Biology subjects

Blair, C.

Publications and source records attributed to Blair, C..

7 recordsLinked to original sources

bioRxiv2025

Targeting Troponin I Phosphorylation for Precision Treatment of Diastolic Dysfunction

BackgroundDefective cardiac relaxation (diastolic dysfunction) is prevalent in heart failure, particularly in the context of diabetes, obesity, hypertension, and ageing, and is associated with increased mortality. Yet there is no available treatment that directly targets this abnormal relaxation. Phosphorylation of troponin I by cAMP-dependent protein kinase A (PKA) promotes relaxation, but global PKA activation has widespread, undesirable effects. Here we investigated whether selectively enhancing troponin I phosphorylation could improve relaxation without engaging the broader PKA signalling network. MethodsTo resolve cAMP signalling at subcellular scales, we combined real-time cAMP measurements with FRET-based genetically encoded reporters targeted to defined nanodomains, alongside biochemical and genetic approaches. Protein-protein interaction surfaces were mapped using peptide-array technology, which guided the design of a disruptor peptide to displace specific interactions. The peptides effects on cardiomyocyte function were assessed in vitro using rodent and human cardiomyocytes with biochemical assays, real-time imaging, and work-loop analysis. In vivo effects were evaluated through echocardiography and haemodynamic measurements. ResultsWe identified a previously unrecognized regulatory mechanism in which the cAMP-hydrolyzing enzyme PDE4D9 interacts with troponin I and restricts local cAMP levels within a nanometre-scale domain, thereby selectively controlling troponin I phosphorylation. We found that PDE4D9-troponin I association is markedly increased in cardiac disease in both rodents and humans. A peptide that displaces PDE4D9 from troponin I in a mouse model of heart failure selectively enhances troponin I phosphorylation, accelerates cardiomyocyte relaxation, and prevents diastolic dysfunction, without compromising systolic contractility. ConclusionsThese findings reveal a highly specific regulatory nanodomain governing troponin I phosphorylation and points to a first-in-class, mechanism-based therapeutic strategy with the potential to address a major unmet contributor to heart failure. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIWe identify a previously unrecognized, nanometre-scale regulatory mechanism in which PDE4D9 binds troponin I to locally suppress cAMP, limit troponin I phosphorylation and attenuate relaxation. C_LIO_LIWe demonstrate that this mechanism becomes maladaptive in disease: PDE4D9-troponin I interaction is markedly upregulated in failing hearts in both rodents and humans, which can mechanistically explain the impaired cardiac relaxation. C_LIO_LIWe show that this interaction is druggable: a rationally designed peptide can selectively displace PDE4D9, restore localized cAMP-PKA signalling, and normalize myocardial relaxation without global cAMP perturbation. C_LIO_LIDisplacement of PDE4D9 from TPNI improves relaxation with no impact on the calcium transient amplitude and without adversely affecting systolic output and cardiac reserve. C_LI What Are the Clinical Implications?O_LIDiastolic dysfunction, a major cause of heart failure with no targeted treatments, may be amenable to therapies that modulate cAMP signalling with subcellular precision rather than global pathway modulation. C_LIO_LISelective disruption of PDE4D9-troponin I binding offers a mechanism-based approach with potential to restore diastolic function while minimizing off-target effects typical of GPCR-directed therapies. C_LIO_LIBy enhancing relaxation while preserving systolic function and cardiac reserve, this approach may overcome limitations of myofilament-targeted therapies currently under evaluation for diastolic dysfunction that exhibit negative inotropic effects. C_LIO_LIMore broadly, this work establishes a platform for targeting localized signalling domains, a concept that may extend beyond cardiovascular disease to other pathological conditions in which dysregulated cAMP contributes to dysfunction. C_LI

cell biology↗

bioRxiv2025

Moderated designs can balance between batch-effect mitigation and cell loss due to hashtag-assisted pooling in single-cell experiments

Minimizing experimental noise is integral to robust data generation in single-cell science. Experimental processing of different samples as a single pool, made possible by hashtag-assisted pooling, helps minimize batch-effects, but the computational demultiplexing of the data can also lead to loss of cells whose hashtags cannot be resolved accurately. Here, we examine four alternate experimental designs that could be used instead of a single-pool approach and quantify the batch effects as well as cell loss in each case. While a reference design offers the best performance, this study can help individual investigators choose one suited for their biological questions.

bioinformatics↗

bioRxiv2025

Genome-wide signatures of reproductive isolation shape the varied genomic landscape of the roundtail horned lizard (Phrynosoma modestum)

Population divergence is promoted and inhibited by gene flow and divergent selection, but the mechanisms of and relationship between these two processes remain poorly understood. Modelling the selective pressures at play in a natural population requires a thorough understanding of species structure and demographic history, which can be helpful in hypothesizing the genetic and evolutionary factors that underlie divergence. In this study, we assess whole genome sequences of round-tailed horned lizards (Phyrnosoma modestum) from throughout the species range and combine phylogenetic analyses with genetic landscape scans to understand how current genetic diversity has been influenced by demographic histories and evolutionary pressures. Maximum likelihood phylogenetic analysis supports two lineages within the species, correlating with a North/South population divide that likely diverged about 7 Ma and displayed little migration. Intermediate gdi values indicate that the two lineages may be in the gray area of speciation, yet our results also support significant isolation-by-distance (IBD). Negative values of Tajimas D offer support for selection acting on P. modestum, but may stem from recent population expansions. Genomic diversity within populations and islands of divergence between populations are also detected across the P. modestum genome, specifically exhibiting differentiation patterns linked to reproductive isolation and within-population selection. We posit potential explanations for the genomic landscape characterized here, namely that allopatry plays a large role in shaping genomic divergence across the species range. Taken together, our results provide a picture of a species currently maintaining species integrity despite significant genomic signatures of geographic structure and reproductive isolation.

evolutionary biology↗

bioRxiv2024

Kinetic patterns of single cell gene expression discriminate between the murine cellular responses to live attenuated and inactivated Yellow Fever vaccines

The success of the live attenuated Yellow Fever vaccine (YF17D) that elicits immunity lasting over thirty years has made it a widely used model to understand the generation of durable protection. We compare the early single-cell level transcriptional response in mice to YF17D and an adjuvanted-inactivated, but less effective version (InYF). Within the first week, we identify 70 kinetic patterns in 45 cellular clusters, majority of which discriminate between the two formulations, some in a tissue and sex-specific manner. Intriguingly, differential transcripts fall into two categories, one whose association with YF17D or InYF is maintained even when decoupled from their cell-type of expression and the other where such cell-plus-gene pairing is critical to maintain differential marker status. We demonstrate applications of this resource, by identifying B cells with varied interferon and antigen responsiveness in relation to each vaccine. This high-resolution dataset is amenable to further biomarker discovery and hypothesis generation.

immunology↗

bioRxiv2024

CountASAP: A Lightweight, Easy to Use Python Package for Processing ASAPseq Data

Declining sequencing costs coupled with the increasing availability of easy-to-use kits for the isolation of DNA and RNA transcripts from single cells have driven a rapid proliferation of studies centered around genomic and transcriptomic data. Simultaneously, a wealth of new techniques have been developed that utilize single cell technologies to interrogate a broad range of cell-biological processes. One recently developed technique, transposase-accessible chromatin with sequencing (ATAC) with select antigen profiling by sequencing (ASAPseq), provides a combination of chromatin accessibility assessments with measurements of cell-surface marker expression levels. While software exists for the characterization of these datasets, there currently exists no tool explicitly designed to reformat ASAP surface marker FASTQ data into a count matrix which can then be used for these downstream analyses. To address this, we created CountASAP, an easy-to-use Python package purposefully designed to transform FASTQ files from ASAP experiments into count matrices compatible with commonly-used downstream bioinformatic analysis packages. CountASAP takes advantage of the independence of the relevant data structures to perform fully parallelized matches of each sequenced read to user-supplied input ASAP oligos and unique cell-identifier sequences.

bioinformatics↗

bioRxiv2023

Multi-scale models reveal hypertrophic cardiomyopathy MYH7 G256E mutation drives hypercontractility and elevated mitochondrial respiration

RationaleOver 200 mutations in the sarcomeric protein {beta}-myosin heavy chain (MYH7) have been linked to hypertrophic cardiomyopathy (HCM). However, different mutations in MYH7 lead to variable penetrance and clinical severity, and alter myosin function to varying degrees, making it difficult to determine genotype-phenotype relationships, especially when caused by rare gene variants such as the G256E mutation. ObjectiveThis study aims to determine the effects of low penetrant MYH7 G256E mutation on myosin function. We hypothesize that the G256E mutation would alter myosin function, precipitating compensatory responses in cellular functions. MethodsWe developed a collaborative pipeline to characterize myosin function at multiple scales (protein to myofibril to cell to tissue). We also used our previously published data on other mutations to compare the degree to which myosin function was altered. ResultsAt the protein level, the G256E mutation disrupts the transducer region of the S1 head and reduces the fraction of myosin in the folded-back state by 50.9%, suggesting more myosins available for contraction. Myofibrils isolated from hiPSC-CMs CRISPR-edited with G256E (MYH7WT/G256E) generated greater tension, had faster tension development and slower early phase relaxation, suggesting altered myosin-actin crossbridge cycling kinetics. This hypercontractile phenotype persisted in single-cell hiPSC-CMs and engineered heart tissues. Single-cell transcriptomic and metabolic profiling demonstrated upregulation of mitochondrial genes and increased mitochondrial respiration, suggesting altered bioenergetics as an early feature of HCM. ConclusionsMYH7 G256E mutation causes structural instability in the transducer region, leading to hypercontractility across scales, perhaps from increased myosin recruitment and altered crossbridge cycling. Hypercontractile function of the mutant myosin was accompanied by increased mitochondrial respiration, while cellular hypertrophy was modest in the physiological stiffness environment. We believe that this multi-scale platform will be useful to elucidate genotype-phenotype relationships underlying other genetic cardiovascular diseases.

molecular biology↗

bioRxiv2020

Phosphodiesterase Type 4 anchoring regulates cAMP signaling to Popeye domain-containing proteins.

Cyclic AMP is a ubiquitous second messenger used to transduce intracellular signals from a variety of Gs-coupled receptors. Compartmentalisation of protein intermediates within the cAMP signaling pathway underpins receptor-specific responses. The cAMP effector proteins protein-kinase A and EPAC are found in complexes that also contain phosphodiesterases whose presence ensures a coordinated cellular response to receptor activation events. Popeye proteins are the most recent class of cAMP effectors to be identified and have crucial roles in cardiac pacemaking and conduction. We report the first observation that Popeye proteins exist in complexes with members of the PDE4 family in cardiac myocytes thus restricting cAMP signaling. We show that POPDC1 preferentially binds the PDE4A sub-family via a specificity motif in the PDE4 UCR1 region and that PDE4s bind to the Popeye domain of POPDC1 in a region known to be susceptible to a mutation that causes human disease. Using a cell-permeable disruptor peptide that displaces the POPDC1-PDE4 complex we show that PDE4 activity localized to POPDC1 is essential to maintain action potential duration in beating cardiac myocytes.

biochemistry↗