Endothelial extracellular vesicles enhance vascular self-assembly in engineered human cardiac tissues.

The fabrication of complex and stable vasculature in engineered cardiac tissues represents a significant hurdle towards building physiologically relevant models of the heart. Here, we implemented a 3D model of cardiac vasculogenesis, incorporating endothelial cells (EC), stromal cells, and human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CM) in a fibrin hydrogel. The presence of CMs disrupted vessel formation in 3D tissues, resulting in the upregulation of endothelial activation markers and altered extracellular vesicle (EV) signaling in engineered tissues as determined by the proteomic analysis of culture supernatant. miRNA sequencing of CM- and EC-secreted EVs highlighted key EV-miRNAs that were postulated to play differing roles in cardiac vasculogenesis, including the let-7 family and miR-126-3p in EC-EVs. In the absence of CMs, the supplementation of CM-EVs to EC monolayers attenuated EC migration and proliferation and resulted in shorter and more discontinuous self-assembling vessels when applied to 3D vascular tissues. In contrast, supplementation of EC-EVs to the tissue culture media of 3D vascularized cardiac tissues mitigated some of the deleterious effects of CMs on vascular self-assembly, enhancing the average length and continuity of vessel tubes that formed in the presence of CMs. Direct transfection validated the effects of the key EC-EV miRNAs let-7b-5p and miR-126-3p in improving the maintenance of continuous vascular networks. EC-EV supplementation to biofabricated cardiac tissues and microfluidic devices resulted in tissue vascularization, illustrating the use of this approach in the engineering of enhanced, perfusable, microfluidic models of the myocardium.

At the heart of inflammation: Unravelling cardiac resident macrophage biology.

Cardiovascular disease remains one of the leading causes of death globally. Recent advancements in sequencing technologies have led to the identification of a unique population of macrophages within the heart, termed cardiac resident macrophages (CRMs), which exhibit self-renewal capabilities and play crucial roles in regulating cardiac homeostasis, inflammation, as well as injury and repair processes. This literature review aims to elucidate the origin and phenotypic characteristics of CRMs, comprehensively outline their contributions to cardiac homeostasis and further summarize their functional roles and molecular mechanisms implicated in the onset and progression of cardiovascular diseases. These insights are poised to pave the way for novel therapeutic strategies centred on targeted interventions based on the distinctive properties of resident macrophages.

Robust Mitochondrial Isolation from Rodent Cardiac Tissue.

Mitochondrial isolation has been practiced for decades, following procedures established by pioneers in the fields of molecular biology and biochemistry to study metabolic impairments and disease. Consistent mitochondrial quality is necessary to properly investigate mitochondrial physiology and bioenergetics; however, many different published isolation methods are available for researchers. Although different experimental strategies require different isolation methods, the basic principles and procedures are similar. This protocol details a method capable of extracting well-coupled mitochondria from a variety of tissue sources, including small animals and cells. The steps outlined include organ dissection, mitochondrial purification, protein quantification, and various quality control checks. The primary quality control metric used to identify high-quality mitochondria is the respiratory control ratio (RCR). The RCR is the ratio of the respiratory rate during oxidative phosphorylation to the rate in the absence of ADP. Alternative metrics are discussed. While high RCR values relative to their tissue source are obtained using this protocol, several steps can be optimized to suit the individual needs of researchers. This procedure is robust and has consistently resulted in isolated mitochondria with above-average RCR values across animal models and tissue sources.

Improving human cardiac organoid design using transcriptomics.

Cardiovascular disease (CVD) is the leading cause of death worldwide. To this end, human cardiac organoids (hCOs) have been developed for improved organotypic CVD modeling over conventional in vivo animal models. Utilizing human cells, hCOs hold great promise to bridge key gaps in CVD research pertaining to human-specific conditions. hCOs are multicellular 3D models which resemble heart structure and function. Varying hCOs fabrication techniques leads to functional and phenotypic differences. To investigate heterogeneity across hCO platforms, we performed a transcriptomic analysis utilizing bulk RNA-sequencing from four previously published unique hCO studies. We further compared selected hCOs to 2D and 3D hiPSC-derived cardiomyocytes (hiPSC-CMs), as well as fetal and adult human myocardium bulk RNA-sequencing samples. Upon investigation utilizing Principal Component Analysis, K-means clustering analysis of key genes, and further downstream analyses such as Gene Set Enrichment (GSEA), Gene Set Variation (GSVA), and GO term enrichment, we found that hCO fabrication method influences maturity and cellular heterogeneity across models. Thus, we propose that adjustment of fabrication method will result in an hCO with a defined maturity and transcriptomic profile to facilitate its specified applications, in turn maximizing its modeling potential.

Cardiac fibroblasts: answering the call.

Cardiac fibroblasts play a pivotal role in maintaining heart homeostasis by depositing extracellular matrix (ECM) to provide structural support for the myocardium, vasculature, and neuronal network and by contributing to essential physiological processes. In response to injury such as myocardial infarction or pressure overload, fibroblasts become activated, leading to increased ECM production that can ultimately drive left ventricular remodeling and progress to heart failure. Recently, the American Journal of Physiology-Heart and Circulatory Physiology issued a call for papers on cardiac fibroblasts that yielded articles with topics spanning fibroblast physiology, technical considerations, signaling pathways, and interactions with other cell types. This mini-review summarizes those articles and places the new findings in the context of what is currently known for cardiac fibroblasts and what future directions remain.

Single-cell analysis of chromatin and expression reveals age- and sex-associated alterations in the human heart.

Sex differences and age-related changes in the human heart at the tissue, cell, and molecular level have been well-documented and many may be relevant for cardiovascular disease. However, how molecular programs within individual cell types vary across individuals by age and sex remains poorly characterized. To better understand this variation, we performed single-nucleus combinatorial indexing (sci) ATAC- and RNA-Seq in human heart samples from nine donors. We identify hundreds of differentially expressed genes by age and sex and find epigenetic signatures of variation in ATAC-Seq data in this discovery cohort. We then scale up our single-cell RNA-Seq analysis by combining our data with five recently published single nucleus RNA-Seq datasets of healthy adult hearts. We find variation such as metabolic alterations by sex and immune changes by age in differential expression tests, as well as alterations in abundance of cardiomyocytes by sex and neurons with age. In addition, we compare our adult-derived ATAC-Seq profiles to analogous fetal cell types to identify putative developmental-stage-specific regulatory factors. Finally, we train predictive models of cell-type-specific RNA expression levels utilizing ATAC-Seq profiles to link distal regulatory sequences to promoters, quantifying the predictive value of a simple TF-to-expression regulatory grammar and identifying cell-type-specific TFs. Our analysis represents the largest single-cell analysis of cardiac variation by age and sex to date and provides a resource for further study of healthy cardiac variation and transcriptional regulation at single-cell resolution.

Intramyocardial Sprouting Tip Cells Specify Coronary Arterialization.

BACKGROUND: The elaborate patterning of coronary arteries critically supports the high metabolic activity of the beating heart. How coronary endothelial cells coordinate hierarchical vascular remodeling and achieve arteriovenous specification remains largely unknown. Understanding the molecular and cellular cues that pattern coronary arteries is crucial to develop innovative therapeutic strategies that restore functional perfusion within the ischemic heart. METHODS: Single-cell transcriptomics and histological validation were used to delineate heterogeneous transcriptional states of the developing and mature coronary endothelium with a focus on sprouting endothelium and arterial cell specification. Genetic lineage tracing and high-resolution 3-dimensional imaging were used to characterize the origin and mechanisms of coronary angiogenic sprouting, as well as to fate-map selective endothelial lineages. Integration of single-cell transcriptomic data from ischemic adult mouse hearts and human embryonic data served to assess the conservation of transcriptional states across development, disease, and species. RESULTS: We discover that coronary arteries originate from cells that have previously transitioned through a specific tip cell phenotype. We identify nonoverlapping intramyocardial and subepicardial tip cell populations with differential gene expression profiles and regulatory pathways. Esm1-lineage tracing confirmed that intramyocardial tip cells selectively contribute to coronary arteries and endocardial tunnels, but not veins. Notably, prearterial cells are detected from development stages to adulthood, increasingly in response to ischemic injury, and in human embryos, suggesting that tip cell-to-artery specification is a conserved mechanism. CONCLUSIONS: A tip cell-to-artery specification mechanism drives arterialization of the intramyocardial plexus and endocardial tunnels throughout life and is reactivated upon ischemic injury. Differential sprouting programs govern the formation and specification of the venous and arterial coronary plexus.

Latest progress of self-healing hydrogels in cardiac tissue engineering.

Cardiovascular diseases represent a significant public health challenge and are responsible for more than 4 million deaths annually in Europe alone (45% of all deaths). Among these, coronary-related heart diseases are a leading cause of mortality, accounting for 20% of all deaths. Cardiac tissue engineering has emerged as a promising strategy to address the limitations encountered after myocardial infarction. This approach aims to improve regulation of the inflammatory and cell proliferation phases, thereby reducing scar tissue formation and restoring cardiac function. In cardiac tissue engineering, biomaterials serve as hosts for cells and therapeutics, supporting cardiac restoration by mimicking the native cardiac environment. Various bioengineered systems, such as 3D scaffolds, injectable hydrogels, and patches play crucial roles in cardiac tissue repair. In this context, self-healing hydrogels are particularly suitable substitutes, as they can restore structural integrity when damaged. This structural healing represents a paradigm shift in therapeutic interventions, offering a more native-like environment compared to static, non-healable hydrogels. Herein, we sharply review the most recent advances in self-healing hydrogels in cardiac tissue engineering and their potential to transform cardiovascular healthcare.

Cardiac Progenitor Cell Metabolism.

Metabolism has emerged recently as an important determinant of stem cell function. Changes in metabolic signaling pathways precede changes in stem cell molecular and functional response. Pluripotent stem cells are highly proliferative and known to exhibit increased glycolysis. Similarly, adult stem cells reside in tissue niches in a quiescent state operating via glycolysis. Upon activation, adult stem cell metabolism transitions from glycolysis to oxidative phosphorylation which coincides with reduced proliferation and multilineage potential. In the heart, different populations of cardiac progenitor cells (CPCs) have been identified. CPCs regenerative potential is linked to changes in metabolic characteristics of cells, impacting cardiac repair following injury. Here, we discuss the methodologies for isolation and characterization of a novel cardiac progenitor cell population from the heart including measurement its metabolic features.

Macrophages: A missing key in cardiac tissue engineering for sustained vascularization.

Macrophages regulate angiogenesis, repair, conduction, and homeostasis in heart tissue. Landau et al.1 demonstrate that incorporating primitive macrophages into engineered heart tissues significantly promotes long-term vascularization and cardiac maturation. This advance demonstrates the importance of resident immune-vascular microenvironments in cardiac tissue engineering, marking an important step forward for heart-on-chip technologies.

Development of a Bmi1+ Cardiac Mouse Progenitor Immortalized Model to Unravel the Relationship with Its Protective Vascular Endothelial Niche.

The adult mammalian heart has been demonstrated to be endowed with low but real turnover capacity, especially for cardiomyocytes, the key functional cell type. The source, however, of that turnover capacity remains controversial. In this regard, we have defined and characterized a resident multipotent cardiac mouse progenitor population, Bmi1+DR (for Bmi1+ Damage-Responsive cells). Bmi1+DR is one of the cell types with the lowest ROS (Reactive Oxygen Species) levels in the adult heart, being particularly characterized by their close relationship with cardiac vessels, most probably involved in the regulation of proliferation/maintenance of Bmi1+DR. This was proposed to work as their endothelial niche. Due to the scarcity of Bmi1+DR cells in the adult mouse heart, we have generated an immortalization/dis-immortalization model using Simian Vacuolating Virus 40-Large Antigen T (SV40-T) to facilitate their in vitro characterization. We have obtained a heterogeneous population of immortalized Bmi1+DR cells (Bmi1+DRIMM) that was validated attending to different criteria, also showing a comparable sensitivity to strong oxidative damage. Then, we concluded that the Bmi1-DRIMM population is an appropriate model for primary Bmi1+DR in vitro studies. The co-culture of Bmi1+DRIMM cells with endothelial cells protects them against oxidative damage, showing a moderate depletion in non-canonical autophagy and also contributing with a modest metabolic regulation.

Protocol for simultaneous isolation of high-quality and high-quantity cardiomyocytes and non-myocyte cells from adult rat hearts.

Isolating high-quality different cell types is a powerful approach for understanding cellular compositions and features in the heart, but it is challenging. The available protocols typically focus on isolating one or two cell types. Here, we present a protocol to simultaneously isolate high-quality and high-quantity cardiomyocytes and non-myocyte cells, including immune cells, from adult rat hearts. We describe steps for purifying cells using bovine serum albumin. We also detail procedures for viability analysis and cell type identification using fluorescence-activated cell sorting. For complete details on the use and execution of this protocol, please refer to Zhang et al.,1 Valkov et al.,2 Vang et al.,3 and Li et al.4.

Generation and Characterization of hiPSC-Derived Vascularized-, Perfusable Cardiac Microtissues-on-Chip.

In the heart in vivo, vasculature forms a semi-permeable endothelial barrier for selective nutrient and (immune) cell delivery to the myocardium and removal of waste products. Crosstalk between the vasculature and the heart cells regulates homeostasis in health and disease. To model heart development and disease in vitro it is important that essential features of this crosstalk are captured. Cardiac organoid and microtissue models often integrate endothelial cells (ECs) to form microvascular networks inside the 3D structure. However, in static culture without perfusion, these networks may fail to show essential functionality. Here, we describe a protocol to generate an in vitro model of human induced pluripotent stem cell (hiPSC)-derived vascularized cardiac microtissues on a microfluidic organ-on-chip platform (VMToC) in which the blood vessels are perfusable. First, prevascularized cardiac microtissues (MT) are formed by combining hiPSC-derived cardiomyocytes, ECs, and cardiac fibroblasts in a pre-defined ratio. Next, these prevascularized MTs are integrated in the chips in a fibrin hydrogel containing additional vascular cells, which self-organize into tubular structures. The MTs become vascularized through anastomosis between the pre-existing microvasculature in the MT and the external vascular network. The VMToCs are then ready for downstream structural and functional assays and basic characterization. Using this protocol, cardiac MTs can be efficiently and robustly vascularized and perfused within 7 days. In vitro vascularized organoid and MT models have the potential to transition current 3D cardiac models to more physiologically relevant organ models that allow the role of the endothelial barrier in drug and inflammatory response to be investigated. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Generation of VMToC Support Protocol 1: Functional Characterization of VMToC Support Protocol 2: Structural Characterization of VMToC.

TGFß1 Regulates Cellular Composition of In Vitro Cardiac Perivascular Niche Based on Cardiospheres.

The cardiac perivascular niche is a cellular microenvironment of a blood vessel. The principles of niche regulation are still poorly understood. We studied the effect of TGFß1 on cells forming the cardiac perivascular niche using 3D cell culture (cardiospheres). Cardiospheres contained progenitor (c-Kit), endothelial (CD31), and mural (αSMA) cells, basement membrane proteins (laminin) and extracellular matrix proteins (collagen I, fibronectin). TGFß1 treatment decreased the length of CD31+ microvasculature, VE cadherin protein level, and proportion of NG2+ cells, and increased proportion of αSMA+ cells and transgelin/SM22α protein level. We supposed that this effect is related to the stabilizing function of TGFß1 on vascular cells: decreased endothelial cell proliferation, as shown for HUVEC, and activation of mural cell differentiation.

Evaluating computational efforts and physiological resolution of mathematical models of cardiac tissue.

Computational techniques have significantly advanced our understanding of cardiac electrophysiology, yet they have predominantly concentrated on averaged models that do not represent the intricate dynamics near individual cardiomyocytes. Recently, accurate models representing individual cells have gained popularity, enabling analysis of the electrophysiology at the micrometer level. Here, we evaluate five mathematical models to determine their computational efficiency and physiological fidelity. Our findings reveal that cell-based models introduced in recent literature offer both efficiency and precision for simulating small tissue samples (comprising thousands of cardiomyocytes). Conversely, the traditional bidomain model and its simplified counterpart, the monodomain model, are more appropriate for larger tissue masses (encompassing millions to billions of cardiomyocytes). For simulations requiring detailed parameter variations along individual cell membranes, the EMI model emerges as the only viable choice. This model distinctively accounts for the extracellular (E), membrane (M), and intracellular (I) spaces, providing a comprehensive framework for detailed studies. Nonetheless, the EMI model's applicability to large-scale tissues is limited by its substantial computational demands for subcellular resolution.

3D matrix stiffness modulation unveils cardiac fibroblast phenotypic switching.

This study investigates how dynamic fluctuations in matrix stiffness affect the behavior of cardiac fibroblasts (CFs) within a three-dimensional (3D) hydrogel environment. Using hybrid hydrogels with tunable stiffness, we created an in vitro model to mimic the varying stiffness of the cardiac microenvironment. By manipulating hydrogel stiffness, we examined CF responses, particularly the expression of α-smooth muscle actin (α-SMA), a marker of myofibroblast differentiation. Our findings reveal that increased matrix stiffness promotes the differentiation of CFs into myofibroblasts, while matrix softening reverses this process. Additionally, we identified the role of focal adhesions and integrin ß1 in mediating stiffness-induced phenotypic switching. This study provides significant insights into the mechanobiology of cardiac fibrosis and suggests that modulating matrix stiffness could be a potential therapeutic strategy for treating cardiovascular diseases.

Impulse initiation in engrafted pluripotent stem cell-derived cardiomyocytes can stimulate the recipient heart.

Transplantation of pluripotent stem cell-derived cardiomyocytes is a novel promising cell-based therapeutic approach for patients with heart failure. However, engraftment arrhythmias are a predictable life-threatening complication and represent a major hurdle for clinical translation. Thus, we wanted to experimentally study whether impulse generation by transplanted cardiomyocytes can propagate to the host myocardium and overdrive the recipient rhythm. We transplanted human induced pluripotent stem cell-derived cardiomyocytes expressing the optogenetic actuator Bidirectional Pair of Opsins for Light-induced Excitation and Silencing (BiPOLES) in a guinea pig injury model. Eight weeks after transplantation ex vivo, Langendorff perfusion was used to assess electrical coupling. Pulsed photostimulation was applied to specifically activate the engrafted cardiomyocytes. Photostimulation resulted in ectopic pacemaking that propagated to the host myocardium, caused non-sustained arrhythmia, and stimulated the recipient heart with higher pacing frequency (4/9 hearts). Our study demonstrates that transplanted cardiomyocytes can (1) electrically couple to the host myocardium and (2) stimulate the recipient heart. Thus, our results provide experimental evidence for an important aspect of engraftment-induced arrhythmia induction and thereby support the current hypothesis that cardiomyocyte automaticity can serve as a trigger for ventricular arrhythmias.