SARS-CoV-2 pathogenesis in an angiotensin II-induced heart-on-a-chip disease model and extracellular vesicle screening.

Adverse cardiac outcomes in COVID-19 patients, particularly those with preexisting cardiac disease, motivate the development of human cell-based organ-on-a-chip models to recapitulate cardiac injury and dysfunction and for screening of cardioprotective therapeutics. Here, we developed a heart-on-a-chip model to study the pathogenesis of SARS-CoV-2 in healthy myocardium established from human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and a cardiac dysfunction model, mimicking aspects of preexisting hypertensive disease induced by angiotensin II (Ang II). We recapitulated cytopathic features of SARS-CoV-2-induced cardiac damage, including progressively impaired contractile function and calcium handling, apoptosis, and sarcomere disarray. SARS-CoV-2 presence in Ang II-treated hearts-on-a-chip decreased contractile force with earlier onset of contractile dysfunction and profoundly enhanced inflammatory cytokines compared to SARS-CoV-2 alone. Toward the development of potential therapeutics, we evaluated the cardioprotective effects of extracellular vesicles (EVs) from human iPSC which alleviated the impairment of contractile force, decreased apoptosis, reduced the disruption of sarcomeric proteins, and enhanced beta-oxidation gene expression. Viral load was not affected by either Ang II or EV treatment. We identified MicroRNAs miR-20a-5p and miR-19a-3p as potential mediators of cardioprotective effects of these EVs.

Primitive macrophages enable long-term vascularization of human heart-on-a-chip platforms.

The intricate anatomical structure and high cellular density of the myocardium complicate the bioengineering of perfusable vascular networks within cardiac tissues. In vivo neonatal studies highlight the key role of resident cardiac macrophages in post-injury regeneration and angiogenesis. Here, we integrate human pluripotent stem-cell-derived primitive yolk-sac-like macrophages within vascularized heart-on-chip platforms. Macrophage incorporation profoundly impacted the functionality and perfusability of microvascularized cardiac tissues up to 2 weeks of culture. Macrophages mitigated tissue cytotoxicity and the release of cell-free mitochondrial DNA (mtDNA), while upregulating the secretion of pro-angiogenic, matrix remodeling, and cardioprotective cytokines. Bulk RNA sequencing (RNA-seq) revealed an upregulation of cardiac maturation and angiogenesis genes. Further, single-nuclei RNA sequencing (snRNA-seq) and secretome data suggest that macrophages may prime stromal cells for vascular development by inducing insulin like growth factor binding protein 7 (IGFBP7) and hepatocyte growth factor (HGF) expression. Our results underscore the vital role of primitive macrophages in the long-term vascularization of cardiac tissues, offering insights for therapy and advancing heart-on-a-chip technologies.

Heart-on-a-Chip Model of Epicardial-Myocardial Interaction in Ischemia Reperfusion Injury.

Epicardial cells (EPIs) form the outer layer of the heart and play an important role in development and disease. Current heart-on-a-chip platforms still do not fully mimic the native cardiac environment due to the absence of relevant cell types, such as EPIs. Here, using the Biowire II platform, engineered cardiac tissues with an epicardial outer layer and inner myocardial structure are constructed, and an image analysis approach is developed to track the EPI cell migration in a beating myocardial environment. Functional properties of EPI cardiac tissues improve over two weeks in culture. In conditions mimicking ischemia reperfusion injury (IRI), the EPI cardiac tissues experience less cell death and a lower impact on functional properties. EPI cell coverage is significantly reduced and more diffuse under normoxic conditions compared to the post-IRI conditions. Upon IRI, migration of EPI cells into the cardiac tissue interior is observed, with contributions to alpha smooth muscle actin positive cell population. Altogether, a novel heart-on-a-chip model is designed to incorporate EPIs through a formation process that mimics cardiac development, and this work demonstrates that EPI cardiac tissues respond to injury differently than epicardium-free controls, highlighting the importance of including EPIs in heart-on-a-chip constructs that aim to accurately mimic the cardiac environment.

Cardiac tissue model of immune-induced dysfunction reveals the role of free mitochondrial DNA and the therapeutic effects of exosomes.

Despite tremendous progress in the development of mature heart-on-a-chip models, human cell-based models of myocardial inflammation are lacking. Here, we bioengineered a vascularized heart-on-a-chip with circulating immune cells to model severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced acute myocarditis. We observed hallmarks of coronavirus disease (COVID-19)-induced myocardial inflammation, as the presence of immune cells augmented the secretion of proinflammatory cytokines, triggered progressive impairment of contractile function, and altered intracellular calcium transients. An elevation of circulating cell-free mitochondrial DNA (ccf-mtDNA) was measured first in the heart-on-a-chip and then validated in COVID-19 patients with low left ventricular ejection fraction, demonstrating that mitochondrial damage is an important pathophysiological hallmark of inflammation-induced cardiac dysfunction. Leveraging this platform in the context of SARS-CoV-2-induced myocardial inflammation, we established that administration of endothelial cell-derived exosomes effectively rescued the contractile deficit, normalized calcium handling, elevated the contraction force, and reduced the ccf-mtDNA and cytokine release via Toll-like receptor-nuclear factor κB signaling axis.

Itaconate and citrate releasing polymer attenuates foreign body response in biofabricated cardiac patches.

Application of cardiac patches to the heart surface can be undertaken to provide support and facilitate regeneration of the damaged cardiac tissue following ischemic injury. Biomaterial composition is an important consideration in the design of cardiac patch materials as it governs host response to ultimately prevent the undesirable fibrotic response. Here, we investigate a novel patch material, poly (itaconate-co-citrate-co-octanediol) (PICO), in the context of cardiac implantation. Citric acid (CA) and itaconic acid (ITA), the molecular components of PICO, provided a level of protection for cardiac cells during ischemic reperfusion injury in vitro. Biofabricated PICO patches were shown to degrade in accelerated and hydrolytic conditions, with CA and ITA being released upon degradation. Furthermore, the host response to PICO patches after implantation on rat epicardium in vivo was explored and compared to two biocompatible cardiac patch materials, poly (octamethylene (anhydride) citrate) (POMaC) and poly (ethylene glycol) diacrylate (PEGDA). PICO patches resulted in less macrophage infiltration and lower foreign body giant cell reaction compared to the other materials, with corresponding reduction in smooth muscle actin-positive vessel infiltration into the implant region. Overall, this work demonstrates that PICO patches release CA and ITA upon degradation, both of which demonstrate cardioprotective effects on cardiac cells after ischemic injury, and that PICO patches generate a reduced inflammatory response upon implantation to the heart compared to other materials, signifying promise for use in cardiac patch applications.

Heart-on-a-chip model of immune-induced cardiac dysfunction reveals the role of free mitochondrial DNA and therapeutic effects of endothelial exosomes.

Cardiovascular disease continues to take more human lives than all cancer combined, prompting the need for improved research models and treatment options. Despite a significant progress in development of mature heart-on-a-chip models of fibrosis and cardiomyopathies starting from induced pluripotent stem cells (iPSCs), human cell-based models of myocardial inflammation are lacking. Here, we bioengineered a vascularized heart-on-a-chip system with circulating immune cells to model SARS-CoV-2-induced acute myocarditis. Briefly, we observed hallmarks of COVID-19-induced myocardial inflammation in the heart-on-a-chip model, as the presence of immune cells augmented the expression levels of proinflammatory cytokines, triggered progressive impairment of contractile function and altered intracellular calcium transient activities. An elevation of circulating cell-free mitochondrial DNA (ccf-mtDNA) was measured first in the in vitro heart-on-a-chip model and then validated in COVID-19 patients with low left ventricular ejection fraction (LVEF), demonstrating that mitochondrial damage is an important pathophysiological hallmark of inflammation induced cardiac dysfunction. Leveraging this platform in the context of SARS-CoV-2 induced myocardial inflammation, we established that administration of human umbilical vein-derived EVs effectively rescued the contractile deficit, normalized intracellular calcium handling, elevated the contraction force and reduced the ccf- mtDNA and chemokine release via TLR-NF-kB signaling axis.

Angiopoietin-1 derived peptide hydrogel promotes molecular hallmarks of regeneration and wound healing in dermal fibroblasts.

By providing an ideal environment for healing, biomaterials can be designed to facilitate and encourage wound regeneration. As the wound healing process is complex, there needs to be consideration for the cell types playing major roles, such as fibroblasts. As a major cell type in the dermis, fibroblasts have a large impact on the processes and outcomes of wound healing. Prevopisly, conjugating the angiopoietin-1 derived Q-peptide (QHREDGS) to a collagen-chitosan hydrogel created a biomaterial with in vivo success in accelerating wound healing. This study utilized solvent cast Q-peptide conjugated collagen-chitosan seeded with fibroblast monolayers to investigate the direct impact of the material on this major cell type. After 24 h, fibroblasts had a significant change in release of anti-inflammatory, pro-healing, and ECM deposition cytokines, with demonstrated immunomodulatory effects on macrophages and upregulated expression of critical wound healing genes.

A New Role for Extracellular Vesicles in Cardiac Tissue Engineering and Regenerative Medicine.

Cardiovascular diseases are the leading cause of death worldwide. Discovering new therapies to treat heart disease requires improved understanding of cardiac physiology at a cellular level. Extracellular vesicles (EVs) are plasma membrane-bound nano- and microparticles secreted by cells and known to play key roles in intercellular communication, often through transfer of biomolecular cargo. Advances in EV research have established techniques for EV isolation from tissue culture media or biofluids, as well as standards for quantitation and biomolecular characterization. EVs released by cardiac cells are known to be involved in regulating cardiac physiology as well as in the progression of myocardial diseases. Due to difficulty accessing the heart in vivo, advanced in vitro cardiac 'tissues-on-a-chip' have become a recent focus for studying EVs in the heart. These physiologically relevant models are producing new insight into the role of EVs in cardiac physiology and disease while providing a useful platform for screening novel EV-based therapeutics for cardiac tissue regeneration post-injury. Numerous hurdles have stalled the clinical translation of EV therapeutics for heart patients, but tissue-on-a-chip models are playing an important role in bridging the translational gap, improving mechanistic understanding of EV signalling in cardiac physiology, disease, and repair.

Dye-Mediated Photo-Oxidation Biomaterial Fixation: Analysis of Bioinductivity and Mechanical Properties of Bovine Pericardium for Use in Cardiac Surgery.

Extracellular matrix bioscaffolds can influence the cardiac microenvironment and modulate endogenous cellular mechanisms. These materials can optimize cardiac surgery for repair and reconstruction. We investigated the biocompatibility and bioinductivity of bovine pericardium fixed via dye-mediated photo-oxidation on human cardiac fibroblast activity. We compared a dye-mediated photo-oxidation fixed bioscaffold to glutaraldehyde-fixed and non-fixed bioscaffolds reported in contemporary literature in cardiac surgery. Human cardiac fibroblasts from consenting patients were seeded on to bioscaffold materials to assess the biocompatibility and bioinductivity. Human cardiac fibroblast gene expression, secretome, morphology and viability were studied. Dye-mediated photo-oxidation fixed acellular bovine pericardium preserves human cardiac fibroblast phenotype and viability; and potentiates a pro-vasculogenic paracrine response. Material tensile properties were compared with biomechanical testing. Dye-mediated photo-oxidation fixed acellular bovine pericardium had higher compliance compared to glutaraldehyde-fixed bioscaffold in response to tensile force. The biocompatibility, bioinductivity, and biomechanical properties of dye-mediated photo-oxidation fixed bovine pericardium demonstrate its feasibility as a bioscaffold for use in cardiac surgery. As a fixed yet bioinductive solution, this bioscaffold demonstrates enhanced compliance and retains bioinductive properties that may leverage endogenous reparative pathways. Dye-mediated photo-oxidation fixed bioscaffold warrants further investigation as a viable tool for cardiac repair and reconstruction.

Extracellular Vesicles in Cardiac Regeneration: Potential Applications for Tissues-on-a-Chip.

Strategies to regenerate cardiac tissue postinjury are limited and heart transplantation remains the only 'cure' for a failing heart. Extracellular vesicles (EVs), membrane-bound cell secretions important in intercellular signaling, have been shown to play a crucial role in regulating heart function. A mechanistic understanding of the role of EVs in the heart remains elusive due to the challenges in studying the native human heart. Tissue-on-a-chip platforms, comprising functional, physiologically relevant human tissue models, are an emerging technology that has yet to be fully applied to the study of EVs. In this review, we summarize recent advances in cardiac tissue-on-a-chip (CTC) platforms and discuss how they are uniquely situated to advance our understanding of EVs in cardiac disease and regeneration.

Acellular bioscaffolds redirect cardiac fibroblasts and promote functional tissue repair in rodents and humans with myocardial injury.

Coronary heart disease is a leading cause of death. Tissue remodeling and fibrosis results in cardiac pump dysfunction and ischemic heart failure. Cardiac fibroblasts may rebuild damaged tissues when prompted by suitable environmental cues. Here, we use acellular biologic extracellular matrix scaffolds (bioscaffolds) to stimulate pathways of muscle repair and restore tissue function. We show that acellular bioscaffolds with bioinductive properties can redirect cardiac fibroblasts to rebuild microvascular networks and avoid tissue fibrosis. Specifically, when human cardiac fibroblasts are combined with bioactive scaffolds, gene expression is upregulated and paracrine mediators are released that promote vasculogenesis and prevent scarring. We assess these properties in rodents with myocardial infarction and observe bioscaffolds to redirect fibroblasts, reduce tissue fibrosis and prevent maladaptive structural remodeling. Our preclinical data confirms that acellular bioscaffold therapy provides an appropriate microenvironment to stimulate pathways of functional repair. We translate our observations to patients with coronary heart disease by conducting a first-in-human observational cohort study. We show that bioscaffold therapy is associated with improved perfusion of infarcted myocardium, reduced myocardial scar burden, and reverse structural remodeling. We establish that clinical use of acellular bioscaffolds is feasible and offers a new frontier to enhance surgical revascularization of ischemic heart muscle.