Cross-species single-cell RNA sequencing reveals divergent phenotypes and activation states of adaptive immunity in human carotid and experimental murine atherosclerosis.

BACKGROUND AND AIMS: The distinct functions of immune cells in atherosclerosis have been mostly defined by preclinical mouse studies. Contrastingly, the immune cell composition of human atherosclerotic plaques and their contribution to disease progression is only poorly understood. It remains uncertain whether genetic animal models allow for valuable translational approaches. METHODS AND RESULTS: Single cell RNA-sequencing (scRNA-seq) was performed to define the immune cell landscape in human carotid atherosclerotic plaques. The human immune cell repertoire demonstrated an unexpectedly high heterogeneity and was dominated by cells of the T-cell lineage, a finding confirmed by immunohistochemistry. Bioinformatical integration with 7 mouse scRNA-seq data sets from adventitial and atherosclerotic vascular tissue revealed a total of 51 identities of cell types and differentiation states, of which some were only poorly conserved between species and exclusively found in humans. Locations, frequencies, and transcriptional programs of immune cells in mouse models did not resemble the immune cell landscape in human carotid atherosclerosis. In contrast to standard mouse models of atherosclerosis, human plaque leukocytes were dominated by several T-cell phenotypes with transcriptional hallmarks of T-cell activation and memory formation, T-cell receptor-, and pro-inflammatory signaling. Only mice at the age of 22 months partially resembled the activated T-cell phenotype. In a validation cohort of 43 patients undergoing carotid endarterectomy, the abundance of activated immune cell subsets in the plaque defined by multi-color flow cytometry associated with the extend of clinical atherosclerosis. CONCLUSIONS: Integrative scRNA-seq reveals a substantial difference in the immune cell composition of murine and human carotid atherosclerosis - a finding that questions the translational value of standard mouse models for adaptive immune cell studies. Clinical associations suggest a specific role for T-cell driven (auto-) immunity in human plaque formation and -instability.

Correction: Greiner et al. Consecutive-Day Ventricular and Atrial Cardiomyocyte Isolations from the Same Heart: Shifting the Cost-Benefit Balance of Cardiac Primary Cell Research. Cells 2022, 11, 233.

In the original publication [...].

Piezo1 stretch-activated channel activity differs between murine bone marrow-derived and cardiac tissue-resident macrophages.

Macrophages (MΦ) play pivotal roles in tissue homeostasis and repair. Their mechanical environment has been identified as a key modulator of various cell functions, and MΦ mechanosensitivity is likely to be critical - in particular in a rhythmically contracting organ such as the heart. Cultured MΦ, differentiated in vitro from bone marrow (MΦBM), form a popular research model. This study explores the activity of mechanosensitive ion channels (MSC) in murine MΦBM and compares it to MSC activity in MΦ enzymatically isolated from cardiac tissue (tissue-resident MΦ; MΦTR). We show that MΦBM and MΦTR have stretch-induced currents, indicating the presence of functional MSC in their plasma membrane. The current profiles in MΦBM and in MΦTR show characteristics of cation non-selective MSC such as Piezo1 or transient receptor potential channels. While Piezo1 ion channel activity is detectable in the plasma membrane of MΦBM using the patch-clamp technique, or by measuring cytosolic calcium concentration upon perfusion with the Piezo1 channel agonist Yoda1, no Piezo1 channel activity was observed in MΦTR. The selective transient receptor potential vanilloid 4 (TRPV4) channel agonist GSK1016790A induces calcium entry in MΦTR and in MΦBM. In MΦ isolated from left-ventricular scar tissue 28 days after cryoablation, stretch-induced current characteristics are not significantly different compared to non-injured control tissue, even though scarred ventricular tissue is expected to be mechanically remodelled and to contain an altered composition of pre-existing cardiac and circulation-recruited MΦ. Our data suggest that the in vitro differentiation protocols used to obtain MΦBM generate cells that differ from MΦ recruited from the circulation during tissue repair in vivo. Further investigations are needed to explore MSC identity in lineage-traced MΦ in scar tissue, and to compare mechanosensitivity of circulating monocytes with that of MΦBM. KEY POINTS: Bone marrow-derived (MΦBM) and tissue resident (MΦTR) macrophages have stretch-induced currents, indicating expression of functional mechanosensitive channels (MSC) in their plasma membrane. Stretch-activated current profiles show characteristics of cation non-selective MSC; and mRNA coding for MSC, including Piezo1 and TRPV4, is expressed in murine MΦBM and in MΦTR. Calcium entry upon pharmacological activation of TRPV4 confirms functionality of the channel in MΦTR and in MΦBM. Piezo1 ion channel activity is detected in the plasma membrane of MΦBM but not in MΦTR, suggesting that MΦBM may not be a good model to study the mechanotransduction of MΦTR. Stretch-induced currents, Piezo1 mRNA expression and response to pharmacological activation are not significantly changed in cardiac MΦ 28 days after cryoinjury compared to sham operated mice.

Optogenetic confirmation of transverse-tubular membrane excitability in intact cardiac myocytes.

T-tubules (TT) form a complex network of sarcolemmal membrane invaginations, essential for well-co-ordinated excitation-contraction coupling (ECC) and thus homogeneous mechanical activation of cardiomyocytes. ECC is initiated by rapid depolarization of the sarcolemmal membrane. Whether TT membrane depolarization is active (local generation of action potentials; AP) or passive (following depolarization of the outer cell surface sarcolemma; SS) has not been experimentally validated in cardiomyocytes. Based on the assessment of ion flux pathways needed for AP generation, we hypothesize that TT are excitable. We therefore explored TT excitability experimentally, using an all-optical approach to stimulate and record trans-membrane potential changes in TT that were structurally disconnected, and hence electrically insulated, from the SS membrane by transient osmotic shock. Our results establish that cardiomyocyte TT can generate AP. These AP show electrical features that differ substantially from those observed in SS, consistent with differences in the density of ion channels and transporters in the two different membrane domains. We propose that TT-generated AP represent a safety mechanism for TT AP propagation and ECC, which may be particularly relevant in pathophysiological settings where morpho-functional changes reduce the electrical connectivity between SS and TT membranes. KEY POINTS: Cardiomyocytes are characterized by a complex network of membrane invaginations (the T-tubular system) that propagate action potentials to the core of the cell, causing uniform excitation-contraction coupling across the cell. In the present study, we investigated whether the T-tubular system is able to generate action potentials autonomously, rather than following depolarization of the outer cell surface sarcolemma. For this purpose, we developed a fully optical platform to probe and manipulate the electrical dynamics of subcellular membrane domains. Our findings demonstrate that T-tubules are intrinsically excitable, revealing distinct characteristics of self-generated T-tubular action potentials. This active electrical capability would protect cells from voltage drops potentially occurring within the T-tubular network.

Disease severity, arrhythmogenesis, and fibrosis are related to longer action potentials in tetralogy of Fallot.

BACKGROUND: Arrhythmias may originate from surgically unaffected right ventricular (RV) regions in patients with tetralogy of Fallot (TOF). We aimed to investigate action potential (AP) remodelling and arrhythmia susceptibility in RV myocardium of patients with repaired and with unrepaired TOF, identify possible correlations with clinical phenotype and myocardial fibrosis, and compare findings with data from patients with atrial septal defect (ASD), a less severe congenital heart disease. METHODS: Intracellular AP were recorded ex vivo in RV outflow tract samples from 22 TOF and three ASD patients. Arrhythmias were provoked by superfusion with solutions containing reduced potassium and barium chloride, or isoprenaline. Myocardial fibrosis was quantified histologically and associations between clinical phenotype, AP shape, tissue arrhythmia propensity, and fibrosis were examined. RESULTS: Electrophysiological abnormalities (arrhythmias, AP duration [APD] alternans, impaired APD shortening at increased stimulation frequencies) were generally present in TOF tissue, even from infants, but rare or absent in ASD samples. More severely diseased and acyanotic patients, pronounced tissue susceptibility to arrhythmogenesis, and greater fibrosis extent were associated with longer APD. In contrast, APD was shorter in tissue from patients with pre-operative cyanosis. Increased fibrosis and repaired-TOF status were linked to tissue arrhythmia inducibility. CONCLUSIONS: Functional and structural tissue remodelling may explain arrhythmic activity in TOF patients, even at a very young age. Surprisingly, clinical acyanosis appears to be associated with more severe arrhythmogenic remodelling. Further research into the clinical drivers of structural and electrical myocardial alterations, and the relation between them, is needed to identify predictive factors for patients at risk.

The challenges of research data management in cardiovascular science: a DGK and DZHK position paper-executive summary.

The sharing and documentation of cardiovascular research data are essential for efficient use and reuse of data, thereby aiding scientific transparency, accelerating the progress of cardiovascular research and healthcare, and contributing to the reproducibility of research results. However, challenges remain. This position paper, written on behalf of and approved by the German Cardiac Society and German Centre for Cardiovascular Research, summarizes our current understanding of the challenges in cardiovascular research data management (RDM). These challenges include lack of time, awareness, incentives, and funding for implementing effective RDM; lack of standardization in RDM processes; a need to better identify meaningful and actionable data among the increasing volume and complexity of data being acquired; and a lack of understanding of the legal aspects of data sharing. While several tools exist to increase the degree to which data are findable, accessible, interoperable, and reusable (FAIR), more work is needed to lower the threshold for effective RDM not just in cardiovascular research but in all biomedical research, with data sharing and reuse being factored in at every stage of the scientific process. A culture of open science with FAIR research data should be fostered through education and training of early-career and established research professionals. Ultimately, FAIR RDM requires permanent, long-term effort at all levels. If outcomes can be shown to be superior and to promote better (and better value) science, modern RDM will make a positive difference to cardiovascular science and practice. The full position paper is available in the supplementary materials.

Age-related structural and functional changes of the intracardiac nervous system.

BACKGROUND: Although aging is known to be associated with an increased incidence of both atrial and ventricular arrhythmias, there is limited knowledge about how Schwann cells (SC) and the intracardiac nervous system (iCNS) remodel with age. Here we investigate the differences in cardiac SC, parasympathetic nerve fibers, and muscarinic acetylcholine receptor M2 (M2R) expression in young and old mice. Additionally, we examine age-related changes in cardiac responses to sympathomimetic and parasympathomimetic drugs. METHODS AND RESULTS: Lower SC density, lower SC proliferation and fewer parasympathetic nerve fibers were observed in cardiac and, as a control sciatic nerves from old (20-24 months) compared to young mice (2-3 months). In old mice, chondroitin sulfate proteoglycan 4 (CSPG4) was increased in sciatic but not cardiac nerves. Expression of M2R was lower in ventricular myocardium and ventricular conduction system from old mice compared to young mice, while no significant difference was seen in M2R expression in sino-atrial or atrio-ventricular node pacemaker tissue. Heart rate was slower and PQ intervals were longer in Langendorff-perfused hearts from old mice. Ventricular tachycardia and fibrillation were more frequently observed in response to carbachol administration in hearts from old mice versus those from young mice. CONCLUSIONS: On the background of reduced presence of SC and parasympathetic nerve fibers, and of lower M2R expression in ventricular cardiomyocytes and conduction system of aged hearts, the propensity of ventricular arrhythmogenesis upon parasympathomimetic drug application is increased. Whether this is caused by an increase in heterogeneity of iCNS structure and function remains to be elucidated.

Meta-Analysis of Mechano-Sensitive Ion Channels in Human Hearts: Chamber- and Disease-Preferential mRNA Expression.

The cardiac cell mechanical environment changes on a beat-by-beat basis as well as in the course of various cardiac diseases. Cells sense and respond to mechanical cues via specialized mechano-sensors initiating adaptive signaling cascades. With the aim of revealing new candidates underlying mechano-transduction relevant to cardiac diseases, we investigated mechano-sensitive ion channels (MSC) in human hearts for their chamber- and disease-preferential mRNA expression. Based on a meta-analysis of RNA sequencing studies, we compared the mRNA expression levels of MSC in human atrial and ventricular tissue samples from transplant donor hearts (no cardiac disease), and from patients in sinus rhythm (underlying diseases: heart failure, coronary artery disease, heart valve disease) or with atrial fibrillation. Our results suggest that a number of MSC genes are expressed chamber preferentially, e.g., CHRNE in the atria (compared to the ventricles), TRPV4 in the right atrium (compared to the left atrium), CACNA1B and KCNMB1 in the left atrium (compared to the right atrium), as well as KCNK2 and KCNJ2 in ventricles (compared to the atria). Furthermore, 15 MSC genes are differentially expressed in cardiac disease, out of which SCN9A (lower expressed in heart failure compared to donor tissue) and KCNQ5 (lower expressed in atrial fibrillation compared to sinus rhythm) show a more than twofold difference, indicative of possible functional relevance. Thus, we provide an overview of cardiac MSC mRNA expression in the four cardiac chambers from patients with different cardiac diseases. We suggest that the observed differences in MSC mRNA expression may identify candidates involved in altered mechano-transduction in the respective diseases.

The heterocellular heart: identities, interactions, and implications for cardiology.

The heterocellular nature of the heart has been receiving increasing attention in recent years. In addition to cardiomyocytes as the prototypical cell type of the heart, non-myocytes such as endothelial cells, fibroblasts, or immune cells are coming more into focus. The rise of single-cell sequencing technologies enables  identification of ever more subtle differences and has reignited the question of what defines a cell's identity. Here we provide an overview of the major cardiac cell types, describe their roles in homeostasis, and outline recent findings on non-canonical functions that may be of relevance for cardiology. We highlight modes of biochemical and biophysical interactions between different cardiac cell types and discuss the potential implications of the heterocellular nature of the heart for basic research and therapeutic interventions.

Nonmyocytes as electrophysiological contributors to cardiac excitation and conduction.

Although cardiac action potential (AP) generation and propagation have traditionally been attributed exclusively to cardiomyocytes (CM), other cell types in the heart are also capable of forming electrically conducting junctions. Interactions between CM and nonmyocytes (NM) enable and modulate each other's activity. This review provides an overview of the current understanding of heterocellular electrical communication in the heart. Although cardiac fibroblasts were initially thought to be electrical insulators, recent studies have demonstrated that they form functional electrical connections with CM in situ. Other NM, such as macrophages, have also been recognized as contributing to cardiac electrophysiology and arrhythmogenesis. Novel experimental tools have enabled the investigation of cell-specific activity patterns in native cardiac tissue, which is expected to yield exciting new insights into the development of novel or improved diagnostic and therapeutic strategies.

Evaluation of cervical lymph nodes using multispectral optoacoustic tomography: a proof-of-concept study.

OBJECTIVES: Examination of lymph nodes is one of the most common indications for imaging in the head and neck region. The purpose of this study is to evaluate whether multispectral optoacoustic tomography can be used to observe chromophore differences between benign and malignant neck lymph nodes. MATERIALS AND METHODS: Proof-of-concept ex vivo study of resected cervical lymph nodes from 11 patients. The examination of lymph nodes included imaging with hybrid ultrasound and multispectral tomography system followed by spectral unmixing to separate signals from the endogenous chromophores water, lipid, hemoglobin and oxygenated hemoglobin; calculation of semi-quantitative parameters (total hemoglobin and relative oxygenation of hemoglobin). Comparison of the results from the hybrid measurement with the histopathological results. RESULTS: Most patients suffered from squamous cell carcinoma (n = 7), also metastasis from salivary gland adenocarcinoma and papillary thyroid carcinoma, were included. The comparison between benign cervical lymph nodes and metastases showed significant differences for the absorbers water, lipid, hemoglobin and oxygenated hemoglobin and total hemoglobin. CONCLUSIONS: Our ex vivo study suggests that multispectral optoacoustic tomography can be used to detect differences between reactive lymph nodes and metastases. The measurement of endogenous chromophores can be used for this purpose. The examinations are non-invasively and thus potentially improve diagnostic prediction. However, potential influences from the ex vivo setting must be considered.

Assessment of Tissue Viability by Functional Imaging of Membrane Potential.

Electrical activity plays a key role in physiology, in particular for signaling and coordination. Cellular electrophysiology is often studied with micropipette-based techniques such as patch clamp and sharp electrodes, but for measurements at the tissue or organ scale, more integrated approaches are needed. Epifluorescence imaging of voltage-sensitive dyes ("optical mapping") is a tissue non-destructive approach to obtain insight into electrophysiology with high spatiotemporal resolution. Optical mapping has primarily been applied to excitable organs, especially the heart and brain. Action potential durations, conduction patterns, and conduction velocities can be determined from the recordings, providing information about electrophysiological mechanisms, including factors such as effects of pharmacological interventions, ion channel mutations, or tissue remodeling. Here, we describe the process for optical mapping of Langendorff-perfused mouse hearts, highlighting potential issues and key considerations.

Simultaneous assessment of radial and axial myocyte mechanics by combining atomic force microscopy and carbon fibre techniques.

Cardiomyocytes sense and shape their mechanical environment, contributing to its dynamics by their passive and active mechanical properties. While axial forces generated by contracting cardiomyocytes have been amply investigated, the corresponding radial mechanics remain poorly characterized. Our aim is to simultaneously monitor passive and active forces, both axially and radially, in cardiomyocytes freshly isolated from adult mouse ventricles. To do so, we combine a carbon fibre (CF) set-up with a custom-made atomic force microscope (AFM). CF allows us to apply stretch and to record passive and active forces in the axial direction. The AFM, modified for frontal access to fit in CF, is used to characterize radial cell mechanics. We show that stretch increases the radial elastic modulus of cardiomyocytes. We further find that during contraction, cardiomyocytes generate radial forces that are reduced, but not abolished, when cells are forced to contract near isometrically. Radial forces may contribute to ventricular wall thickening during contraction, together with the dynamic re-orientation of cells and sheetlets in the myocardium. This new approach for characterizing cell mechanics allows one to obtain a more detailed picture of the balance of axial and radial mechanics in cardiomyocytes at rest, during stretch, and during contraction. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.