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.

BeatProfiler: Multimodal In Vitro Analysis of Cardiac Function Enables Machine Learning Classification of Diseases and Drugs.

Goal: Contractile response and calcium handling are central to understanding cardiac function and physiology, yet existing methods of analysis to quantify these metrics are often time-consuming, prone to mistakes, or require specialized equipment/license. We developed BeatProfiler, a suite of cardiac analysis tools designed to quantify contractile function, calcium handling, and force generation for multiple in vitro cardiac models and apply downstream machine learning methods for deep phenotyping and classification. Methods: We first validate BeatProfiler's accuracy, robustness, and speed by benchmarking against existing tools with a fixed dataset. We further confirm its ability to robustly characterize disease and dose-dependent drug response. We then demonstrate that the data acquired by our automatic acquisition pipeline can be further harnessed for machine learning (ML) analysis to phenotype a disease model of restrictive cardiomyopathy and profile cardioactive drug functional response. To accurately classify between these biological signals, we apply feature-based ML and deep learning models (temporal convolutional-bidirectional long short-term memory model or TCN-BiLSTM). Results: Benchmarking against existing tools revealed that BeatProfiler detected and analyzed contraction and calcium signals better than existing tools through improved sensitivity in low signal data, reduction in false positives, and analysis speed increase by 7 to 50-fold. Of signals accurately detected by published methods (PMs), BeatProfiler's extracted features showed high correlations to PMs, confirming that it is reliable and consistent with PMs. The features extracted by BeatProfiler classified restrictive cardiomyopathy cardiomyocytes from isogenic healthy controls with 98% accuracy and identified relax90 as a top distinguishing feature in congruence with previous findings. We also show that our TCN-BiLSTM model was able to classify drug-free control and 4 cardiac drugs with different mechanisms of action at 96% accuracy. We further apply Grad-CAM on our convolution-based models to identify signature regions of perturbations by these drugs in calcium signals. Conclusions: We anticipate that the capabilities of BeatProfiler will help advance in vitro studies in cardiac biology through rapid phenotyping, revealing mechanisms underlying cardiac health and disease, and enabling objective classification of cardiac disease and responses to drugs.

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.

Macrophages enhance contractile force in iPSC-derived human engineered cardiac tissue.

Resident cardiac macrophages are critical mediators of cardiac function. Despite their known importance to cardiac electrophysiology and tissue maintenance, there are currently no stem-cell-derived models of human engineered cardiac tissues (hECTs) that include resident macrophages. In this study, we made an induced pluripotent stem cell (iPSC)-derived hECT model with a resident population of macrophages (iM0) to better recapitulate the native myocardium and characterized their impact on tissue function. Macrophage retention within the hECTs was confirmed via immunofluorescence after 28 days of cultivation. The inclusion of iM0s significantly impacted hECT function, increasing contractile force production. A potential mechanism underlying these changes was revealed by the interrogation of calcium signaling, which demonstrated the modulation of ß-adrenergic signaling in +iM0 hECTs. Collectively, these findings demonstrate that macrophages significantly enhance cardiac function in iPSC-derived hECT models, emphasizing the need to further explore their contributions not only in healthy hECT models but also in the contexts of disease and injury.

Modeling the Effects of Protracted Cosmic Radiation in a Human Organ-on-Chip Platform.

Galactic cosmic radiation (GCR) is one of the most serious risks posed to astronauts during missions to the Moon and Mars. Experimental models capable of recapitulating human physiology are critical to understanding the effects of radiation on human organs and developing radioprotective measures against space travel exposures. The effects of systemic radiation are studied using a multi-organ-on-a-chip (multi-OoC) platform containing engineered tissue models of human bone marrow (site of hematopoiesis and acute radiation damage), cardiac muscle (site of chronic radiation damage) and liver (site of metabolism), linked by vascular circulation with an endothelial barrier separating individual tissue chambers from the vascular perfusate. Following protracted neutron radiation, the most damaging radiation component in deep space, a greater deviation of tissue function is observed as compared to the same cumulative dose delivered acutely. Further, by characterizing engineered bone marrow (eBM)-derived immune cells in circulation, 58 unique genes specific to the effects of protracted neutron dosing are identified, as compared to acutely irradiated and healthy tissues. It propose that this bioengineered platform allows studies of human responses to extended radiation exposure in an "astronaut-on-a-chip" model that can inform measures for mitigating cosmic radiation injury.

An engineered human cardiac tissue model reveals contributions of systemic lupus erythematosus autoantibodies to myocardial injury.

Systemic lupus erythematosus (SLE) is a highly heterogenous autoimmune disease that affects multiple organs, including the heart. The mechanisms by which myocardial injury develops in SLE, however, remain poorly understood. Here we engineered human cardiac tissues and cultured them with IgG fractions containing autoantibodies from SLE patients with and without myocardial involvement. We observed unique binding patterns of IgG from two patient subgroups: (i) patients with severe myocardial inflammation exhibited enhanced binding to apoptotic cells within cardiac tissues subjected to stress, and (ii) patients with systolic dysfunction exhibited enhanced binding to the surfaces of viable cardiomyocytes. Functional assays and RNA sequencing (RNA-seq) revealed that IgGs from patients with systolic dysfunction exerted direct effects on engineered tissues in the absence of immune cells, altering tissue cellular composition, respiration and calcium handling. Autoantibody target characterization by phage immunoprecipitation sequencing (PhIP-seq) confirmed distinctive IgG profiles between patient subgroups. By coupling IgG profiling with cell surface protein analyses, we identified four pathogenic autoantibody candidates that may directly alter the function of cells within the myocardium. Taken together, these observations provide insights into the cellular processes of myocardial injury in SLE that have the potential to improve patient risk stratification and inform the development of novel therapeutic strategies.

Modeling and countering the effects of cosmic radiation using bioengineered human tissues.

Cosmic radiation is the most serious risk that will be encountered during the planned missions to the Moon and Mars. There is a compelling need to understand the effects, safety thresholds, and mechanisms of radiation damage in human tissues, in order to develop measures for radiation protection during extended space travel. As animal models fail to recapitulate the molecular changes in astronauts, engineered human tissues and "organs-on-chips" are valuable tools for studying effects of radiation in vitro. We have developed a bioengineered tissue platform for studying radiation damage in individualized settings. To demonstrate its utility, we determined the effects of radiation using engineered models of two human tissues known to be radiosensitive: engineered cardiac tissues (eCT, a target of chronic radiation damage) and engineered bone marrow (eBM, a target of acute radiation damage). We report the effects of high-dose neutrons, a proxy for simulated galactic cosmic rays, on the expression of key genes implicated in tissue responses to ionizing radiation, phenotypic and functional changes in both tissues, and proof-of-principle application of radioprotective agents. We further determined the extent of inflammatory, oxidative stress, and matrix remodeling gene expression changes, and found that these changes were associated with an early hypertrophic phenotype in eCT and myeloid skewing in eBM. We propose that individualized models of human tissues have potential to provide insights into the effects and mechanisms of radiation during deep-space missions and allow testing of radioprotective measures.

Effect of mechanical unloading on genome-wide DNA methylation profile of the failing human heart.

Heart failure (HF) is characterized by global alterations in myocardial DNA methylation, yet little is known about the epigenetic regulation of the noncoding genome and potential reversibility of DNA methylation with left ventricular assist device (LVAD) therapy. Genome-wide mapping of myocardial DNA methylation in 36 patients with HF at LVAD implantation, 8 patients at LVAD explantation, and 7 nonfailing (NF) donors using a high-density bead array platform identified 2,079 differentially methylated positions (DMPs) in ischemic cardiomyopathy (ICM) and 261 DMPs in nonischemic cardiomyopathy (NICM). LVAD support resulted in normalization of 3.2% of HF-associated DMPs. Methylation-expression correlation analysis yielded several protein-coding genes that are hypomethylated and upregulated (HTRA1, FBXO16, EFCAB13, and AKAP13) or hypermethylated and downregulated (TBX3) in HF. A potentially novel cardiac-specific super-enhancer long noncoding RNA (lncRNA) (LINC00881) is hypermethylated and downregulated in human HF. LINC00881 is an upstream regulator of sarcomere and calcium channel gene expression including MYH6, CACNA1C, and RYR2. LINC00881 knockdown reduces peak calcium amplitude in the beating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). These data suggest that HF-associated changes in myocardial DNA methylation within coding and noncoding genomes are minimally reversible with mechanical unloading. Epigenetic reprogramming strategies may be necessary to achieve sustained clinical recovery from heart failure.

Engineered cardiac tissue model of restrictive cardiomyopathy for drug discovery.

Restrictive cardiomyopathy (RCM) is defined as increased myocardial stiffness and impaired diastolic relaxation leading to elevated ventricular filling pressures. Human variants in filamin C (FLNC) are linked to a variety of cardiomyopathies, and in this study, we investigate an in-frame deletion (c.7416_7418delGAA, p.Glu2472_Asn2473delinAsp) in a patient with RCM. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with this variant display impaired relaxation and reduced calcium kinetics in 2D culture when compared with a CRISPR-Cas9-corrected isogenic control line. Similarly, mutant engineered cardiac tissues (ECTs) demonstrate increased passive tension and impaired relaxation velocity compared with isogenic controls. High-throughput small-molecule screening identifies phosphodiesterase 3 (PDE3) inhibition by trequinsin as a potential therapy to improve cardiomyocyte relaxation in this genotype. Together, these data demonstrate an engineered cardiac tissue model of RCM and establish the translational potential of this precision medicine approach to identify therapeutics targeting myocardial relaxation.

Engineering and Characterization of an Optogenetic Model of the Human Neuromuscular Junction.

Many neuromuscular diseases, such as myasthenia gravis (MG), are associated with dysfunction of the neuromuscular junction (NMJ), which is difficult to characterize in animal models due to physiological differences between animals and humans. Tissue engineering offers opportunities to provide in vitro models of functional human NMJs that can be used to diagnose and investigate NMJ pathologies and test potential therapeutics. By incorporating optogenetic proteins into induced pluripotent stem cells (iPSCs), we generated neurons that can be stimulated with specific wavelengths of light. If the NMJ is healthy and functional, a neurochemical signal from the motoneuron results in muscle contraction. Through the integration of optogenetics and microfabrication with tissue engineering, we established an unbiased and automated methodology for characterizing NMJ function using video analysis. A standardized protocol was developed for NMJ formation, optical stimulation with simultaneous video recording, and video analysis of tissue contractility. Stimulation of optogenetic motoneurons by light to induce skeletal muscle contractions recapitulates human NMJ physiology and allows for repeated functional measurements of NMJ over time and in response to various inputs. We demonstrate this platform's ability to show functional improvements in neuromuscular connectivity over time and characterize the damaging effects of patient MG antibodies or neurotoxins on NMJ function.

STK25 inhibits PKA signaling by phosphorylating PRKAR1A.

In the heart, protein kinase A (PKA) is critical for activating calcium handling and sarcomeric proteins in response to beta-adrenergic stimulation leading to increased myocardial contractility and performance. The catalytic activity of PKA is tightly regulated by regulatory subunits that inhibit the catalytic subunit until released by cAMP binding. Phosphorylation of type II regulatory subunits promotes PKA activation; however, the role of phosphorylation in type I regulatory subunits remain uncertain. Here, we utilize human induced pluripotent stem cell cardiomyocytes (iPSC-CMs) to identify STK25 as a kinase of the type Iα regulatory subunit PRKAR1A. Phosphorylation of PRKAR1A leads to inhibition of PKA kinase activity and increased binding to the catalytic subunit in the presence of cAMP. Stk25 knockout in mice diminishes Prkar1a phosphorylation, increases Pka activity, and augments contractile response to beta-adrenergic stimulation. Together, these data support STK25 as a negative regulator of PKA signaling through phosphorylation of PRKAR1A.

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.

Bioengineered optogenetic model of human neuromuscular junction.

Functional human tissues engineered from patient-specific induced pluripotent stem cells (hiPSCs) hold great promise for investigating the progression, mechanisms, and treatment of musculoskeletal diseases in a controlled and systematic manner. For example, bioengineered models of innervated human skeletal muscle could be used to identify novel therapeutic targets and treatments for patients with complex central and peripheral nervous system disorders. There is a need to develop standardized and objective quantitative methods for engineering and using these complex tissues, in order increase their robustness, reproducibility, and predictiveness across users. Here we describe a standardized method for engineering an isogenic, patient specific human neuromuscular junction (NMJ) that allows for automated quantification of NMJ function to diagnose disease using a small sample of blood serum and evaluate new therapeutic modalities. By combining tissue engineering, optogenetics, microfabrication, optoelectronics and video processing, we created a novel platform for the precise investigation of the development and degeneration of human NMJ. We demonstrate the utility of this platform for the detection and diagnosis of myasthenia gravis, an antibody-mediated autoimmune disease that disrupts the NMJ function.

Daily blue-light exposure shortens lifespan and causes brain neurodegeneration in Drosophila.

Light is necessary for life, but prolonged exposure to artificial light is a matter of increasing health concern. Humans are exposed to increased amounts of light in the blue spectrum produced by light-emitting diodes (LEDs), which can interfere with normal sleep cycles. The LED technologies are relatively new; therefore, the long-term effects of exposure to blue light across the lifespan are not understood. We investigated the effects of light in the model organism, Drosophila melanogaster, and determined that flies maintained in daily cycles of 12-h blue LED and 12-h darkness had significantly reduced longevity compared with flies maintained in constant darkness or in white light with blue wavelengths blocked. Exposure of adult flies to 12 h of blue light per day accelerated aging phenotypes causing damage to retinal cells, brain neurodegeneration, and impaired locomotion. We report that brain damage and locomotor impairments do not depend on the degeneration in the retina, as these phenotypes were evident under blue light in flies with genetically ablated eyes. Blue light induces expression of stress-responsive genes in old flies but not in young, suggesting that cumulative light exposure acts as a stressor during aging. We also determined that several known blue-light-sensitive proteins are not acting in pathways mediating detrimental light effects. Our study reveals the unexpected effects of blue light on fly brain and establishes Drosophila as a model in which to investigate long-term effects of blue light at the cellular and organismal level.

Modulating bacterial and gut mucosal interactions with engineered biofilm matrix proteins.

Extracellular appendages play a significant role in mediating communication between bacteria and their host. Curli fibers are a class of bacterial fimbria that is highly amenable to engineering. We demonstrate the use of engineered curli fibers to rationally program interactions between bacteria and components of the mucosal epithelium. Commensal E. coli strains were engineered to produce recombinant curli fibers fused to the trefoil family of human cytokines. Biofilms formed from these strains bound more mucins than those producing wild-type curli fibers, and modulated mucin rheology as well. When treated with bacteria producing the curli-trefoil fusions mammalian cells behaved identically in terms of their migration behavior as when they were treated with the corresponding soluble trefoil factors. Overall, this demonstrates the potential utility of curli fibers as a scaffold for the display of bioactive domains and an untapped approach to rationally modulating host-microbe interactions using bacterial matrix proteins.