Integrated transcriptomics and histopathology approach identifies a subset of rejected donor livers with potential suitability for transplantation.

BACKGROUND: Liver transplantation is an effective treatment for liver failure. There is a large unmet demand, even as not all donated livers are transplanted. The clinical selection criteria for donor livers based on histopathological evaluation and liver function tests are variable. We integrated transcriptomics and histopathology to characterize donor liver biopsies obtained at the time of organ recovery. We performed RNA sequencing as well as manual and artificial intelligence-based histopathology (10 accepted and 21 rejected for transplantation). RESULTS: We identified two transcriptomically distinct rejected subsets (termed rejected-1 and rejected-2), where rejected-2 exhibited a near-complete transcriptomic overlap with the accepted livers, suggesting acceptability from a molecular standpoint. Liver metabolic functional genes were similarly upregulated, and extracellular matrix genes were similarly downregulated in the accepted and rejected-2 groups compared to rejected-1. The transcriptomic pattern of the rejected-2 subset was enriched for a gene expression signature of graft success post-transplantation. Serum AST, ALT, and total bilirubin levels showed similar overlapping patterns. Additional histopathological filtering identified cases with borderline scores and extensive molecular overlap with accepted donor livers. CONCLUSIONS: Our integrated approach identified a subset of rejected donor livers that are likely suitable for transplantation, demonstrating the potential to expand the pool of transplantable livers.

Dynamic dysregulation of transcriptomic networks in brainstem autonomic nuclei during hypertension development in the female spontaneously hypertensive rat.

Neurogenic hypertension stems from an imbalance in autonomic function that shifts the central cardiovascular control circuits toward a state of dysfunction. Using the female spontaneously hypertensive rat and the normotensive Wistar-Kyoto rat model, we compared the transcriptomic changes in three autonomic nuclei in the brainstem, nucleus of the solitary tract (NTS), caudal ventrolateral medulla, and rostral ventrolateral medulla (RVLM) in a time series at 8, 10, 12, 16, and 24 wk of age, spanning the prehypertensive stage through extended chronic hypertension. RNA-sequencing data were analyzed using an unbiased, dynamic pattern-based approach that uncovered dominant and several subtle differential gene regulatory signatures. Our results showed a persistent dysregulation across all three autonomic nuclei regardless of the stage of hypertension development as well as a cascade of transient dysregulation beginning in the RVLM at the prehypertensive stage that shifts toward the NTS at the hypertension onset. Genes that were persistently dysregulated were heavily enriched for immunological processes such as antigen processing and presentation, the adaptive immune response, and the complement system. Genes with transient dysregulation were also largely region-specific and were annotated for processes that influence neuronal excitability such as synaptic vesicle release, neurotransmitter transport, and an array of neuropeptides and ion channels. Our results demonstrate that neurogenic hypertension is characterized by brainstem region-specific transcriptomic changes that are highly dynamic with significant gene regulatory changes occurring at the hypertension onset as a key time window for dysregulation of homeostatic processes across the autonomic control circuits.NEW & NOTEWORTHY Hypertension is a major disease and is the primary risk factor for cardiovascular complications and stroke. The gene expression changes in the central nervous system circuits driving hypertension are understudied. Here, we show that coordinated and region-specific gene expression changes occur in the brainstem autonomic circuits over time during the development of a high blood pressure phenotype in a rat model of human essential hypertension.

Acyl-CoA thioesterase-2 facilitates ß-oxidation in glycolytic skeletal muscle in a lipid supply dependent manner.

Acyl-Coenzyme A (acyl-CoA) thioesters are compartmentalized intermediates that participate in in multiple metabolic reactions within the mitochondrial matrix. The limited availability of free CoA (CoASH) in the matrix raises the question of how the local acyl-CoA concentration is regulated to prevent trapping of CoASH from overload of any specific substrate. Acyl-CoA thioesterase-2 (ACOT2) hydrolyzes long-chain acyl-CoAs to their constituent fatty acids and CoASH, and is the only mitochondrial matrix ACOT refractory to inhibition by CoASH. Thus, we reasoned that ACOT2 may constitutively regulate matrix acyl-CoA levels. Acot2 deletion in murine skeletal muscle (SM) resulted in acyl-CoA build-up when lipid supply and energy demands were modest. When energy demand and pyruvate availability were elevated, lack of ACOT2 activity promoted glucose oxidation. This preference for glucose over fatty acid oxidation was recapitulated in C2C12 myotubes with acute depletion of Acot2 , and overt inhibition of ß-oxidation was demonstrated in isolated mitochondria from Acot2 -depleted glycolytic SM. In mice fed a high fat diet, ACOT2 enabled the accretion of acyl-CoAs and ceramide derivatives in glycolytic SM, and this was associated with worse glucose homeostasis compared to when ACOT2 was absent. These observations suggest that ACOT2 supports CoASH availability to facilitate ß-oxidation in glycolytic SM when lipid supply is modest. However, when lipid supply is high, ACOT2 enables acyl-CoA and lipid accumulation, CoASH sequestration, and poor glucose homeostasis. Thus, ACOT2 regulates matrix acyl-CoA concentration in glycolytic muscle, and its impact depends on lipid supply.

Closed-loop modeling of central and intrinsic cardiac nervous system circuits underlying cardiovascular control.

The baroreflex is a multi-input, multi-output control physiological system that regulates blood pressure by modulating nerve activity between the brainstem and the heart. Existing computational models of the baroreflex do not explictly incorporate the intrinsic cardiac nervous system (ICN), which mediates central control of the heart function. We developed a computational model of closed-loop cardiovascular control by integrating a network representation of the ICN within central control reflex circuits. We examined central and local contributions to the control of heart rate, ventricular functions, and respiratory sinus arrhythmia (RSA). Our simulations match the experimentally observed relationship between RSA and lung tidal volume. Our simulations predicted the relative contributions of the sensory and the motor neuron pathways to the experimentally observed changes in the heart rate. Our closed-loop cardiovascular control model is primed for evaluating bioelectronic interventions to treat heart failure and renormalize cardiovascular physiology.

Unpacking the multimodal, multi-scale data of the fast and slow lanes of the cardiac vagus through computational modelling.

NEW FINDINGS: What is the topic of this review? The vagus nerve is a crucial regulator of cardiovascular homeostasis, and its activity is linked to heart health. Vagal activity originates from two brainstem nuclei: the nucleus ambiguus (fast lane) and the dorsal motor nucleus of the vagus (slow lane), nicknamed for the time scales that they require to transmit signals. What advances does it highlight? Computational models are powerful tools for organizing multi-scale, multimodal data on the fast and slow lanes in a physiologically meaningful way. A strategy is laid out for how these models can guide experiments aimed at harnessing the cardiovascular health benefits of differential activation of the fast and slow lanes. ABSTRACT: The vagus nerve is a key mediator of brain-heart signaling, and its activity is necessary for cardiovascular health. Vagal outflow stems from the nucleus ambiguus, responsible primarily for fast, beat-to-beat regulation of heart rate and rhythm, and the dorsal motor nucleus of the vagus, responsible primarily for slow regulation of ventricular contractility. Due to the high-dimensional and multimodal nature of the anatomical, molecular and physiological data on neural regulation of cardiac function, data-derived mechanistic insights have proven elusive. Elucidating insights has been complicated further by the broad distribution of the data across heart, brain and peripheral nervous system circuits. Here we lay out an integrative framework based on computational modelling for combining these disparate and multi-scale data on the two vagal control lanes of the cardiovascular system. Newly available molecular-scale data, particularly single-cell transcriptomic analyses, have augmented our understanding of the heterogeneous neuronal states underlying vagally mediated fast and slow regulation of cardiac physiology. Cellular-scale computational models built from these data sets represent building blocks that can be combined using anatomical and neural circuit connectivity, neuronal electrophysiology, and organ/organismal-scale physiology data to create multi-system, multi-scale models that enable in silico exploration of the fast versus slow lane vagal stimulation. The insights from the computational modelling and analyses will guide new experimental questions on the mechanisms regulating the fast and slow lanes of the cardiac vagus toward exploiting targeted vagal neuromodulatory activity to promote cardiovascular health.

Conditional expression of endorepellin in the tumor vasculature attenuates breast cancer growth, angiogenesis and hyaluronan deposition.

The tumor stroma of most solid malignancies is characterized by a pathological accumulation of pro-angiogenic and pro-tumorigenic hyaluronan driving tumorigenesis and metastatic potential. Of all three hyaluronan synthase isoforms, HAS2 is the primary enzyme that promotes the build-up of tumorigenic HA in breast cancer. Previously, we discovered that endorepellin, the angiostatic C-terminal fragment of perlecan, evokes a catabolic mechanism targeting endothelial HAS2 and hyaluronan via autophagic induction. To explore the translational implications of endorepellin in breast cancer, we created a double transgenic, inducible Tie2CreERT2;endorepellin(ER)Ki mouse line that expresses recombinant endorepellin specifically from the endothelium. We investigated the therapeutic effects of recombinant endorepellin overexpression in an orthotopic, syngeneic breast cancer allograft mouse model. First, adenoviral delivery of Cre evoking intratumor expression of endorepellin in ERKi mice suppressed breast cancer growth, peritumor hyaluronan and angiogenesis. Moreover, tamoxifen-induced expression of recombinant endorepellin specifically from the endothelium in Tie2CreERT2;ERKi mice markedly suppressed breast cancer allograft growth, hyaluronan deposition in the tumor proper and perivascular tissues, and tumor angiogenesis. These results provide insight into the tumor suppressing activity of endorepellin at the molecular level and implicate endorepellin as a promising cancer protein therapy that targets hyaluronan in the tumor microenvironment.

Longitudinal ultrasound imaging and network modeling in rats reveal sex-dependent suppression of liver regeneration after resection in alcoholic liver disease.

Liver resection is an important surgical technique in the treatment of cancers and transplantation. We used ultrasound imaging to study the dynamics of liver regeneration following two-thirds partial hepatectomy (PHx) in male and female rats fed via Lieber-deCarli liquid diet protocol of ethanol or isocaloric control or chow for 5-7 weeks. Ethanol-fed male rats did not recover liver volume to the pre-surgery levels over the course of 2 weeks after surgery. By contrast, ethanol-fed female rats as well as controls of both sexes showed normal volume recovery. Contrary to expectations, transient increases in both portal and hepatic artery blood flow rates were seen in most animals, with ethanol-fed males showing higher peak portal flow than any other experimental group. A computational model of liver regeneration was used to evaluate the contribution of physiological stimuli and estimate the animal-specific parameter intervals. The results implicate lower metabolic load, over a wide range of cell death sensitivity, in matching the model simulations to experimental data of ethanol-fed male rats. However, in the ethanol-fed female rats and controls of both sexes, metabolic load was higher and in combination with cell death sensitivity matched the observed volume recovery dynamics. We conclude that adaptation to chronic ethanol intake has a sex-dependent impact on liver volume recovery following liver resection, likely mediated by differences in the physiological stimuli or cell death responses that govern the regeneration process. Immunohistochemical analysis of pre- and post-resection liver tissue validated the results of computational modeling by associating lack of sensitivity to cell death with lower rates of cell death in ethanol-fed male rats. Our results illustrate the potential for non-invasive ultrasound imaging to assess liver volume recovery towards supporting development of clinically relevant computational models of liver regeneration.

Elucidating the Mechanisms of Dynamic and Robust Control of the Liver Homeostatic Renewal Process: Cell Network Modeling and Analysis.

Recent experimental investigations of liver homeostatic renewal have identified high replication capacity hepatocyte populations as the primary maintainers of liver mass. However, the molecular and cellular processes controlling liver homeostatic renewal remain unknown. To address this problem, we developed and analyzed a mathematical model describing cellular network interactions underlying liver homeostatic renewal. Model simulation results demonstrate that without feedback control, basic homeostatic renewal is not robust to disruptions, leading to tissue loss under persistent/repetitive insults. Consequently, we extended our basic model to incorporate putative regulatory interactions and investigated how such interactions may confer robustness on the homeostatic renewal process. We utilized a Design of Experiments approach to identify the combination of feedback interactions that yields a cell network model of homeostatic renewal capable of maintaining liver mass robustly during persistent/repetitive injury. Simulations of this robust model indicate that repeated injury destabilizes liver homeostasis within several months, which differs from epidemiological observations of a much slower decay of liver function occurring over several years. To address this discrepancy, we extended the model to include feedback control by liver nonparenchymal cells. Simulations and analysis of the final multicellular feedback control network suggest that achieving robust liver homeostatic renewal requires intrinsic stability in a hepatocellular network combined with feedback control by nonparenchymal cells.

Genome-Scale Metabolic Modeling Reveals Sequential Dysregulation of Glutathione Metabolism in Livers from Patients with Alcoholic Hepatitis.

Alcoholic hepatitis (AH) is the most severe form of alcoholic liver disease for which there is no efficacious treatment aiding most patients. AH manifests differently in individuals, with some patients showing debilitating symptoms more so than others. Previous studies showed significant metabolic dysregulation associated with AH. Therefore, we sought to analyze how the activity of metabolic pathways differed in the liver of patients with varying degrees of AH severity. We utilized a genome-scale metabolic modeling approach that allowed for integration of a generic human cellular metabolic model with specific RNA-seq data corresponding to healthy and multiple liver disease states to predict the metabolic fluxes within each disease state. Additionally, we performed a systems-level analysis of the transcriptomic data and predicted metabolic flux data to identify the regulatory and functional differences in liver metabolism with increasing severity of AH. Our results provide unique insights into the sequential dysregulation of the solute transport mechanisms underlying the glutathione metabolic pathway with increasing AH disease severity. We propose targeting of the solute transporters in the glutathione pathway to mimic the flux activity of the healthy liver state as a potential therapeutic intervention for AH.

Investigating Drivers of Antireward in Addiction Behavior with Anatomically Specific Single-Cell Gene Expression Methods.

Increasing rates of addiction behavior have motivated mental health researchers and clinicians alike to understand antireward and recovery. This shift away from reward and commencement necessitates novel perspectives, paradigms, and hypotheses along with an expansion of the methods applied to investigate addiction. Here, we provide an example: A systems biology approach to investigate antireward that combines laser capture microdissection (LCM) and high-throughput microfluidic reverse transcription quantitative polymerase chain reactions (RT-qPCR). Gene expression network dynamics were measured and a key driver of neurovisceral dysregulation in alcohol and opioid withdrawal, neuroinflammation, was identified. This combination of technologies provides anatomic and phenotypic specificity at single-cell resolution with high-throughput sensitivity and specific gene expression measures yielding both hypothesis-generating datasets and mechanistic possibilities that generate opportunities for novel insights and treatments.

From Seeing to Simulating: A Survey of Imaging Techniques and Spatially-Resolved Data for Developing Multiscale Computational Models of Liver Regeneration.

Liver regeneration, which leads to the re-establishment of organ mass, follows a specifically organized set of biological processes acting on various time and length scales. Computational models of liver regeneration largely focused on incorporating molecular and signaling detail have been developed by multiple research groups in the recent years. These modeling efforts have supported a synthesis of disparate experimental results at the molecular scale. Incorporation of tissue and organ scale data using noninvasive imaging methods can extend these computational models towards a comprehensive accounting of multiscale dynamics of liver regeneration. For instance, microscopy-based imaging methods provide detailed histological information at the tissue and cellular scales. Noninvasive imaging methods such as ultrasound, computed tomography and magnetic resonance imaging provide morphological and physiological features including volumetric measures over time. In this review, we discuss multiple imaging modalities capable of informing computational models of liver regeneration at the organ-, tissue- and cellular level. Additionally, we discuss available software and algorithms, which aid in the analysis and integration of imaging data into computational models. Such models can be generated or tuned for an individual patient with liver disease. Progress towards integrated multiscale models of liver regeneration can aid in prognostic tool development for treating liver disease.

Single Cell Scale Neuronal and Glial Gene Expression and Putative Cell Phenotypes and Networks in the Nucleus Tractus Solitarius in an Alcohol Withdrawal Time Series.

Alcohol withdrawal syndrome (AWS) is characterized by neuronal hyperexcitability, autonomic dysregulation, and severe negative emotion. The nucleus tractus solitarius (NTS) likely plays a prominent role in the neurological processes underlying these symptoms as it is the main viscerosensory nucleus in the brain. The NTS receives visceral interoceptive inputs, influences autonomic outputs, and has strong connections to the limbic system and hypothalamic-pituitary-adrenal axis to maintain homeostasis. Our prior analysis of single neuronal gene expression data from the NTS shows that neurons exist in heterogeneous transcriptional states that form distinct functional subphenotypes. Our working model conjectures that the allostasis secondary to alcohol dependence causes peripheral and central biological network decompensation in acute abstinence resulting in neurovisceral feedback to the NTS that substantially contributes to the observed AWS. We collected single noradrenergic and glucagon-like peptide-1 (GLP-1) neurons and microglia from rat NTS and measured a subset of their transcriptome as pooled samples in an alcohol withdrawal time series. Inflammatory subphenotypes predominate at certain time points, and GLP-1 subphenotypes demonstrated hyperexcitability post-withdrawal. We hypothesize such inflammatory and anxiogenic signaling contributes to alcohol dependence via negative reinforcement. Targets to mitigate such dysregulation and treat dependence can be identified from this dataset.

A Spatial Model of Hepatic Calcium Signaling and Glucose Metabolism Under Autonomic Control Reveals Functional Consequences of Varying Liver Innervation Patterns Across Species.

Rapid breakdown of hepatic glycogen stores into glucose plays an important role during intense physical exercise to maintain systemic euglycemia. Hepatic glycogenolysis is governed by several different liver-intrinsic and systemic factors such as hepatic zonation, circulating catecholamines, hepatocellular calcium signaling, hepatic neuroanatomy, and the central nervous system (CNS). Of the factors regulating hepatic glycogenolysis, the extent of lobular innervation varies significantly between humans and rodents. While rodents display very few autonomic nerve terminals in the liver, nearly every hepatic layer in the human liver receives neural input. In the present study, we developed a multi-scale, multi-organ model of hepatic metabolism incorporating liver zonation, lobular scale calcium signaling, hepatic innervation, and direct and peripheral organ-mediated communication between the liver and the CNS. We evaluated the effect of each of these governing factors on the total hepatic glucose output and zonal glycogenolytic patterns within liver lobules during simulated physical exercise. Our simulations revealed that direct neuronal stimulation of the liver and an increase in circulating catecholamines increases hepatic glucose output mediated by mobilization of intracellular calcium stores and lobular scale calcium waves. Comparing simulated glycogenolysis between human-like and rodent-like hepatic innervation patterns (extensive vs. minimal) suggested that propagation of calcium transients across liver lobules acts as a compensatory mechanism to improve hepatic glucose output in sparsely innervated livers. Interestingly, our simulations suggested that catecholamine-driven glycogenolysis is reduced under portal hypertension. However, increased innervation coupled with strong intercellular communication can improve the total hepatic glucose output under portal hypertension. In summary, our modeling and simulation study reveals a complex interplay of intercellular and multi-organ interactions that can lead to differing calcium dynamics and spatial distributions of glycogenolysis at the lobular scale in the liver.

Retinoic Acid Fluctuation Activates an Uneven, Direction-Dependent Network-Wide Robustness Response in Early Embryogenesis.

Robustness is a feature of regulatory pathways to ensure signal consistency in light of environmental changes or genetic polymorphisms. The retinoic acid (RA) pathway, is a central developmental and tissue homeostasis regulatory signal, strongly dependent on nutritional sources of retinoids and affected by environmental chemicals. This pathway is characterized by multiple proteins or enzymes capable of performing each step and their integration into a self-regulating network. We studied RA network robustness by transient physiological RA signaling disturbances followed by kinetic transcriptomic analysis of the recovery during embryogenesis. The RA metabolic network was identified as the main regulated module to achieve signaling robustness using an unbiased pattern analysis. We describe the network-wide responses to RA signal manipulation and found the feedback autoregulation to be sensitive to the direction of the RA perturbation: RA knockdown exhibited an upper response limit, whereas RA addition had a minimal feedback-activation threshold. Surprisingly, our robustness response analysis suggests that the RA metabolic network regulation exhibits a multi-objective optimization, known as Pareto optimization, characterized by trade-offs between competing functionalities. We observe that efficient robustness to increasing RA is accompanied by worsening robustness to reduced RA levels and vice versa. This direction-dependent trade-off in the network-wide feedback response, results in an uneven robustness capacity of the RA network during early embryogenesis, likely a significant contributor to the manifestation of developmental defects.

Dysregulation of miR-21-associated miRNA regulatory networks by chronic ethanol consumption impairs liver regeneration.

Impaired liver regeneration has been considered as a hallmark of progression of alcohol-associated liver disease. Our previous studies demonstrated that in vivo inhibition of the microRNA (miRNA) miR21 can restore regenerative capacity of the liver in chronic ethanol-fed animals. The present study focuses on the role of microRNA regulatory networks that are likely to mediate the miR-21 action. Rats were chronically fed an ethanol-enriched diet along with pair-fed control animals and treated with AM21 (anti-miR-21), a locked nucleic acid antisense to miR-21. Partial hepatectomy (PHx) was performed and miRNA expression profiling over the course of liver regeneration was assessed. Our results showed dynamic expression changes in several miRNAs after PHx, notably with altered miRNA expression profiles between ethanol and control groups. We found that in vivo inhibition of miR-21 led to correlated differential expression of miR-340-5p and anticorrelated expression of miR-365, let-7a, miR-1224, and miR-146a across all sample groups after PHx. Gene set enrichment analysis identified a miRNA signature significantly associated with hepatic stellate cell activation within whole liver tissue data. We hypothesized that at least part of the PHx-induced miRNA network changes responsive to miR-21 inhibition is localized to hepatic stellate cells. We validated this hypothesis using AM21 and TGF-ß treatments in LX-2 human hepatic stellate cells in culture and measured expression levels of select miRNAs by quantitative RT-PCR. Based on the in vivo and in vitro results, we propose a hepatic stellate cell miRNA regulatory network as contributing to the restoration of liver regenerative capacity by miR-21 inhibition.

Analyzing the relationships between city opioid deaths and socioeconomic factors.

BACKGROUND: The opioid crisis has been increasing in severity over the past several decades. Every year, thousands of people die due to opioid-related issues. The factors that play into the causes of this are complex. Our study aimed to see how commute times, city budget for roads, and city budget for arts and culture contribute to the city's overall opioid death rates. We know that overdose rates are more common in cities rather than rural areas, therefore making one major city per state as part of our design. METHODS: We collected data from one city per state (n = 50) and ran two ANOVA tests and 11 logistical regression tests. Both types of tests were run on IBM SPSS Statistical software version 25 at its default settings with a confidence interval set to 95 percent. Opioid deaths were the dependent variable, whereas commute time, budget for roads, and budget for arts and culture were the independent variables. RESULTS: Commute time yielded a significant result in almost all the tests it was included in: Table 1, 0.033; Table 3, 0.000; Figure 4, 0.000; Figure 5, 0.000. Budget for roads also showed significant results in most of its tests as well: Table 1, 0.003; Table 2, 0.047; Figure 3, 0.001. Budget for arts and culture showed significant results but not in a pattern that we could interpret: Table 1, 0.002; Table 2, 0.021; Table 4, 0.013. CONCLUSIONS: Commute time and the budget for roads are likely to play a role in their city's opioid crisis. Understanding where a city fits in relation to these results may better help them prepare and reduce opioid death rates.

A single cell transcriptomics map of paracrine networks in the intrinsic cardiac nervous system.

We developed a spatially-tracked single neuron transcriptomics map of an intrinsic cardiac ganglion, the right atrial ganglionic plexus (RAGP) that is a critical mediator of sinoatrial node (SAN) activity. This 3D representation of RAGP used neuronal tracing to extensively map the spatial distribution of the subset of neurons that project to the SAN. RNA-seq of laser capture microdissected neurons revealed a distinct composition of RAGP neurons compared to the central nervous system and a surprising finding that cholinergic and catecholaminergic markers are coexpressed, suggesting multipotential phenotypes that can drive neuroplasticity within RAGP. High-throughput qPCR of hundreds of laser capture microdissected single neurons confirmed these findings and revealed a high dimensionality of neuromodulatory factors that contribute to dynamic control of the heart. Neuropeptide-receptor coexpression analysis revealed a combinatorial paracrine neuromodulatory network within RAGP informing follow-on studies on the vagal control of RAGP to regulate cardiac function in health and disease.

3D single cell scale anatomical map of sex-dependent variability of the rat intrinsic cardiac nervous system.

We developed and analyzed a single cell scale anatomical map of the rat intrinsic cardiac nervous system (ICNS) across four male and three female hearts. We find the ICNS has a reliable structural organizational plan across individuals that provide the foundation for further analyses of the ICNS in cardiac function and disease. The distribution of the ICNS was evaluated by 3D visualization and data-driven clustering. The pattern, distribution, and clustering of ICNS neurons across all male and female rat hearts is highly conserved, demonstrating a coherent organizational plan where distinct clusters of neurons are consistently localized. Female hearts had fewer neurons, lower packing density, and slightly reduced distribution, but with identical localization. We registered the anatomical data from each heart to a geometric scaffold, normalizing their 3D coordinates for standardization of common anatomical planes and providing a path where multiple experimental results and data types can be integrated and compared.

Input-output signal processing plasticity of vagal motor neurons in response to cardiac ischemic injury.

Vagal stimulation is emerging as the next frontier in bioelectronic medicine to modulate peripheral organ health and treat disease. The neuronal molecular phenotypes in the dorsal motor nucleus of the vagus (DMV) remain largely unexplored, limiting the potential for harnessing the DMV plasticity for therapeutic interventions. We developed a mesoscale single-cell transcriptomics data from hundreds of DMV neurons under homeostasis and following physiological perturbations. Our results revealed that homeostatic DMV neuronal states can be organized into distinguishable input-output signal processing units. Remote ischemic preconditioning induced a distinctive shift in the neuronal states toward diminishing the role of inhibitory inputs, with concomitant changes in regulatory microRNAs miR-218a and miR-495. Chronic cardiac ischemic injury resulted in a dramatic shift in DMV neuronal states suggestive of enhanced neurosecretory function. We propose a DMV molecular network mechanism that integrates combinatorial neurotransmitter inputs from multiple brain regions and humoral signals to modulate cardiac health.