call the "Hernandez-Hernandez model", of electrical and Ca2+ signaling in arterial myocytes based on new experimental data indicating sex-specific differences in male and female arterial myocytes from murine resistance arteries. The model suggests the fundamental ionic mechanisms underlying membrane potential and intracellular Ca2+ signaling during the development of myogenic tone in arterial blood vessels. Although experimental data suggest that KV1.5 channel currents have similar amplitudes, kinetics, and voltage dependencies in male and female myocytes, simulations suggest that the KV1.5 current is the dominant current regulating membrane potential in male myocytes. In female cells, which have larger KV2.1 channel expression and longer time constants for activation than male myocytes, predictions from simulated female myocytes suggest that KV2.1 plays a primary role in the control of membrane potential. Over the physiological range of membrane potentials, the gating of a small number of voltage-gated K+ channels and L-type Ca2+ channels are predicted to drive sex-specific differences in intracellular Ca2+ and excitability. We also show that in an idealized computational model of a vessel, female arterial smooth muscle exhibits heightened sensitivity to commonly used Ca2+ channel blockers compared to male. In summary, we present a new model framework to investigate the potential sex-specific impact of anti-hypertensive drugs.

physiology↗

bioRxiv2023

SanPy: A whole-cell electrophysiology analysis pipeline.

The analysis of action potentials and other membrane voltage fluctuations provide a powerful approach for interrogating the function of excitable cells. Yet, a major bottleneck in the interpretation of this critical data is the lack of intuitive, agreed upon software tools for its analysis. Here, we present SanPy, a Python-based open-source and freely available software pipeline for the analysis and exploration of whole-cell current-clamp recordings. SanPy provides a robust computational engine with an application programming interface. Using this, we have developed a cross-platform graphical user interface that does not require programming. SanPy is designed to extract common parameters from action potentials including threshold time and voltage, peak, half-width, and interval statistics. In addition, several cardiac parameters are measured including the early diastolic duration and rate. SanPy is built to be fully extensible by providing a plugin architecture for the addition of new file loaders, analysis, and visualizations. A key feature of SanPy is its focus on quality control and data exploration. In the desktop interface, all plots of the data and analysis are linked allowing simultaneous data visualization from different dimensions with the goal of obtaining ground truth analysis. We provide documentation for all aspects of SanPy including several use cases and examples. To test SanPy, we have performed analysis on current-clamp recordings from heart and brain cells. Taken together, SanPy is a powerful tool for whole-cell current-clamp analysis and lays the foundation for future extension by the scientific community. News and NoteworthyWhole-cell current-clamp recording is a critical technique to understand detailed biophysical mechanisms at the cellular and network levels. Yet, the analysis of this data is by no means standardized. Here, we present SanPy, a Python based open-source software pipeline for the exploration and analysis of current-clamp recordings. SanPy provides a powerful computational engine with an application programming interface. Finally, SanPy provides a cross-platform point-and-click graphical user interface that does not require programming.

neuroscience↗

bioRxiv2022

Aging impairs the clustering and reduces the activity of L-type calcium channels in cardiac pacemaker cells

Every heartbeat is initiated by a spontaneous electrical signal generated inside the cardiac pacemaker. The generation of this electrical signal depends on the coordinated opening and closing of different ion channels, where voltage-gated L-type calcium channels play a central role. Despite the reliability of the pacemaker, all mammals experience a linear slowdown of the pacemaker rate with age. In humans, this slowing can become pathological and constitutes the main cause for the requirement of the implantation of artificial pacemakers. However, the mechanisms behind the age-associated slowdown of the pacemaker are not well understood. Here, we show that age alters L-type calcium channels in pacemaker cells from mice. The age-associated alterations include: i) a reduction in the density of the channels at the plasma membrane, ii) a reduction in the clustering of the channels, and iii) a decrease in channel open probability. Altogether, these age-associated alterations result in a global reduction of the L-type calcium current density and in a slowdown of the pacemaker diastolic depolarization. Remarkably, increasing the open probability of L-type calcium channels pharmacologically was enough to restore pacemaker rate in old cells to the same levels observed in the young. Overall, our findings provide evidence that proper organization and function of L-type calcium channels is impaired by aging and that this dysfunction contributes to the slowdown of pacemaker cells in old animals.

biophysics↗

bioRxiv2021

Genetically Engineered Strains for Combinatorial Cardiovascular Optobiology

Optogenetic effectors and sensors provide a novel real-time window into complex physiological processes, enabling determination of molecular signaling processes within functioning cellular networks. However, the combination of these optical tools in mice is made practical by construction of genetic lines that are optically compatible and genetically tractable. We present a new toolbox of 21 mouse lines with lineage -specific expression of optogenetic effectors and sensors for direct biallelic combination, avoiding the multiallelic requirement of Cre recombinase -mediated DNA recombination, focusing on models relevant for cardiovascular biology. Optogenetic effectors (11 lines) or Ca2+ sensors (10 lines) were selectively expressed in cardiac pacemaker cells, cardiomyocytes, vascular endothelial and smooth muscle cells, alveolar epithelial cells, lymphocytes, glia, and other cell types. Optogenetic effector and sensor function was demonstrated in numerous tissues. Arterial/arteriolar tone was modulated by optical activation of the second messengers InsP3 (opto1AR) and cAMP (opto{beta}2AR), or Ca2+- permeant membrane channels (CatCh2) in smooth muscle (Acta2) and endothelium (Cdh5). Cardiac activation was separately controlled through activation of nodal/conducting cells or cardiac myocytes. We demonstrate combined effector and sensor function in biallelic mouse crosses: optical cardiac pacing and simultaneous cardiomyocyte Ca2+ imaging in HCN4BAC-CatCh2/MHC-GCaMP8 crosses. These experiments highlight the potential of these mice to explore cellular signaling in vivo, in complex tissue networks.

physiology↗

bioRxiv2019

Kv2.1 mediates spatial and functional coupling of L-type calcium channels and ryanodine receptors in neurons

The voltage-gated K+ channel Kv2.1 serves a major structural role in the soma and proximal dendrites of brain neurons, tethering the plasma membrane (PM) to the endoplasmic reticulum (ER). Although Kv2.1 clustering at neuronal ER-PM junctions (EPJs) is tightly regulated and conserved across species, its function at these sites is unclear. By identifying and evaluating proteins in close spatial proximity to Kv2.1-containing EPJs, we discovered that a significant role of Kv2.1 at EPJs is to promote the clustering and functional coupling of PM L-type Ca2+ channels (LTCCs) to ryanodine receptor (RyR) ER Ca2+ release channels. Kv2.1 clustering also unexpectedly enhanced LTCC opening at polarized membrane potentials. This enabled Kv2.1-LTCC-RyR triads to generate localized Ca2+ release events (i.e., Ca2+ sparks) independently of action potentials. Together, these findings uncover a novel mode of LTCC regulation and establish a unique mechanism whereby Kv2.1-associated EPJs provide a molecular platform for localized somatodendritic Ca2+ signals.

neuroscience↗

bioRxiv2019

A toolbox of nanobodies developed and validated for diverse neuroscience research applications

Nanobodies (nAbs) are small, minimal antibodies that have distinct attributes that make them uniquely suited for certain biomedical research, diagnostic and therapeutic applications. Prominent uses include as intracellular antibodies or intrabodies to bind and deliver cargo to specific proteins and/or subcellular sites within cells, and as nanoscale immunolabels for enhanced tissue penetration and improved spatial imaging resolution. Here, we report the generation and validation of nAbs against a set of proteins prominently expressed at specific subcellular sites in brain neurons. We describe a novel hierarchical validation pipeline to systematically evaluate nAbs isolated by phage display for effective and specific use as intrabodies and immunolabels in mammalian cells including brain neurons. These nAbs form part of a robust toolbox for targeting proteins with distinct and highly spatially-restricted subcellular localization in mammalian brain neurons, allowing for visualization and/or modulation of structure and function at those sites.

neuroscience↗