Biology subjects

Cox, C.

Publications and source records attributed to Cox, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Fibroblast TGF-β3 promotes tissue-residency and survival of CD8 T cells in barrier tissues and tumors

Fibroblasts are key organizers of tissue architecture and immune cell homeostasis, yet how they shape adaptive immune function within non-lymphoid tissues remains incompletely understood. CD8+ tissue-resident memory T cells (TRM) provide localized protection against pathogens and contribute to tumor control, but the microenvironmental signals that maintain their persistence and survival are poorly defined. Here, we identify fibroblast-derived TGF-{beta}3 as a conserved stromal niche factor that specifically sustains CD8+ TRM in both steady-state and disease settings. Across human single-cell cross-tissue atlases, CD8+ TRM preferentially correlated with fibroblast abundance in healthy barrier tissues and multiple tumor types, and TGFB3 emerged as a key fibroblast-enriched candidate mediator. In human and murine co-culture systems, fibroblast-derived TGF-{beta}3 promoted CD8 TRM-like differentiation in vitro. Using a novel genetic in vivo model, inducible fibroblast-specific deletion of Tgfb3 reduced CD8 TRM across barrier tissues at steady state and impaired antigen-specific CD8 TRM formation following viral infection. In tumor models, genetic loss or antibody mediated neutralization of TGF-{beta}3 impaired CD8 T cell residency and cytotoxicity, induced dysfunction via proteotoxic stress and apoptotic programs, and accelerated tumor growth. These findings provide mechanistic insight into the limited efficacy of pan-TGF-{beta} blockade in cancer therapy. Collectively, we describe a novel fibroblast-CD8 T cell axis mediated by TGF-{beta}3 that sustains residency and restrains proteotoxic stress in barrier tissues and tumors.

immunology↗

bioRxiv2026

Scalable longitudinal imaging and transcriptomics of cells in dynamic enclosures

Dynamic transitions between cell states underlie both normal physiology and disease. However, most single-cell technologies capture only static snapshots. To address this gap, we developed a platform that integrates light-guided hydrogel polymerization with computer vision to generate on-demand compartments around live cells, enabling longitudinal imaging of cellular behavior paired with whole-transcriptome profiling of the same cells at scale. These data link dynamic phenotypes with molecular programs, enabling deeper characterization of cellular states. This approach revealed an adaptive, drug-resistant state in lung cancer cells characterized by potassium channel upregulation and p53-dependent quiescence. In models of adipogenesis and microglial phagocytosis, joint analysis of imaging and transcriptomic data identified key drivers of cellular function that were missed by transcriptomic clustering alone. These results establish the value of paired functional and transcriptomic analysis to resolve molecular drivers of complex cellular behaviors.

systems biology↗

bioRxiv2026

Linking live-cell behavior to transcriptional responses across perturbations using dynamic caging

Single-cell technologies, encompassing molecular, morphological, and functional assays, have emerged as cornerstones of modern biological research and discovery. However, current experimental methods often fail to explicitly link these omic modalities, especially in live cells or longitudinally through time, impeding the study of multi-scale interactions and mechanisms of regulation. CellCage Enclosure (CCE) technology overcomes these limitations by dynamically compartmentalizing cells, allowing for scalable, live-cell, longitudinal exploration and simultaneous analysis of transcriptomic, proteomic, and morphological profiles. Using this novel technology, we generate previously inaccessible insights across various in vitro cellular systems under a diverse set of perturbations, including the discovery of morphological and proteomic features linked to immune suppressive gene set expression in human primary regulatory T cells (Tregs), as well as direct association of morphological and proteomic features with inflammatory gene modules in human colonic fibroblasts. We then develop a novel pooled CRISPR genetic screening technology using CCEs, PERTURB-LINK (PERTURBational LINKage of transcriptomics and imaging in single cells via enclosure-based screening) and apply this approach in murine bone marrow derived macrophages (BMDMs), enabling multiomic dissection of NF-{kappa}B pathway regulation in response to lipopolysaccharide (LPS) stimulation. Together, these findings demonstrate the broad impact that advancements in live-cell, paired multimodal technologies, especially upon perturbation, may offer in deepening our understanding of cellular biology.

cell biology↗

bioRxiv2025

MDFIC2 is a sensory neuron-specific PIEZO channel auxiliary subunit

PIEZO channels are critical for sensory mechanotransduction. While MyoD-family inhibitor proteins were identified as PIEZO1 auxiliary subunits, their broader regulatory roles, particularly in sensory cells, remained unclear. Here we demonstrate native MDFIC and MDFI regulate endogenous PIEZO channel currents in various non-sensory cell types. However, neither MDFIC nor MDFI are expressed in primary sensory neurons. In these cell types we identified an uncharacterised member of this family, Mdfic2/Gm765, that shares the ability to physically bind to PIEZO1 and PIEZO2. MDFIC2 is selectively expressed in subsets of mechanosensitive neurons, including dorsal root ganglia, trigeminal ganglia, and vagal sensory neurons. Like its paralogues, MDFIC2 alters PIEZO1/2 mechanosensitivity and inactivation kinetics, converting them into high-threshold slowly inactivating mechanoreceptors. Extensive cryo-EM reveals a conserved binding pocket for these auxiliary subunits in the pore modules of both PIEZO1 and PIEZO2 mediated by the post-translationally modified distal C-termini of MyoD-family inhibitor proteins. This provides a comprehensive structural and functional characterisation of MyoD-family inhibitor proteins as PIEZO1/2 channel regulators and offers new insights into sensory physiology and mechanical pain mechanisms.

biophysics↗

bioRxiv2025

Inversions Dominate Evolution in the Highly Admixed European Sardine (Sardina pilchardus)

Inversions can play key roles in the genetic architecture of adaptation, but the scale of their effects across different species remains poorly understood. Here, we use whole- genome sequencing to investigate the influence of inversions on the population genomics of the r-selected European sardine (Sardina pilchardus). Allele frequency differences from millions of SNPs across 34 populations spanning the species range were analyzed. Genomic scans identified several extreme outlier regions overlapping chromosome-scale inversions, collectively representing over half the genome. Our findings suggest these inversions are associated with locally adapted life history strategies. First, SNPs within outlier regions containing inversions exhibited striking allele frequency differences between Atlantic and Mediterranean sardines, which differ in key adaptive life history traits. In the Atlantic, inversion allele frequencies varied latitudinally, while in the Mediterranean, they shifted longitudinally, aligning with temperature and oceanographic features that influence sardine life history strategies. Moreover, adjacent populations in contrasting environments displayed pronounced allele frequency differences in inversions. These spatial patterns of allele frequencies sharply contrasted with those based on neutral loci, indicating they are driven by selection. After rigorously filtering SNPs affected by selection and inversions, sardine populations showed high admixture across their range but significant population structure and isolation by distance, especially in the Mediterranean. This study demonstrates that inversions can shape genome-wide patterns of genetic diversity and population structure in highly admixed r-selected marine species. These findings also offer crucial insights for stock delimitation and management of this commercially valuable species in the face of climate change.

genomics↗

bioRxiv2024

Divergent CD8 memory T cell subsets in an encephalitis model of Toxoplasma gondii infection

CD8 T cell exhaustion has been reported in mice susceptible to Toxoplasma gondii infection. While the differentiation of CD8 exhausted subsets has been extensively reported, most of these studies have been conducted in chronic viral and cancer models. During chronic T. gondii infection, phenotypic and transcriptomic analyses of the polyclonal antigen-specific CD8 T cell response characterize four populations based on KLRG1 and CD62L expression. Pop1 (KLRG1+CD62Llo) bears the attributes of a terminal effector subset, and pop2 (KLRG1-CD62Llo) is similar to effector memory CD8 T cells. Akin to chronic viral infection and cancer systems, pop3 (KLRG1-CD62Lhi) exhibits the characteristics of stem-like progenitor CD8 T cells (high Tcf7, Slamf6, and Cxcr5 expression), whereas pop4 (KLRG1+CD62Lhi) closely resembles a transitory subset (elevated Tbx21, low Tcf1, and Tox expression). During chronic viral infection, the stem-like progenitor CD8 T cells transition into a terminally differentiated exhausted subset via an intermediate population. However, in our system, pop3 generates pop4, which does not convert into a conventional terminally differentiated exhausted subset but instead transitions into effector pop1. Notably, during the chronic phase of the infection, pop1 cannot retain its functionality, irrespective of its origin, which may hamper its ability to control reactivation. Our observations emphasize that the differentiation of exhausted CD8 T cells in non-viral infections, like chronic toxoplasmosis, follows a different pattern than established models and highlights the need to develop new immune strategies better tailored for a broad range of pathogens.

immunology↗

bioRxiv2024

Systematic perturbation screens decode regulators of inflammatory macrophage states and identify a role for TNF mRNA m6A modification

Macrophages adopt dynamic cell states with distinct effector functions to maintain tissue homeostasis and respond to environmental challenges. During chronic inflammation, macrophage polarization is subverted towards sustained inflammatory states which contribute to disease, but there is limited understanding of the regulatory mechanisms underlying these disease-associated states. Here, we describe a systematic functional genomics approach that combines genome-wide phenotypic screening in primary murine macrophages with transcriptional and cytokine profiling of genetic perturbations in primary human monocyte-derived macrophages (hMDMs) to uncover regulatory circuits of inflammatory macrophage states. This process identifies regulators of five distinct inflammatory states associated with key features of macrophage function. Among these, the mRNA m6A writer components emerge as novel inhibitors of a TNF-driven cell state associated with multiple inflammatory pathologies. Loss of m6A writer components in hMDMs enhances TNF transcript stability, thereby elevating macrophage TNF production. A PheWAS on SNPs predicted to impact m6A installation on TNF revealed an association with cystic kidney disease, implicating an m6A-mediated regulatory mechanism in human disease. Thus, systematic phenotypic characterization of primary human macrophages describes the regulatory circuits underlying distinct inflammatory states, revealing post-transcriptional control of TNF mRNA stability as an immunosuppressive mechanism in innate immunity.

immunology↗

bioRxiv2023

Pathogenic Leptospira are widespread in the urban wildlife of southern California

Leptospirosis is the most widespread zoonotic disease in the world, yet it is broadly understudied in multi-host wildlife systems. Knowledge gaps regarding Leptospira circulation in wildlife, particularly in densely populated areas, contribute to frequent misdiagnoses in humans and domestic animals. We assessed Leptospira prevalence levels and risk factors in five target wildlife species across the greater Los Angeles region: striped skunks (Mephitis mephitis), Northern raccoons (Procyon lotor), coyotes (Canis latrans), Virginia opossums (Didelphis virginiana), and fox squirrels (Sciurus niger). We sampled more than 960 individual animals, including over 700 from target species in the greater Los Angeles region, and an additional 260 sampled opportunistically from other regions and species. In the five target species, seroprevalences ranged from 5-60% and active infection prevalences ranged from 0.8-15.2% in all except fox squirrels (0%). Patterns of serologic reactivity suggest that mainland terrestrial wildlife, particularly mesocarnivores, could be the source of repeated observed introductions of Leptospira into local marine and island ecosystems. Overall, we found evidence of widespread Leptospira exposure in wildlife across Los Angeles and surrounding regions. This indicates exposure risk for humans and domestic animals and highlights that this pathogen can circulate endemically in many wildlife species even in densely populated urban areas.

ecology↗

bioRxiv2021

Alpha-Gal Syndrome: Involvement of Amblyomma americanum α-D-galactosidase and β-1,4 Galactosyltransferase enzymes in α-gal metabolism

Alpha-Gal Syndrome (AGS) is an IgE-mediated delayed-type hypersensitivity reaction to the oligosaccharide galactose--1,3-galactose (-gal) injected into humans from the lone star tick (Amblyomma americanum) bite. This study aims at the functional characterization of two tick enzymes, -D-galactosidase (ADGal) and -1,4 galactosyltransferase ({beta}-1,4GalT) in -gal metabolism. The ADGal enzyme cleaves terminal -galactose moieties from glycoproteins and glycolipids, whereas {beta}-1,4GalT transfers -galactose to a {beta}1,4 terminal linkage acceptor sugars: GlcNAc, Glc, and Xyl in various processes of glycoconjugate synthesis. An RNA interference approach was utilized to silence ADGal and {beta}-1,4GalT in Am. americanum to examine their functional role in -gal metabolism and AGS onset. Silencing of ADGal led to the significant down regulation of genes involved in galactose metabolism and transport in Am. americanum. Immunoblot and N-glycan analysis of the Am. americanum salivary glands showed a significant reduction in -gal levels in silenced tissues. However, there was no significant difference in the level of -gal in {beta}-1,4GalT silenced tick salivary glands. A basophil-activation test showed a decrease in the frequency of activated basophil by ADGal silenced salivary glands. These results provide an insight into the role of -D galactosidase & {beta}-1,4GalT in tick biology and the probable involvement in the onset of AGS.

molecular biology↗

bioRxiv2021

Twenty years of change in benthic communities across the Belizean Barrier Reef

Disease, ocean warming, and pollution have caused catastrophic declines in the cover of living coral on reefs across the Caribbean. Subsequently, reef-building corals have been replaced by invertebrates and macroalgae, leading to changes in ecological functioning. We describe changes in benthic community composition and cover at 15 sites across the Belizean Barrier Reef (BBR) following numerous major disturbances--bleaching, storms, and disease outbreaks--over the 20-year period 1997-2016. We tested the role of potential drivers of change on coral reefs, including local human impacts and ocean temperature. From 1997 to 2016, mean coral cover significantly declined from 26.3% to 10.7%, while macroalgal cover significantly increased from 12.9% to 39.7%. We documented a significant decline over time of the reef-building corals Orbicella spp. and described a major shift in benthic composition between early sampling years (1997-2005) and later years (2009-2016). The covers of hard-coral taxa, including Acropora spp., M. cavernosa, Orbicella spp., and Porites spp., were negatively related to marine heatwave frequency. Only gorgonian cover was related, negatively, to our metric of the magnitude of local impacts (the Human Influence Index). Changes in benthic composition and cover were not associated with local protection or fishing. This result is concordant with studies throughout the Caribbean that have documented living coral decline and shifts in reef-community composition following disturbances, regardless of local fisheries restrictions. Our results suggest that benthic communities along the BBR have experienced disturbances that are beyond the capacity of the current management structure to mitigate. We recommend that managers devote greater resources and capacity to enforce and expand existing marine protected areas and that government, industry, and the public act to reduce global carbon emissions.

ecology↗

bioRxiv2021

Cyclodextrins increase membrane tension and are universal activators of mechanosensitive channels

The bacterial mechanosensitive channel of small conductance, MscS, has been extensively studied to understand how mechanical forces are converted into the conformational changes that underlie mechanosensitive (MS) channel gating. We showed that lipid removal by {beta}-cyclodextrin can mimic membrane tension. Here, we show that all cyclodextrins (CDs) can activate reconstituted E. coli MscS, that MscS activation by CDs depends on CD-mediated lipid removal, and that the CD amount required to gate MscS scales with the channels sensitivity to membrane tension. CD-mediated lipid removal ultimately causes MscS desensitization, which we show is affected by the lipid environment. CDs can also activate the structurally unrelated MscL. While many MS channels respond to membrane forces, generalized by the force-from-lipids principle, their different molecular architectures suggest that they use unique ways to convert mechanical forces into conformational changes. CDs emerge as a universal tool for the structural and functional characterization of unrelated MS channels.

biophysics↗

bioRxiv2020

The Ca2+-activated cation channel TRPM4 is a positive regulator of pressure overload-induced cardiac hypertrophy

Pathological left ventricular hypertrophy (LVH) is a consequence of pressure overload caused by systemic hypertension or aortic stenosis and is a strong predictor of cardiac failure and mortality. Understanding the molecular pathways in the development of pathological LVH may lead to more effective treatment. Here, we show that the transient receptor potential cation channel subfamily melastatin 4 (TRPM4) ion channel is an important contributor to the mechanosensory transduction of pressure overload that induces LVH. In mice with pressure overload induced by transverse aortic constriction (TAC) for two weeks, cardiomyocyte TRPM4 expression was reduced, as compared to control mice. Cardiomyocyte-specific TRPM4 inactivation reduced by ~50% the degree of TAC-induced LVH, as compared with wild type (WT). In WT mice, TAC activated the CaMKII{delta}-HDAC4-MEF2A but not the calcineurin-NFAT-GATA4 pathway. In TRPM4 knock-out mice, activation of the CaMKII{delta}-HDAC4-MEF2A pathway by TAC was significantly reduced. However, consistent with a reduction in the known inhibitory effect of CaMKII{delta} on calcineurin activity, reduction in the CaMKII{delta}-HDAC4-MEF2A pathway was associated with partial activation of the calcineurin-NFAT-GATA4 pathway. These findings indicate that the TRPM4 channel and its cognate signalling pathway are potential novel therapeutic targets for the prevention of pathological pressure overload-induced LVH. Significance statementPathological left ventricular hypertrophy (LVH) occurs in response to pressure overload and remains the single most important clinical predictor of cardiac mortality. Preventing pressure overload LVH is a major goal of therapeutic intervention. Current treatments aim to remove the stimulus for LVH by lowering elevated blood pressure or replacing a stenotic aortic valve. However, neither of these interventions completely reverses adverse cardiac remodelling. Although numerous molecular signalling steps in the induction of LVH have been identified, the initial step by which mechanical stretch associated with cardiac pressure overload is converted into a chemical signal that initiates hypertrophic signalling, remains unresolved. Here, we demonstrate that the TRPM4 channel is a component of the mechanosensory transduction pathway that ultimately leads to LVH.

physiology↗

bioRxiv2020

Efficacy of Biofilm Disrupters Against Candida auris and Other Candida species

BackgroundC. auris has become a globally emerging fungal pathogen, frequently reported to be multi-drug resistant, commonly found with Staphylococcus aureus in polymicrobial nosocomial infections. Although chlorhexidine (CHD) has been shown to be effective, it is associated with serious anaphylaxis reactions. Biofilm disrupters (BDs) are novel agents with a broad spectrum of antimicrobial activity. BDs have been used in the management of chronic wounds and to sterilize environmental surfaces. The goal of this study was to evaluate BDs against polymicrobial biofilms compared to CHD. MethodologyWe evaluated various BDs (BlastX, Torrent, NSSD) and CHD against Candida spp and S. aureus polymicrobial biofilms by zone of inhibition, biofilm, and time-kill assays. Effectiveness was based on the inhibition zone and the reduction of CFU, respectively, compared to the drug-free control. ResultsAll BDs and CHD inhibited C. auris growth effectively in a concentration-dependent manner. Additionally, CHD and the BDs all showed excellent antimicrobial activity against polymicrobial biofilms. BDs were all highly effective against both C. auris isolates, whereas CHD was only moderately effective against C. auris 0386, suggesting resistance/tolerance. A comparative analysis of the BDs and CHD against C. auris and C. albicans by biofilm kill-curves showed at least 99.999% killing. ConclusionsAll three BDs and CHD have excellent activity against different Candida species, including C. auris. However, certain isolates of C. auris showed resistance/tolerance to CHD, but not to the BDs. The fungicidal activity of these novel agents will be valuable in eradicating surface colonization of Candida spp, including C. auris.

microbiology↗

bioRxiv2020

The extent and impact of variation in ADME genes in sub-Saharan African populations

Investigating variation in genes involved in the absorption, distribution, metabolism, and excretion (ADME) of drugs are key to characterising pharmacogenomic (PGx) relationships. ADME gene variation is relatively well characterised in European and Asian populations, but African populations are under-studied - which has implications for safe and effective drug use in Africa. The genetic diversity of ADME genes across sub-Saharan African populations is large. The Southern African population cluster is most distinct from that of far West Africa. PGx strategies based on European variants will be of limited use in African populations. Although established variants are important, PGx must take into account the full range of African variation. This work urges further characterisation of variants in African populations including in vitro and in silico studies, and to consider the unique African ADME landscape when developing precision medicine guidelines and tools for African populations. Author summaryThe ADME genes are a group of genes that play a key role in absorption, distribution, metabolism and excretion of drugs. Variations in these genes can have a significant impact on drug safety and efficacy. Africa has a high level of genetic variation and is under-studied in drug development, which makes study of variations in these genes in African populations very important. Using a new data set of 458 high-coverage genomes from across Africa, we characterise the extent and impact of variation in the ADME genes, looking at both single nucleotide and copy number variations. We identified 343,368 variants, including 40,692 novel variants, and 930 coding variants which are predicted to have high impact on function. Our discovery curves indicate that there will be considerable value in sequencing more African genomes. Moreover, relatively few of these novel variants are captured on common genotyping arrays. We show that there is considerable diversity within Africa in some important genes, and this will have significant consequences for the emerging field of precision medicine in Africa.

genetics↗

bioRxiv2019

Piezo1 Induces Local Curvature in a Mammalian Membrane and Forms Specific Protein-Lipid Interactions

Touch, hearing, and blood pressure control require mechanically-gated ion channels that convert mechanical stimuli into electrical currents. Piezo1 and Piezo2 were recently identified as essential eukaryotic mechanically-gated ion channels, yet how they respond to physical forces remains poorly understood. Here we use a multi-disciplinary approach to interrogate the interaction of Piezo1 with its lipid environment. We show that individual Piezo1 channels induce significant local curvature in the membrane that is magnified in a cooperative manner to generate larger curved Piezo1 pits. Curvature decreases under lateral membrane tension, consistent with a hypothesis that force detection can involve sensing changes to local curvature. The protein alters its local membrane composition, enriching specific lipids and forming essential binding sites for phosphoinositides and cholesterol that are functionally relevant and often related to Piezo1-mediated pathologies. Finally, we show that Piezo1 alters the expression of lipid-regulating proteins and modifies the cellular lipidome. In short, we find that lipids influence Piezo1 activity and Piezo1 influences the local morphology and composition of the bilayer as well as the cellular lipidome.

biophysics↗

bioRxiv2019

Finding and extending ancient simple sequence repeat-derived regions in the human genome

BackgroundPreviously, 3% of the human genome has been annotated as simple sequence repeats (SSRs), similar to the proportion annotated as protein coding. The origin of much of the genome is not well annotated, however, and some of the unidentified regions are likely to be ancient SSR-derived regions not identified by current methods. The identification of these regions is complicated because SSRs appear to evolve through complex cycles of expansion and contraction, often interrupted by mutations that alter both the repeated motif and mutation rate. We applied an empirical, kmer-based, approach to identify genome regions that are likely derived from SSRs.\n\nResultsThe sequences flanking annotated SSRs are enriched for similar sequences and for SSRs with similar motifs, suggesting that the evolutionary remains of SSR activity abound in regions near obvious SSRs. Using our previously described P-clouds approach, we identified SSR-clouds, groups of similar kmers (or oligos) that are enriched near a training set of unbroken SSR loci, and then used the SSR-clouds to detect likely SSR-derived regions throughout the genome.\n\nConclusionsOur analysis indicates that the amount of likely SSR-derived sequence in the human genome is 6.77%, over twice as much as previous estimates, including millions of newly identified ancient SSR-derived loci. SSR-clouds identified poly-A sequences adjacent to transposable element termini in over 74% of the oldest class of Alu (roughly, AluJ), validating the sensitivity of the approach. Poly-As annotated by SSR-clouds also had a length distribution that was more consistent with their poly-A origins, with mean about 35 bp even in older Alus. This work demonstrate that the high sensitivity provided by SSR-Clouds improves the detection of SSR-derived regions and will enable deeper analysis of how decaying repeats contribute to genome structure.

genomics↗

bioRxiv2019

Evidence for a deep, distributed and dynamic semantic code in human ventral anterior temporal cortex

How does the human brain encode semantic information about objects? This paper reconciles two seemingly contradictory views. The first proposes that local neural populations independently encode semantic features; the second, that semantic representations arise as a dynamic distributed code that changes radically with stimulus processing. Combining simulations with a well-known neural network model of semantic memory, multivariate pattern classification, and human electrocorticography, we find that both views are partially correct: semantic information is distributed across ventral temporal cortex in a dynamic code that possesses stable feature-like elements in posterior regions but with elements that change rapidly and nonlinearly in anterior regions. This pattern is consistent with the view that anterior temporal lobes serve as a deep cross-modal "hub" in an interactive semantic network, and more generally suggests that tertiary association cortices may adopt dynamic distributed codes difficult to detect with common brain imaging methods.

neuroscience↗

bioRxiv2019

Circadian control of intrinsic heart rate via a sinus node clock and the pacemaker channel

In the human, there is a circadian rhythm in the resting heart rate and it is higher during the day in preparation for physical activity. Conversely, slow heart rhythms (bradyarrhythmias) occur primarily at night. Although the lower heart rate at night is widely assumed to be neural in origin (the result of high vagal tone), the objective of the study was to test whether there is an intrinsic change in heart rate driven by a local circadian clock. In the mouse, there was a circadian rhythm in the heart rate in vivo in the conscious telemetrized animal, but there was also a circadian rhythm in the intrinsic heart rate in denervated preparations: the Langendorff-perfused heart and isolated sinus node. In the sinus node, experiments (qPCR and bioluminescence recordings in mice with a Per1 luciferase reporter) revealed functioning canonical clock genes, e.g. Bmal1 and Per1. We identified a circadian rhythm in the expression of key ion channels, notably the pacemaker channel Hcn4 (mRNA and protein) and the corresponding ionic current (funny current, measured by whole cell patch clamp in isolated sinus node cells). Block of funny current in the isolated sinus node abolished the circadian rhythm in the intrinsic heart rate. Incapacitating the local clock (by cardiac-specific knockout of Bmal1) abolished the normal circadian rhythm of Hcn4, funny current and the intrinsic heart rate. Chromatin immunoprecipitation demonstrated that Hcn4 is a transcriptional target of BMAL1 establishing a pathway by which the local clock can regulate heart rate. In conclusion, there is a circadian rhythm in the intrinsic heart rate as a result of a local circadian clock in the sinus node that drives rhythmic expression of Hcn4. The data reveal a novel regulator of heart rate and mechanistic insight into the occurrence of bradyarrhythmias at night.

physiology↗