Journal of General Physiology

The cardiac sodium channel NaV1.5 initiates each heartbeat by generating the rapid depolarizing upstroke of the action potential. Dysregulation of NaV1.5 gating can produce cardiac arrhythmias by slowing inactivation, increasing late sodium current (INa,L), and impairing electrical stability. Here, we show that phosphatidylinositol-4,5-bisphosphate (PIP2) is a critical membrane cofactor that stabilizes NaV1.5 gating. Acute PIP2 depletion in human iPSC-derived cardiomyocytes, produced by activation of endogenous AT1 receptors, activation of an engineered M3q-DREADD, or optogenetic recruitment of CRY2-pseudojanin, shifted voltage dependence, slowed fast inactivation, and increased INa,L. These effects were prevented by augmenting intracellular PIP2, required PLC activity when driven by Gq-coupled receptors, and were independent of downstream Ca2+ or PKC signaling. Unlike the skeletal-muscle isoform NaV1.4, NaV1.5 displayed PIP2-dependent shifts in both activation and steady-state inactivation, indicating isoform-specific lipid coupling. Induced-fit docking and molecular dynamics simulations identified a PIP2-interaction interface between the domain IV voltage sensor and pore that contains disease-linked residues. The disease-reported variant R1644C weakened and redistributed the predicted PIP2-contact network, produced elevated basal INa,L, showed enhanced sensitivity to PIP2 depletion, and caused an approximately 30-fold reduction in apparent functional PIP2 sensitivity in excised patches. These findings define a lipid-dependent mechanism that stabilizes NaV1.5 gating and reveal how physiological Gq signaling and inherited channel variants can converge on the channel-PIP2 axis to promote proarrhythmic late sodium current.

Predictions of skeletal muscle energy consumption under a diverse range of muscle contractile conditions are critical for improving our understanding of locomotion. Existing mathematical models, while capturing the mechanical dependence of energy consuming processes, neglect the time-dependent behaviour and recovery costs associated with regenerating ATP. This time-dependence is important for predicting the energetic response of muscles during repetitive or cyclical tasks like locomotion, where muscle undergoes many contraction cycles. This study presents a novel model to predict energetic rates based on physiological processes: Ca2+ transport costs, cross-bridge cycling costs, and ATP regeneration. Previous mathematical models include the dependence on Ca2+ transport and cross-bridge cycling, but neglect the time-dependent response and the subsequent recovery of ATP following the contraction. Model parameters were obtained from existing data on isolated muscle preparations, and predicted energetic rates were validated on separate datasets across a range of contractile conditions including dynamic, sub-maximal, and twitch contractions. The time-dependent model was able to capture the influence of contraction frequency on peak energetic rates and the time-course of energetic recovery observed experimentally. The model captures key physiological processes while maintaining a minimal number of free parameters and low computational cost. This enables generalisability across muscles and species, and implementation into larger scale musculoskeletal models.

Epithelial sheets must maintain robust barrier function while enduring severe mechanical deformations across various physiological environments. While baseline actomyosin contractility is understood to stabilize intercellular junctions and hence cell-cell contact integrity, how cell-generated active forces interact with external physical strain to dictate contact integrity remains poorly understood. In this study, we investigated the biophysical trade-offs between actomyosin contractility and barrier resilience when Madin-Darby Canine Kidney (MDCK) cell islands are subject to large stretch. In contrast to a high concentration (50 M) of the non-muscle myosin II inhibitor blebbistatin that disrupted cell-cell contacts, we first identified a lower concentration (10 M) that maintained cell-cell contact integrity in the absence of any stretch. Such moderate inhibition of non-muscle myosin II reduced, but preserved some level of actin bundle organization. Remarkably, when challenged with a pathological 38% linear stretch using a custom-built biaxial stretching device, 10 M blebbistatin treated epithelial islands exhibited significantly fewer cell-cell contact ruptures than untreated controls, demonstrating a potent protective effect against mechanical strain. Traction force microscopy revealed diminished cell-generated strain energy by over 60% indicating a partial but significant reduction in contractility upon 10 M blebbistatin treatment. Nanoindentation measurements revealed that moderate contractility inhibition decreased the cellular Young's modulus by more than 40%. Consequently, moderate contractility inhibition safeguards epithelial junctions through a dual mechanical effect: it simultaneously reduces baseline active tensile stresses due to cell contractility and lowers the passive elastic forces generated within the softened cell island during external stretch. Our findings indicate that this systemic reduction in forces dominates over any loss of biochemical adhesion strength at cell-cell contacts. We propose that shifting the epithelium from a rigid, highly stressed continuum to a more compliant, relaxed state by moderate contractility inhibition can serve as a general biophysical mechanism to preserve barrier integrity under severe mechanical challenge.

Synaptotagmins (Syts) are Ca{superscript 2}-sensing exocytosis regulators whose tandem C2 domains interact with phosphoinositides and membranes to trigger neurotransmitter and hormone release. Although Ca{superscript 2} binding is known to enhance C2 domain-membrane interactions, the sequence determinants governing lipid binding and membrane penetration across Syt isoforms remain incompletely understood. Here, we performed MARTINI coarse-grained molecular dynamics simulations of isolated C2A and C2B domains from eight Ca{superscript 2}-sensing Syt isoforms (Syt1, Syt2, Syt3, Syt5, Syt6, Syt7, Syt9, and Syt10) interacting with phosphatidylinositol 4,5-bisphosphate (PIP2)-containing plasma membranes. To systematically modulate electrostatic properties, we introduced partial and full charge-flip mutations at conserved acidic residues within the calcium-binding loops (CBLs). By integrating simulations across multiple isoforms and charge states, we sought to identify the dominant sequence determinants governing membrane interactions. We found that PIP2 binding to both, CBLs and polybasic patches (PBs), is associated with loop net charge, yielding correlations > 0.95 across all isoforms. However, membrane penetration is not sufficiently explained by loop net charge alone. The local phenylalanines additionally increase membrane penetration independent of loop net charge. Together, these findings establish a comprehensive electrostatic-aromatic framework where loop net charge governs PIP2 binding, whereas loop net charge and local phenylalanine enrichment jointly govern membrane penetration across Syt C2 domains.

There is a vast literature on the voltage gating of ion channels, with a fairly large fraction concerned with potassium channels, especially of the KV1 family, including Shaker. Experimental evidence derived from protein structure has been interpreted to give gating mechanisms that largely disregard water. We propose that the K+ ion, in order to pass through the gating region and enter the cavity pore, must be largely dehydrated. Competitive interactions of each single hydration shell water at the gate, with counterions, protein, or other water molecules, can remove one water at a time. There are several such interactions for the ion hydration shell; for the ion to pass through the gating region, there must be enough such interactions to leave the ion with at most two hydrating water molecules, in which case the gate is open. Protein conformational changes are secondary, small, and mostly unimportant. The hypothesis has a second part: protons, previously shown to be candidate carriers of the gating current (Kariev and Green, JPC B, 2019, Membranes, 2022, 2024) are capable of reaching the gate; adding four protons to the gate prevents dehydration, leaving the ion with at least three hydrating water molecules, enough to block passage. Quantum calculations presented here support the dehydration part of the hypothesis; they also mostly support the second part, concerning the protons, but further work will be required to fully confirm this. The hypothesis explains the experimental finding that the P475D mutant is essentially constitutively open, while the P475S mutant, with a wider gate opening, is closed at all relevant potentials; the computations presented here show the mechanism for this in detail, further confirming the first part of the hypothesis, and largely but not completely confirming the second part, concerning protons, while showing where further work is needed. This mechanism can also qualitatively account for flicker noise and fluctuations, and their consequences.

BackgroundNeprilysin (NEP) is a zinc-dependent metalloprotease targeted in heart failure therapy to prevent it degrading circulating cardioprotective vasoactive peptides. NEP can also cleave sarcolipin (SLN), the skeletal- and atrial muscle-specific micropeptide regulator of the sarcoplasmic reticulum Ca2+-ATPase (SERCA). A direct pathophysiological role of NEP in ventricular muscle has not been established. MethodsProteomics and immunoblot analysis of human myocardial specimens were used to quantify NEP abundance in failing and non-failing hearts. Heterologous protein expression and biochemical binding assays assessed NEP-mediated cleavage of phospholamban (PLB) and its impact on PLB-SERCA interactions. Functional consequences of NEP expression or inhibition were evaluated in neonatal rat ventricular myocytes and in a human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model of heart failure. ResultsWe observed increased NEP abundance in failing human myocardium relative to non-failing controls. We demonstrated that NEP cleaves phospholamban (PLB), disrupting PLB-SERCA interactions. Mutation of PLB (V49A), prevented NEP cleavage and preserved PLB-SERCA binding, indicating V49 is critical for NEP substrate recognition. In neonatal rat ventricular myocytes, NEP expression was associated with faster Ca2+ transient decay kinetics and increased SR Ca2+ load, consistent with reduced SERCA inhibition. Inhibition of NEP in a hiPSC-CM heart failure model attenuated the hypertrophic transcriptional responses and reversed Ca2+-transport dysregulation. ConclusionsThese findings implicate increased NEP expression in the sarcoplasmic reticulum of cardiomyocytes as previously unrecognized maladaptive consequence of heart failure contributing to cardiac dysfunction. In this novel pathophysiological mechanism, increased NEP results in PLB cleavage and loss of regulation of SERCA. While this may relieve SERCA inhibition and augment cellular Ca2+ handling, loss of PLB chronically disrupts hearts dynamic response to adrenergic stress, changing heart rate, or other physiological challenges. The data provide new insight into the cardioprotective effects of pharmacological NEP inhibition in clinical practice, reveal a novel mechanism of action of neprilysin inhibition in cardiomyocytes and may help inform future therapeutic strategies for patients with heart failure. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/732949v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@4daf93org.highwire.dtl.DTLVardef@41ef8aorg.highwire.dtl.DTLVardef@d568daorg.highwire.dtl.DTLVardef@d6d213_HPS_FORMAT_FIGEXP M_FIG C_FIG

After myocardial infarction (MI), pathological autonomic remodeling, including vagal dysfunction and sympathoexcitation, occurs and predisposes to ventricular arrhythmias (VT/VF). The underlying factors that drive this remodeling, including the observed neuroinflammation and glial activation, remain unknown. We hypothesized that sympathetic nociceptive afferents underlie this remodeling post-MI. Epidural resiniferatoxin (RTX, to ablate sympathetic cardiac afferent neurons) vs. saline was administered in pigs prior to MI and autonomic and electrophysiological effects assessed four to six weeks post-infarction. Acute effects of afferent ablation after chronic MI were also assessed in a separate group of animals. Baroreflex sensitivity and vagal tone, as measured by parasympathetic neuronal activity and cardiac nociceptive responses, were improved in infarcted animals which received epidural RTX prior to MI. These animals also demonstrated reduced spinal cord inflammation and glial activation, downregulation of circulating stress and inflammatory pathways, and stabilization of electrophysiological parameters, with reduced VT/VF-inducibility. Epidural RTX after chronic MI also acutely restored vagal function and decreased VT/VF. These data suggest that cardiac spinal nociceptive afferents directly contribute to VT/VF susceptibility and MI-induced autonomic remodeling, including oxidative stress, inflammation, glial activation, and reduced vagal function, providing novel insights into the causal role of these afferents in driving sympathovagal imbalance after MI.

CO2 opens a subset of connexin hemichannels by binding to a site in the cytoplasmic domain of the channel. From outside the cell, CO2 must cross at least one membrane to reach this site. We have used Neuro-2A cells, which exhibit very low expression of CO2 permeable aquaporins (AQPs) and do not express any of the connexins (Cxs) known to be CO2 sensitive, to evaluate the minimal complement of molecular components required to recapitulate whole cell CO2 sensitivity mediated by connexins (assayed by either whole cell patch clamp recordings or real time recordings of ATP release via a co-expressed genetically encoded ATP sensor). Neuro-2A cells that expressed either Cx26, Cx32 or Cx43 on their own did not exhibit CO2-dependent connexin hemichannel gating. Expression of AQP1 or AQP5 either with or without carbonic anhydrase 2 (CA2) did not reveal any endogenous CO2 sensitivity of Neuro-2A cells. Only by expressing one of Cx26, Cx32 or Cx43 with either AQP1 or AQP5, plus CA2 were we able to reconstitute whole cell CO2 sensitivity. We found that expression of Cx26 with either AQP1 or AQP5 resulted in high levels of cell death. This was prevented by co-expression of CA2. Simulations of the influx and diffusion of CO2 show that CA2 prevents accumulation of intracellular CO2 and excessive activation of Cx26, thus protecting the cells from death. Surveying the transcriptome of cells that express CO2 sensitive connexins shows that many also express CO2 permeable aquaporins and CA2. We suggest that connexins, aquaporins and carbonic anhydrases represent the minimal trifecta of components required for cellular CO2 sensing.

Skeletal muscles secrete a variety of cytokines in response to inflammatory stimuli such as lipopolysaccharide (LPS); however, the contributions of resident macrophages or other non-muscle cells to the secretory responses are not well understood. To determine the potential impact of resident macrophages to inflammatory cytokine production, we tested the LPS responsiveness of isolated mouse soleus muscle when a critical toll receptor adapter protein (Myd88) was knocked down only in myeloid-derived cells within the muscle (e.g. resident macrophages). The phenotype is referred to as LyzMyd88-/- ; the litter mate controls were Myd88fl/fl. In solei from LyzMyd88-/- mice, cytokine secretory rates for interleukin-6 (IL-6) and keratinocyte-derived cytokine (KC, CXCL1) were significantly reduced to 56.3%, and 60.6% of control, respectively, over the first hour of LPS exposure. In the second hour, secretion of granulocyte colony stimulating factor (G-CSF), IL-6, KC(CXCL1) and monocyte chemoattractant protein-1 (MCP-1, CCL2) were greatly elevated by 5-10-fold in both phenotypes compared to the first hour. However, only MCP-1 secretion was decreased to 70.6% of control in the second hour. We also tested the secretory response to buffer containing 1% sterile mouse plasma because dilute plasma is known to amplify the responses of macrophages to LPS. Treatment with 1% plasma alone affected baseline measures of some cytokines but resulted in no further increases in secretion during either hour of exposure. However, small and gradual increases in secretory rates were observed for several cytokines over the study period, with or without plasma, with the largest responses seen in IL-6 and KC. Overall, the results are consistent with a significant early contribution of myeloid-derived, resident immune cells to the cytokine secretory responses of intact oxidative skeletal muscle. In addition, small quantities of plasma in the buffer have no independent stimulatory effects on cytokine secretion

Reaction diffusion (RD) systems play a fundamental role in numerous biochemical and biophysical processes. Here, we present a novel analytical framework for solving RD equations by applying the Wentzel Kramers Brillouin Jeffreys (WKBJ) formalism to Ca nanodomains generated by individual membrane channels, a widely used paradigm for intracellular Ca signaling. Previous models have primarily focused on stationary Ca nanodomains while neglecting diffusion and saturation of intracellular Ca buffers and sensors. In contrast, we derive analytical solutions without these simplifying assumptions. Our analysis demonstrates that sustained Ca influx generates continuously expanding distributions of free Ca, whereas Ca bound buffers and sensors propagate as traveling waves. These predictions are supported experimentally by measurements of one-dimensional fluorescence profiles produced by single-channel activity and two-dimensional profiles generated by whole cell Ca currents. The analytical framework developed here readily extends Michaelis Menten type kinetics to reaction diffusion systems and may therefore be broadly applicable to biochemical and biophysical processes in which diffusion cannot be neglected.

Obesity is associated with an elevated risk of pathological cardiac hypertrophy, whereas exercise confers cardioprotective effects; however, the cellular mechanisms underlying these opposing influences remain incompletely defined, particularly in females. We investigated how obesity and exercise affect cardiomyocyte ultrastructure, Ca{superscript 2} release, and contractility in female Zucker Diabetic Fatty rats and their lean littermates. Animals were assigned at 12 weeks to sedentary or aerobic exercise-trained groups and maintained on a standard diet. By 18 weeks, obese rats exhibited increased body mass and myocardial hypertrophy in the absence of diabetes. Sedentary obese animals showed a reduced fraction of compact dyads and diminished stimulated and caffeine-induced Ca{superscript 2} release, while contractility remained preserved. In lean rats, exercise increased dyad density but reduced Ca{superscript 2} release, whereas in obese rats, exercise enhanced both dyad compactness and Ca{superscript 2} release. Across all groups, global cardiomyocyte ultrastructure and contractile function were similar. Type III ANOVA revealed a significant obesity x exercise interaction for dyadic structure and Ca{superscript 2} release. These findings demonstrate that obesity itself, independent of diabetes, triggers early dyadic remodeling and altered Ca{superscript 2} handling in female myocardium before detectable impairment of global cardiomyocyte structure or contractile function. Furthermore, exercise exerts beneficial effects on dyadic ultrastructure and Ca{superscript 2} signaling in obese animals. New & NoteworthyUsing a female rat model of obesity without diabetes, we demonstrate that obesity induces early remodeling of the dyadic system and impairs Ca{superscript 2} release in cardiac myocytes. We further show that the effects of aerobic exercise on dyadic structure and function are obesity-dependent, improving both dyad organization and Ca{superscript 2} signaling. These findings identify the dyadic microdomain as a vulnerable cellular site in obesity and a potential target for exercise-induced recovery.

The discovery of sensory ion channels, such as thermosensitive transient receptor potential (TRP) channels and mechanosensitive PIEZOs, have transformed our understanding of mammalian sensory biology. However, the sensory receptor landscape remains incomplete, as many physiologically relevant sensory stimuli still lack identified molecular targets. Here, we describe a novel screening strategy utilizing FM 1-43, a fluorescent marker for activity of various cation channels, with a CRISPRa library (MPCL) targeting multi-transmembrane domain proteins. We validate this method by focusing on allyl isothiocyanate (AITC) and its putative receptor TRPA1. Specifically, we show that CRISPRa-mediated overexpression of TRPA1 is sufficient for FM 1-43 labeling when co-treated with AITC. Furthermore, we show that using FM 1-43 and AITC, we can efficiently FACS enrich TRPA1-expressing cells from a pool of MPCL-expressing cells. Collectively, this presents a novel method for rapidly screening select cation-dependent sensory stimuli.

Mitochondrial Ca2+ uptake through the mitochondrial calcium uniporter complex (MCUcx) is a critical determinant of cellular metabolism, integrating Ca2+ signaling with ATP production and redox control. Yet how MCUcx activity is constrained to prevent Ca2+ overload and cell injury, and how the essential MCU regulator (EMRE), a subunit required for channel activity, mechanistically supports MCUcx function remains incompletely defined. Here, using a newly developed high-sensitivity assay to quantify MCUcx function in intact mitochondria, we uncover two fundamental roles of EMRE. First, EMRE is required for robust matrix Ca2+-dependent inhibition of MCUcx, acting through a juxtamembrane site via a mechanism distinct from MICU1-mediated inhibition at low cytosolic Ca2+. Second, by decoupling channel function from regulation, we demonstrate that EMRE promotes robust ion permeation through MCUcx, elevating its role from a structural scaffold to an active determinant of channel throughput. Together, our findings refine current models of mitochondrial Ca2+ regulation, establish EMRE as an essential multifunctional regulator of uniporter activity, and highlight the utility of our assay for probing MCUcx biophysical mechanisms and enabling the discovery of uniporter modulators. Significance StatementMitochondria use Ca2+ signals to adjust energy production to cellular demand, but excessive Ca2+ entry can trigger cell death. How the mitochondrial calcium uniporter balances these opposing needs remains fundamentally unresolved. Using a high-sensitivity approach that isolates uniporter permeation from Ca2+-dependent confounders in intact mitochondria, we characterize a matrix Ca2+-dependent inhibitory mechanism that depends on EMRE and is functionally distinct from MICU1-mediated regulation. We further show that EMRE, a small regulatory subunit unique to higher organisms, not only enables channel function but promotes robust ion permeation through the pore. Together, these findings refine current models of mitochondrial Ca2+ regulation and provide a unified framework for understanding EMRE-dependent uniporter regulation in intact mitochondria.

BackgroundCatecholamines play a central role in cardiac performance, coordinating myocardial contractility, conduction, metabolism, and electrophysiological stability. In the heart, their actions have been attributed primarily to sympathetic nerve terminals and circulating adrenal catecholamines. The discovery of an intrinsic non-neuronal cholinergic system within cardiomyocytes challenges this neurocentric paradigm and raises the possibility that cardiomyocytes also possess an intrinsic catecholaminergic programme. Here, we investigated whether cardiomyocytes possess an intrinsic catecholaminergic programme and its contribution to cardiomyocyte homeostasis and stress responses. MethodsWe investigated catecholamine biosynthesis and handling in human induced pluripotent stem cell-derived cardiomyocytes, adult mouse cardiomyocytes, H9C2 cells, rat ventricular tissue, and Langendorff-perfused mouse hearts. Protein expression of catecholamine biosynthetic enzymes and vesicular monoamine transporters was assessed by immunoblotting and immunohistochemistry, while vesicular monoamine uptake was evaluated using fluorescent false neurotransmitters. Functional consequences of catecholamine biosynthesis inhibition were examined using pharmacological approaches, assessing cell viability, apoptosis, organelle homeostasis, metabolic signalling, and cardiac electrophysiology. ResultsTyrosine hydroxylase, aromatic L-amino acid decarboxylase, dopamine {beta}-hydroxylase, and vesicular monoamine transporters were detected in cardiomyocytes across models. Expression of catecholamine biosynthetic enzymes increased following ischaemia-reperfusion injury in rat heart tissue (TH p=0.008, AADC p=0.031, DBH p=0.008). Pharmacological inhibition of catecholamine biosynthesis caused dose-dependent reductions in cardiomyocyte viability (p<0.0001), increased apoptosis, organelle stress, and mitochondrial dysfunction, with greater effects under oxidative stress. Mechanistically, catecholamine depletion suppressed mTORC1 signalling and activated LKB1-AMPK-ULK1 pathways. In Langendorff-perfused hearts, tyrosine hydroxylase inhibition induced ventricular arrhythmias in 5 of 6 hearts, including sustained ventricular tachycardia, polymorphic ventricular tachycardia, and ventricular fibrillation. ConclusionsThese findings identify cardiomyocytes as previously unrecognised catecholamine-competent cells expressing intrinsic machinery for catecholamine biosynthesis and vesicular handling. Disruption of this pathway compromises metabolic and organelle homeostasis, activates energy-stress and autophagy-related signalling, and promotes malignant ventricular arrhythmias. Intrinsic cardiomyocyte catecholamine biology therefore represents a non-neuronal regulatory axis essential for myocardial resilience and electrical stability, with potential relevance to ischaemic injury and stress-induced dysfunction.

Synthetic opioids like fentanyl are a leading cause of overdose mortality. Although the hallmark of fentanyl overdose is ventilatory depression, fentanyl also induces tonic activation of skeletal musculature, including the diaphragm, which may advance progression of overdose towards death. While tonicity may further restrict diaphragmatic contractility, phase-specific dysregulation may also reflect a larger state of discoordination in respiratory control. Using urethane-anesthetized mice exposed to fentanyl, we test the hypothesis that fentanyl-induced diaphragm tonicity results from a loss of coordinated motor activity. Fentanyl produced two distinct phases: an initial phase of maximal ventilatory depression with preserved phasic activity, and a later phase characterized by unstable ventilation that partially rebounds, tonic diaphragmatic activation with loss of inspiratory phase dominance in EMG activity, and diminished bilateral diaphragmatic coordination. Carotid body denervation eliminated tonic activity and expiratory-phase EMG elevation, but it did not prevent hemi-diaphragm discoordination or ventilatory instability. Rhythmic brainstem slice recordings showed that bilateral preBotzinger complex burst-amplitude coupling was disrupted by u-opioid receptor (MOR) agonism. Furthermore, disordered diaphragm activity was reversed by administration of the MOR antagonist, Naloxone. Our findings reframe fentanyl overdose as a temporally evolving syndrome that involves distinct mechanisms to disrupt respiratory motor coordination.

While bioelectricity is increasingly recognized as an important regulator of cell function and morphogenesis, the field has almost exclusively focused on plasma membrane states. Voltage across the inner nuclear membrane (INM) has been proposed as a potential regulator of nuclear function, but how it responds to extracellular ionic perturbations and whether it relates to chromatin organization remain unclear. Here, we targeted the ratiometric genetically encoded voltage indicator ASAP3-R3 to SUN2-associated nuclear membranes in intact NRK cells and combined INM voltage measurements with Gray-Level Co-Occurrence Matrix (GLCM)-based chromatin texture analysis. Reporter localization was confirmed by fluorescence imaging and electron microscopy, and functional validation in isolated nuclei showed that sodium-potassium pump inhibition produced INM depolarization consistent with Goldman-Hodgkin-Katz (GHK)-based prediction. We then used our validated construct to determine the response of Vnuc and chromatin texture to changing ionic conditions via two exposure methods, gradual (ramped) exposure or direct application. In intact cells, ramping different sets of ionic solutions of decreasing sodium/increasing potassium, decreasing sodium, increasing potassium, or decreasing chloride induced INM hyperpolarization and coordinated changes in chromatin texture, including increased contrast and entropy, reduced homogeneity, and reduced nuclear area. These effects were strongly path-dependent, with nuclear responses shaped by the history and order of ionic exposure: sodium and potassium responses emerged most clearly during ramping exposure, whereas reducing chloride by direct exposure showed a more pronounced response profile. Direct changes in sodium exposure produced limited electrical and chromatin-texture effects, while direct potassium exposure altered chromatin texture and nuclear area without significantly changing VNuc. Importantly, shifting baseline chromatin state in either direction, through Trichostatin-A (TSA)-induced chromatin relaxation or sodium azide/2-deoxy-D-glucose-induced compaction, blunted ion-associated Vnuc and chromatin responses across sodium, potassium, and chloride conditions. Together, these findings identify the nucleus as a dynamic, ion-responsive electro-structural system in which INM voltage and chromatin organization are functionally coupled, and in which both ionic trajectory and pre-existing chromatin state shape the magnitude of the nuclear response.

Regular exercise induces adaptations in skeletal muscle and other organ systems to improve physical performance and overall health. Exercise results in phosphorylation of 5 AMP-activated protein kinase (AMPK) at threonine 172 (T172) of the 2 subunit; however, the role of this activation in cellular and functional adaptations has not been elucidated. To this end, we subjected non-activatable Ampk2(T172A) knock-in (KI) adult mice and wild-type (WT) littermates to 4 weeks of voluntary wheel running (VWR). Exercise training led to significant improvements in endurance capacity, maximal oxygen consumption ([Formula]O2max), and glucose tolerance, as well as skeletal muscle IIb-to-IIa fiber type shift in both WT and KI mice. Contrastingly, VWR resulted in increased mitochondrial OxPhos protein expression, mitochondrial volume density, and capillary density in skeletal muscle of WT but not KI mice. Exercise-induced improvements of mitochondrial respiration and conductance revealed by high-resolution respirometry of isolated mitochondria were blunted in KI mice. Therefore, for the first time, we reveal that AMPK2 T172 activation is required for exercise training-induced mitochondrial biogenesis, improvement of mitochondrial respiratory function, and angiogenesis in skeletal muscle, but that these adaptations are not solely responsible for improved [Formula]O2max and exercise endurance capacity. Significance StatementExercise is the most effective lifestyle intervention for promoting health and preventing chronic diseases through adaptive changes in skeletal muscle and many other tissues/organs. AMPK is an energy sensor and signaling regulator for exercise-induced skeletal muscle adaptation, yet its functional role and the impact on exercise capacity have been studied in mouse genetic models wherein protein stoichiometry is disrupted. Using non-activatable Ampk2(T172A) knock-in mice, we ascertained that AMPK2 activation via T172 phosphorylation is required for endurance training-induced mitochondrial and angiogenic adaptations in skeletal muscle. Importantly, these adaptations are not required for improved exercise capacity, challenging the prevailing concept that increased mitochondrial content and function and microvasculature are the sole driving factors for the performance gains with endurance training.

Mechanotransduction via the actin cytoskeleton is linked to fundamental cellular processes such as morphogenesis, cell division, and motility, requiring the control of tensile forces mediated by the motor protein non-muscle myosin 2 (NM2). Formins such as mDia1 have been shown to elongate actin structures that are under mechanical tension; conversely, mDia1s elongation rates are modulated by the applied force. Despite their relevance at the membrane/cortex interface, reported values for tension in formin-elongated actin filaments stem from theoretical estimates and simulations, but have not been amenable experimentally so far. Thus, we developed a Forster resonance energy transfer (FRET)-based, tension-sensitive probe (mDia1TS) and quantified the measured tension in live U2OS cells using fluorescence lifetime imaging microscopy (FLIM). Through whole-cell ROI analysis we show a short and long lifetime component, reporting an intensity-weighted, averaged lifetime corresponding to [~]3.5 pN. Upon mitogen stimulation of cells using EGF, we show that the tension homeostasis changed significantly, with a measurable increase in tension in the cells periphery and relaxation in its center. Furthermore, the reported average tension relaxed by 2 pN after adding the NM2 inhibitor para-nitroblebbistatin. We utilized siRNA knockdowns of individual NM2 paralogs (NM2-A, NM2-B, or NM2-C) to measure their individual contribution, revealing NM2-A as the main paralog to produce tensile force in this system. Taken together, we demonstrate that mDia1TS is able to directly determine that active mDia1 in cells is under tension, and that subcellular quantification with pN precision is possible. SignificanceDespite the fundamental importance of formins in regulating actin-based processes, reported values for tension in formin-mediated actin structures stem from simulations and theoretical estimates. In this study we developed a FRET-based, tension-sensitive reporter probe for formin mDia1, which we termed mDia1TS. Given the expanding clinical spectrum of DIAPH1/mDia1 mutations, our tool mDia1TS provides a quantitative tool for elucidation of changes in cytoskeletal assemblies.

Decreases in brain pH are associated with numerous neurological and neuropsychiatric conditions yet the molecular mechanisms linking decreased brain pH with these disorders are incomplete. The ionotropic glutamate receptors (iGluRs) mediate the majority of excitatory neurotransmission in the brain and are inhibited by extracellular protons; however, the proton sensitivity of the delta-glutamate receptor subclass of iGluRs is unknown. Using whole-cell patch-clamp recordings of serotonin neurons in mouse brain slices and activating alpha 1-adrenergic receptors to induce delta 1 glutamate receptor (GluD1R) current, we demonstrated that GluD1R current is inhibited by physiological drops in extracellular pH. Unlike other iGluRs, protons inhibited GluD1R current via a voltage-independent decrease in unitary current. Moreover, mice lacking GluD1R showed impaired behavioral responses to inhalation of CO2. Taken together, this study continues to expand on the growing body of evidence positing GluD1R as functional ion channels and suggests that GluD1R facilitate pH sensing in vivo.

Resistance training lasting a few weeks increases maximal force mainly through neural adaptations that enhance the drive from the nervous system to muscle. While these adaptations have been well documented at the motor unit (MU) level during submaximal force contractions, the mechanisms underlying force increases during maximal voluntary contractions are poorly understood. This is due to a classic technical limitation in tracking MUs longitudinally during maximal force tasks. Here, we solved this technical challenge, enabling the investigation of MU adaptations during MVCs in both the trained and untrained limbs following unilateral resistance training. High-density surface electromyography was recorded from the biceps brachii of both limbs before and after a 4-week unilateral resistance-training intervention, and the same MUs were longitudinally tracked across sessions during MVCs by concatenation of three MVC trials of ~5-s each.Unilateral training increased maximal force in the trained limb (+16%) and induced strength transfer to the untrained limb (+8%). In both limbs, maximal contractions after training were characterized by greater EMG amplitude, faster muscle-fiber conduction velocity, and higher MU discharge rates, indicating enhanced neural drive to the motoneuron pool. These adaptations were strongly associated with improvements in maximal force (R2 > 0.7 for all). Importantly, longitudinal MU tracking revealed a non-uniform adaptation across the MU pool: MUs with higher baseline conduction velocity, indicative of higher recruitment threshold, exhibited the largest pre-post increases in discharge rate, whereas lower-threshold units showed smaller changes. Collectively, these findings demonstrate that gains in maximal force and their transfer to the untrained limb are primarily mediated by enhanced rate coding of higher-threshold MUs during MVCs.