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Introduction: Three dimensional engineered cardiac tissues (3D ECTs) have become indispensable as in vitro models to assess drug cardiotoxicity, a leading cause of failure in pharmaceutical development. A current bottleneck is the relatively low throughput of assays that measure spontaneous contractile forces exerted by millimeter-scale ECTs typically recorded through precise optical measurement of deflection of the polymer scaffolds that support them. The required resolution and speed limit the field of view to at most a few ECTs at a time using conventional imaging.Methods: To balance the inherent tradeoff among imaging resolution, field of view and speed, an innovative mosaic imaging system was designed, built, and validated to sense contractile force of 3D ECTs seeded on a 96-well plate. Results: The system performance was validated through real-time, parallel contractile force monitoring for up to 3 weeks. Pilot drug testing was conducted using isoproterenol.Discussion: The described tool increases contractile force sensing throughput to 96 samples per measurement; significantly reduces cost, time and labor needed for preclinical cardiotoxicity assay using 3D ECT. More broadly, our mosaicking approach is a general way to scale up image-based screening in multi-well formats.

Engineered microsystems for in vitro studies of cultured cells are evolving from simple two-dimensional (2D) platforms to three-dimensional (3D) architectures and organoid cultures. Despite advances in reproducing ever more sophisticated biology in these systems, there remain foundational challenges in recreating key aspects of tissue composition, architecture, and mechanics that are critical to recapitulating in vivo processes. Against the backdrop of current progress in 3D fabrication methods, we evaluate the key requirements for the next generation of cellular platforms. We postulate that these future platforms—apart from building tissue-like structures—will need to have the ability to readily sense and autonomously modulate tissue responses over time, as occurs in natural microenvironments. Such interactive, robotic platforms that report and guide cellular events will enable us to probe a previously inaccessible class of questions in cell biology.

The Notch pathway regulates complex patterning events in many species and is critical for the proper formation and function of the vasculature. Despite this importance, how the various components of the Notch pathway work in concert is still not well understood. For example, NOTCH1 stabilizes homotypic endothelial junctions, but the role of NOTCH1 in heterotypic interactions is not entirely clear. NOTCH3, on the other hand, is essential for heterotypic interactions of pericytes with the endothelium, but how NOTCH3 signaling in pericytes impacts the endothelium remains elusive. Here, we use in vitro vascular models to investigate whether pericyte-induced stabilization of the vasculature requires the cooperation of NOTCH1 and NOTCH3. We observe that both pericyte NOTCH3 and endothelial NOTCH1 are required for the stabilization of the endothelium. Loss of either NOTCH3 or NOTCH1 decreases the accumulation of VE-cadherin at endothelial adherens junctions and increases the frequency of wider, more motile junctions. We found that DLL4 was the key ligand for simulating NOTCH1 activation in endothelial cells and observed that DLL4 expression in pericytes is dependent on NOTCH3. Altogether, these data suggest that an interplay between pericyte NOTCH3 and endothelial NOTCH1 is critical for pericyte-induced vascular stabilization.

Tumor accumulation of AS750 after PEG-NR heat exposure with varying delta T (S1); Diminished window of tumor transport after re-exposure to PEG-NR heating (S2); Animal weights during PEG-NR and doxorubicin liposome therapeutic trial (S3); Transvascular transport in tumors after single exposure and re-exposure to PEGNR heating (S4); Vascular endothelial growth factor (VEGF) expression in PEG-NR heated tumors (S5); Endothelial cell F-Actin dynamics during heat exposure and recovery (S6); Visualizing PEG-NRs in the tumor microenvironment using multiphoton microscopy (S7); Cargo accumulation in tumors with Hsf1+/+ and Hsf1-/- vasculature after single exposure to PEG-NR heating (S8); HSF1 expression in tumor-associated vasculature following PEG-NR heating (S9); Size-dependent macromolecular extravasation from the unheated vasculature (S10).

Legend for Supplementary Figures S1-S10.

A proper understanding of disease etiology will require longitudinal systems-scale reconstruction of the multitiered architecture of eukaryotic signaling. Here we combine state-of-the-art data acquisition platforms and bioinformatics tools to devise PAMAF, a workflow that simultaneously examines twelve omics modalities, i.e., protein abundance from whole-cells, nucleus, exosomes, secretome and membrane; N-glycosylation, phosphorylation; metabolites; mRNA, miRNA; and, in parallel, single-cell transcriptomes. We apply PAMAF in an established in vitro model of TGFβ-induced epithelial to mesenchymal transition (EMT) to quantify >61,000 molecules from 12 omics and 10 timepoints over 12 days. Bioinformatics analysis of this EMT-ExMap resource allowed us to identify; –topological coupling between omics, –four distinct cell states during EMT, –omics-specific kinetic paths, –stage-specific multi-omics characteristics, –distinct regulatory classes of genes, –ligand–receptor mediated intercellular crosstalk by integrating scRNAseq and subcellular proteomics, and –combinatorial drug targets (e.g., Hedgehog signaling and CAMK-II) to inhibit EMT, which we validate using a 3D mammary duct-on-a-chip platform. Overall, this study provides a resource on TGFβ signaling and EMT.

Rationale: Dominant heterozygous variants in filamin C ( FLNC ) cause diverse cardiomyopathies, although the underlying molecular mechanisms remain poorly understood. Objective: We aimed to define the molecular mechanisms by which FLNC variants altered human cardiomyocyte gene and protein expression, sarcomere structure, and contractile performance. Methods and Results: Using CRISPR/Cas9, we introduced FLNC variants into human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs). We compared isogenic hiPSC-CMs with normal (wild-type), ablated expression ( FLNC −/− ), or haploinsufficiency ( FLNC +/− ) that causes dilated cardiomyopathy. We also studied a heterozygous in-frame deletion ( FLNC +/Δ7aa ) which did not affect FLNC expression but caused aggregate formation, similar to FLNC variants associated with hypertrophic cardiomyopathy. FLNC −/− hiPSC-CMs demonstrated profound sarcomere misassembly and reduced contractility. Although sarcomere formation and function were unaffected in FLNC +/ − and FLNC +/Δ7aa hiPSC-CMs, these heterozygous variants caused increases in lysosome content, enhancement of autophagic flux, and accumulation of FLNC-binding partners and Z-disc proteins. Conclusions: FLNC expression is required for sarcomere organization and physiological function. Variants that produce misfolded FLNC proteins cause the accumulation of FLNC and FLNC-binding partners which leads to increased lysosome expression and activation of autophagic pathways. Surprisingly, similar pathways were activated in FLNC haploinsufficient hiPSC-CMs, likely initiated by the loss of stoichiometric FLNC protein interactions and impaired turnover of proteins at the Z-disc. These results indicate that both FLNC haploinsufficient variants and variants that produce misfolded FLNC protein cause disease by similar proteotoxic mechanisms and indicate the therapeutic potential for augmenting protein degradative pathways to treat a wide range of FLNC -related cardiomyopathies.

After a myocardial infarction (MI), the heart undergoes changes including local remodeling that can lead to regional abnormalities in mechanical and electrical properties, ultimately increasing the risk of arrhythmias and heart failure. Although these responses have been successfully recapitulated in animal models of MI, local changes in tissue and cell-level mechanics caused by MI remain difficult to study in vivo. Here, we developed an in vitro cardiac microtissue (CMT) injury system that through acute focal injury recapitulates aspects of the regional responses seen following an MI. With a pulsed laser, cell death was induced in the center of the microtissue causing a loss of calcium signaling and a complete loss of contractile function in the injured region and resulting in a 39% reduction in the CMT's overall force production. After 7 days, the injured area remained void of cardiomyocytes (CMs) and showed increased expression of vimentin and fibronectin, two markers for fibrotic remodeling. Interestingly, although the injured region showed minimal recovery, calcium amplitudes in uninjured regions returned to levels comparable with control. Furthermore, overall force production returned to preinjury levels despite the lack of contractile function in the injured region. Instead, uninjured regions exhibited elevated contractile function, compensating for the loss of function in the injured region, drawing parallels to changes in tissue-level mechanics seen in vivo. Overall, this work presents a new in vitro model to study cardiac tissue remodeling and electromechanical changes after injury.

Background: Cardiac contractility modulation (CCM) is a medical device-based therapy delivering non-excitatory electrical simulations to the heart during the absolute refractory period to enhance cardiac function. We previously evaluated the acute effects of CCM in isolated rabbit-CM and 2D hiPSC-CM monolayers, on flexible substrate, and found enhanced calcium and contractility. In the present study, we sought to extend these results to chronic studies in 3D human engineered cardiac tissues (ECTs) to develop a robust model to evaluate the long-term effects of CCM stimulation in vitro on intact human cardiac tissues. Methods: HiPSC-CMs and cardiac fibroblasts in a fibrin-based gel were combined to form ECTs (Figure 1). Morphology and contractility were evaluated. We found ECTs displayed baseline peak contraction amplitude of 194.8±80.8 a.u., (n=5) and peak force and stress of 26.5±6.8 uN, (n=5) and 87.9±22.0.8 Pa, (n=5) respectively. ECT displayed robust baseline electrophysiological (AP) properties and calcium handling properties. Chronic CCM pulses (i.e., 30 minutes) were applied using a commercial pulse generator. Under these conditions ECTs displayed enhanced contractile kinetics 29.0±0.04 %, (P Conclusion: This study provides a comprehensive characterization of chronic CCM effects on 3D ECTs. Future studies will investigate prolonged time points. These data provide an in vitro model to assess physiologically relevant mechanisms and evaluate safety and efficacy of future cardiac electrophysiology medical devices. Disclaimer: The mention of commercial products, their sources, or their use in connection with material reported herein is not to be construed as either an actual or implied endorsement of such products by the Department of Health and Human Services.

Mechanical control is essential for adaptive regulation in biological systems. This work presents a magnetic, non-contact approach to simultaneous detection and actuation in a microscale tissue testbed. The platform builds upon previously developed passive mechanical platforms, where tissues self-assemble on flexible pillars. Standard detection is typically derived from microscope images and actuation is often conducted using invasive approaches. In the presented platform, actuation and detection, both derived from magnetic fields, are demonstrated with high spatial, temporal, and force resolution resulting in a detectable $0.6~\mu \text{m}$ step-size in air. The forced deflection range is +/−125 microns (+/−10% strain), and an arbitrary magnitude step-and-settle actuation is achieved in less than 1 ms. Engineered human cardiac microtissue is used to demonstrate the capabilities of the system, as cardiac tissues generate contractions and adapt to external forces. Spontaneous contractions are monitored for an hour by a built-in sensor with a signal-to-noise of 2. Cyclic actuation at 1 Hz using a 1 mT/mm magnetic field is demonstrated, where max tissue strain is 0.3%. All this is achieved in a moderate-throughput, compact device, which is easily integrated into the typical flow of biological experimentation. Simultaneous control of actuation and detection enables decisions to be made on a sample-specific basis, and in future developments, will enable arbitrary design of the mechanical environment for 3D tissue conditioning, maturation, and control. [2020-0296]

Liver regeneration is a well-orchestrated process that is typically studied in animal models. Although previous animal studies have offered many insights into liver regeneration, human biology is less well understood. To this end, we developed a three-dimensional (3D) platform called structurally vascularized hepatic ensembles for analyzing regeneration (SHEAR) to model multiple aspects of human liver regeneration. SHEAR enables control over hemodynamic alterations to mimic those that occur during liver injury and regeneration and supports the administration of biochemical inputs such as cytokines and paracrine interactions with endothelial cells. We found that exposing the endothelium-lined channel to fluid flow led to increased secretion of regeneration-associated factors. Stimulation with relevant cytokines not only amplified the secretory response, but also induced cell-cycle entry of primary human hepatocytes (PHHs) embedded within the device. Further, we identified endothelial-derived mediators that are sufficient to initiate proliferation of PHHs in this context. Collectively, the data presented here underscore the importance of multicellular models that can recapitulate high-level tissue functions and demonstrate that the SHEAR device can be used to discover and validate conditions that promote human liver regeneration.

Spatial regulation of angiogenesis is important for the generation of functional engineered vasculature in regenerative medicine. The Notch ligands Jag1 and Dll4 show distinct expression patterns in endothelial cells and, respectively, promote and inhibit endothelial sprouting. Therefore, patterns of Notch ligands may be utilized to spatially control sprouting, but their potential and the underlying mechanisms of action are unclear. Here, we coupled in vitro and in silico models to analyze the ability of micropatterned Jag1 and Dll4 ligands to spatially control endothelial sprouting. Dll4 patterns, but not Jag1 patterns, elicited spatial control. Computational simulations of the underlying signaling dynamics suggest that different timing of Notch activation by Jag1 and Dll4 underlie their distinct ability to spatially control sprouting. Hence, Dll4 patterns efficiently direct the sprouts, whereas longer exposure to Jag1 patterns is required to achieve spatial control. These insights in sprouting regulation offer therapeutic handles for spatial regulation of angiogenesis.

Biomimetic on-chip tissue models serve as a powerful tool for studying human physiology and developing therapeutics; however, their modeling power is hindered by our inability to develop highly ordered functional structures in small length scales. Here, we demonstrate how high-precision fabrication can enable scaled-down modeling of organ-level cardiac mechanical function. We use two-photon direct laser writing (TPDLW) to fabricate a nanoscale-resolution metamaterial scaffold with fine-tuned mechanical properties to support the formation and cyclic contraction of a miniaturized, induced pluripotent stem cell–derived ventricular chamber. Furthermore, we fabricate microfluidic valves with extreme sensitivity to rectify the flow generated by the ventricular chamber. The integrated microfluidic system recapitulates the ventricular fluidic function and exhibits a complete pressure-volume loop with isovolumetric phases. Together, our results demonstrate a previously unexplored application of high-precision fabrication that can be generalized to expand the accessible spectrum of organ-on-a-chip models toward structurally and biomechanically sophisticated tissue systems.

Supplemental Digital Content is available in the text., Background: To assess the genetic architecture of hypertrophic cardiomyopathy (HCM) in patients of predominantly Chinese ancestry. Methods: We sequenced HCM disease genes in Singaporean patients (n=224) and Singaporean controls (n=3634), compared findings with additional populations and White HCM cohorts (n=6179), and performed in vitro functional studies. Results: Singaporean HCM patients had significantly fewer confidently interpreted HCM disease variants (pathogenic/likely pathogenic: 18%, P

Background: Alpelisib, a PI3Kα inhibitor, was recently approved by the FDA for the treatment of metastatic PIK3CA-mutated HR+ breast cancer in combination with fulvestrant. This therapeutic breakthrough came after years of disappointing efforts to develop solid tumor drugs targeting PIK3CA, one of the most commonly altered oncogenes in cancer, including 40% of HR+ breast and 30% of head and neck squamous cell cancer. The limited success of PI3Kinhibitors outside of breast cancer may be partly because cancers may harbor heterogeneous PIK3CA mutations and/or co-occurring mutations that confer varying degrees of sensitivity to PI3Kα inhibition. Methods: To identify biomarkers of sensitivity and resistance to PI3Kinhibitors, we assessed all plasma samples sent to a CLIA-certified commercial laboratory (Guardant Health) for a targeted NGS assay (Guardant360) from November 2016 to March 2019. The assay included 73 genes for single-nucleotide variants, 23 genes for insertions / deletions, 18 genes for amplifications, and 6 genes for fusions. In total, 53,118 cancer patients were sequenced, of whom 8,464 patients (15.9%) had a PIK3CA point mutation or amplification. This PIK3CA-mutated subset served as our analysis cohort. Results: The four most common tumor types in our PIK3CA-mutated cohort were breast (n=2060), non-small cell lung (n=2667), colorectal (n=999), and prostate cancer (n=713); head and neck cancer patients (n=129) were also included given the high frequency of PIK3CA mutations in this disease. Breast cancer patients had a median of 6 plasma mutations detected (range 1-92) and median maximum variant allele frequency (VAF) of 7.6% (range 0.05-96.5%). PIK3CA mutations were clonal in 68.2% of breast cancer patients (as defined by a VAF>50% of the maximum VAF), compared to 18-44% in patients of other tumor types (p Conclusions: PIK3CA-mutated breast cancer patients were more likely to have clonal PIK3CA mutations and multiple PIK3CA and PI3K pathway mutations compared to patients with other tumor types, which may suggest greater PI3Kpathway dependency and sensitivity to treatment with PI3Kα inhibitors. Ongoing whole exome and RNA sequencing of 21 pairs of pre- and post-treatment tissue biopsies of breast cancer patients treated with PI3Kα inhibitors will provide additional genomic and transcriptomic context to these plasma-based results. Breast (n=2060)NSCLC (n=2667)Colorectal (n=999)Prostate (n=713)Head and neck (n=129)Median age (range)60 (25-101)68 (25-97)62 (17-94)71 (39-97)60 (21-88)% male0%49%56%100%72.7%Median # mutations (range)6 (1-92)6 (1-51)8 (1-207)7 (1-76)6 (1-66)Median max VAF (range)7.6% (0.05-96.5)9.2% (0.04-96.1)17.7% (0.08-90.1)12.2% (0.01-94.5)3.7% (0.08-84.5)% PIK3CA clonal mutation a68.2% (1404)27.1% (724)44.1% (441)18.2% (130)40.3% (52)5 most frequent co-mutated genes, among patients with clonal PIK3CA mutationsTP53 (50.7%)TP53 (66.3%)APC (81.0%)AR (48.5%)TP53 (36.6%)ESR1 (31.6%)EGFR (34.9%)TP53 (70.5%)TP53 (40%)ARID1A (25%)EGFR (24.7%)KRAS (21.7%) bKRAS (67.3%) bEGFR (21.5%)NF1 (21.2%)ERBB2 (20.8%)NF1 (18.0%)EGFR (31.1%)MYC (19.2%)EGFR (21.2%)FGFR1 (18.7%)ARID1A(15.0%)BRAF (23.4%)CDK6 (18.5%)TERT (17.3%)5 most frequent co-mutated genes, among patients with subclonal PIK3CA mutationsTP53 (60.7%)TP53 (80.6%)APC (81.6%)TP53 (63.9%)TP53 (60%)ESR1 (28.1%)EGFR (43.3%)TP53 (76.2%)AR (63.0%)NOTCH1 (36.7%)ERBB2 (24.7%)KRAS (24.0%)KRAS (64.7%)EGFR (30.3%)EGFR 33.3%)NF1 (24.0%)NF1 (18.3%)EGFR (41.3%)BRAF (26.9%)MTOR (26.7%)EGFR (23.0%)MET (18.3%)BRAF (32.0%)APC (26.0%)ERBB2 (26.7%)>1 PIK3CA mutation c 29.7% (612)14.2% (378)17.3% (173)10.8% (77)31.0% (40)>1 PI3K pathway mutation c38.2% (787)21.0% (561)26.6% (266)20.9% (149)36.4% (47)% helical mutations36.8% (1079)16.1% (504)19.9% (245)10.7% (87)12.9% (25)% kinase mutations a15.1% (444)16.0% (501)24.1% (296)9.5% (77)16.0% (31)% other point mutations15.1% (444)16.0% (501)24.1% (296)9.5% (77)16.0% (31)% amplifications18.4% (538)37.7% (1182)14.6% (180)64.9% (526)37.6% (73)a p Citation Format: Christopher Chen, Avinash Sahu, Kristin Price, Christopher Pinto, Read Allen, Xiaoman Wang, Annamaria Szabolcs, Lipika Goyal, David Ting, Shirley Liu, Dejan Juric. Plasma sequencing demonstrates that breast cancer patients have a higher prevalence of clonal and multiple PIK3CA mutations than other solid tumor patients [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-10-34.

Background: Hypertrophic cardiomyopathy (HCM) is caused by pathogenic variants in sarcomere protein genes that evoke hypercontractility, poor relaxation, and increased energy consumption by the heart and increased patient risks for arrhythmias and heart failure. Recent studies show that pathogenic missense variants in myosin, the molecular motor of the sarcomere, are clustered in residues that participate in dynamic conformational states of sarcomere proteins. We hypothesized that these conformations are essential to adapt contractile output for energy conservation and that pathophysiology of HCM results from destabilization of these conformations. Methods: We assayed myosin ATP binding to define the proportion of myosins in the super relaxed state (SRX) conformation or the disordered relaxed state (DRX) conformation in healthy rodent and human hearts, at baseline and in response to reduced hemodynamic demands of hibernation or pathogenic HCM variants. To determine the relationships between myosin conformations, sarcomere function, and cell biology, we assessed contractility, relaxation, and cardiomyocyte morphology and metabolism, with and without an allosteric modulator of myosin ATPase activity. We then tested whether the positions of myosin variants of unknown clinical significance that were identified in patients with HCM, predicted functional consequences and associations with heart failure and arrhythmias. Results: Myosins undergo physiological shifts between the SRX conformation that maximizes energy conservation and the DRX conformation that enables cross-bridge formation with greater ATP consumption. Systemic hemodynamic requirements, pharmacological modulators of myosin, and pathogenic myosin missense mutations influenced the proportions of these conformations. Hibernation increased the proportion of myosins in the SRX conformation, whereas pathogenic variants destabilized these and increased the proportion of myosins in the DRX conformation, which enhanced cardiomyocyte contractility, but impaired relaxation and evoked hypertrophic remodeling with increased energetic stress. Using structural locations to stratify variants of unknown clinical significance, we showed that the variants that destabilized myosin conformations were associated with higher rates of heart failure and arrhythmias in patients with HCM. Conclusions: Myosin conformations establish work-energy equipoise that is essential for life-long cellular homeostasis and heart function. Destabilization of myosin energy-conserving states promotes contractile abnormalities, morphological and metabolic remodeling, and adverse clinical outcomes in patients with HCM. Therapeutic restabilization corrects cellular contractile and metabolic phenotypes and may limit these adverse clinical outcomes in patients with HCM.

Gap closure is a dynamic process in wound healing, in which a wound contracts and a provisional matrix is laid down, to restore structural integrity to injured tissues. The efficiency of wound closure has been found to depend on the shape of a wound, and this shape dependence has been echoed in various in vitro studies. While wound shape itself appears to contribute to this effect, it remains unclear whether the alignment of the surrounding extracellular matrix (ECM) may also contribute. In this study, we investigate the role both wound curvature and ECM alignment have on gap closure in a 3D culture model of fibrous tissue. Using microfabricated flexible micropillars positioned in rectangular and octagonal arrangements, seeded 3T3 fibroblasts embedded in a collagen matrix formed microtissues with different ECM alignments. Wounding these microtissues with a microsurgical knife resulted in wounds with different shapes and curvatures that closed at different rates. Observing different regions around the wounds, we noted local wound curvature did not impact the rate of production of provisional fibronectin matrix assembled by the fibroblasts. Instead, the rate of provisional matrix assembly was lowest emerging from regions of high fibronectin alignment and highest in the areas of low matrix alignment. Our data suggest that the underlying ECM structure affects the shape of the wound as well as the ability of fibroblasts to build provisional matrix, an important step in the process of tissue closure and restoration of tissue architecture. The study highlights an important interplay between ECM alignment, wound shape, and tissue healing that has not been previously recognized and may inform approaches to engineer tissues. IMPACT STATEMENT: Current models of tissue growth have identified a role for curvature in driving provisional matrix assembly. However, most tissue repair occurs in fibrous tissues with different levels of extracellular matrix (ECM) alignment. Here, we show how this underlying ECM alignment may affect the ability of fibroblasts to build new provisional matrix, with implications for in vivo wound healing and providing insight for engineering of new tissues.

Background: Heterozygous truncating variants in the sarcomere protein titin (TTN) are the most common genetic cause of heart failure, a major cause of morbidity and mortality. This causality indicates that even two-fold changes in the amount of TTN can profoundly disturb cardiac physiology. Although a critical role of TTN in sarcomere formation and cardiomyocyte contractility is well established, the mechanisms regulating transcription of the TTN gene remain poorly understood. Methods: We performed bioinformatics analysis to identify a putative transcriptional enhancer of TTN . Next, we created biallelic deletion of the enhancer in human induced pluripotent stem cell derived cardiomyocytes and performed enhancer reporter assays both in vitro and in vivo to demonstrate necessity and sufficiency of the enhancer in TTN gene expression, respectively. Furthermore, we performed massive parallel reporter assay to define critical transcriptional factors of the TTN enhancer activity and analyzed whole genome sequencing (WGS) data of human patients with unexplained dilated cardiomyopathy (DCM). Results: We identified an intron mediated enhancer that promotes cardiac-specific TTN expression. Global deletion of this element downregulated TTN expression in cardiomyocytes and impaired sarcomere development, while transgenic expression promoted cardiac expression in mice. Using mutational scanning we defined key transcription factor binding sites, including NKX2-5 and MEF2 motifs that promote TTN expression in cardiomyocytes. Consistent with these functions, analyses of WGS data in 69 patients with unexplained DCM revealed one rare variant that disrupted the conserved MEF2 transcriptional factor binding motif. Conclusions: Discovery of a TTN enhancer advances our understanding of cardiomyocyte development, provides an opportunity to modulate TTN transcriptional activity, and ultimately develop therapeutic strategies to treat dilated cardiomyopathy caused by TTN haploinsufficiency.

Patients with Alagille syndrome carry monogenic mutations in the Notch signaling pathway and face complications such as jaundice and cholestasis. Given the presence of intrahepatic ductopenia in these patients, Notch2 receptor signaling is implicated in driving normal biliary development and downstream branching morphogenesis. As a result, in vitro model systems of liver epithelium are needed to further mechanistic insight of biliary tissue assembly. Here, primary human intrahepatic cholangiocytes as a candidate population for such a platform are systematically evaluated, and conditions that direct their branching morphogenesis are described. It is found that extracellular matrix presentation, coupled with mitogen stimulation, promotes biliary branching in a Notch‐dependent manner. These results demonstrate the utility of using 3D scaffolds for mechanistic investigation of cholangiocyte branching and provide a gateway to integrate biliary architecture in additional in vitro models of liver tissue., The utility of natural polymer blends, coupled with developmentally relevant chemical cues, is appraised for their ability to direct branching morphogenesis in primary human cholangiocytes. The resulting structures from crosstalk between EGF and Notch signaling, can be ablated using CRISPR‐Cas9 editing, and organized within a 3D microfluidic device.

Description, PKP2 mutations lead to instability in cell-cell junctions and sarcomeres that impairs cardiac tissue contractility in ACM., Progressive loss of cardiac systolic function in arrhythmogenic cardiomyopathy (ACM) has recently gained attention as an important clinical consideration in managing the disease. However, the mechanisms leading to reduction in cardiac contractility are poorly defined. Here, we use CRISPR gene editing to generate human induced pluripotent stem cells (iPSCs) that harbor plakophilin-2 truncating variants (PKP2tv), the most prevalent ACM-linked mutations. The PKP2tv iPSC–derived cardiomyocytes are shown to have aberrant action potentials and reduced systolic function in cardiac microtissues, recapitulating both the electrical and mechanical pathologies reported in ACM. By combining cell micropatterning with traction force microscopy and live imaging, we found that PKP2tvs impair cardiac tissue contractility by destabilizing cell-cell junctions and in turn disrupting sarcomere stability and organization. These findings highlight the interplay between cell-cell adhesions and sarcomeres required for stabilizing cardiomyocyte structure and function and suggest fundamental pathogenic mechanisms that may be shared among different types of cardiomyopathies.

Three-dimensional engineered heart tissues (EHTs) derived from human induced pluripotent stem cells (iPSCs) have become an important resource for both drug toxicity screening and research on heart disease. A key metric of EHT phenotype is the contractile force with which the tissue spontaneously beats. It is well-known that cardiac muscle contractility – its ability to do mechanical work – depends on tissue prestrain (preload) and external resistance (afterload). Objectives: Here, we demonstrate a technique to control both preload and afterload dynamically while monitoring contractile force exerted by EHTs. Methods: We developed an apparatus that uses real-time feedback control to monitor and regulate EHT forces. The system is comprised of a pair of high-speed piezoelectric actuators that can strain the EHT scaffold and a fast optical measurement tool to provide EHT contractile force feedback while monitoring tissue strain. Results: The system was used to regulate the effective stiffness of the scaffold. When controlled to have effectively isometric boundary conditions, EHTs exerted a contractile force that was almost twice as large as that observed under auxotonic conditions. Conclusion: These experimental results demonstrate that EHT contractility can be increased through feedback control to regulate boundary stiffness. Significance: The work advances our understanding of the role that mechanical environment plays in EHT contractility. This could be used to help study or alter EHT phenotype and potentially EHT maturation through controlled mechanical conditioning.

SARS-CoV-2 can infect multiple organs, including lung, intestine, kidney, heart, liver, and brain. The molecular details of how the virus navigates through diverse cellular environments and establishes replication are poorly defined. Here, we generated a panel of phenotypically diverse, SARS-CoV-2-infectible human cell lines representing different body organs and performed longitudinal survey of cellular proteins and pathways broadly affected by the virus. This revealed universal inhibition of interferon signaling across cell types following SARS-CoV-2 infection. We performed systematic analyses of the JAK-STAT pathway in a broad range of cellular systems, including immortalized cells and primary-like cardiomyocytes, and found that SARS-CoV-2 targeted the proximal pathway components, including Janus kinase 1 (JAK1), tyrosine kinase 2 (Tyk2), and the interferon receptor subunit 1 (IFNAR1), resulting in cellular desensitization to type I IFN. Detailed mechanistic investigation of IFNAR1 showed that the protein underwent ubiquitination upon SARS-CoV-2 infection. Furthermore, chemical inhibition of JAK kinases enhanced infection of stem cell-derived cultures, indicating that the virus benefits from inhibiting the JAK-STAT pathway. These findings suggest that the suppression of interferon signaling is a mechanism widely used by the virus to evade antiviral innate immunity, and that targeting the viral mediators of immune evasion may help block virus replication in patients with COVID-19. IMPORTANCE SARS-CoV-2 can infect various organs in the human body, but the molecular interface between the virus and these organs remains unexplored. In this study, we generated a panel of highly infectible human cell lines originating from various body organs and employed these cells to identify cellular processes commonly or distinctly disrupted by SARS-CoV-2 in different cell types. One among the universally impaired processes was interferon signaling. Systematic analysis of this pathway in diverse culture systems showed that SARS-CoV-2 targets the proximal JAK-STAT pathway components, destabilizes the type I interferon receptor though ubiquitination, and consequently renders the infected cells resistant to type I interferon. These findings illuminate how SARS-CoV-2 can continue to propagate in different tissues even in the presence of a disseminated innate immune response.

Human pluripotent stem cell (hPSCs)-derived ventricular (V) cardiomyocytes (CMs) display immature Ca2+–handing properties with smaller transient amplitudes and slower kinetics due to such differences in crucial Ca2+-handling proteins as the poor sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pump but robust Na+-Ca2+ exchanger (NCX) activities in human embryonic stem cell (ESC)-derived VCMs compared with adult. Despite their fundamental importance in excitation-contraction coupling, the relative contribution of SERCA and NCX to Ca2+-handling of hPSC-VCMs remains unexplored. We systematically altered the activities of SERCA and NCX in human embryonic stem cell-derived ventricular cardiomyocytes (hESC-VCMs) and their engineered microtissues, followed by examining the resultant phenotypic consequences. SERCA overexpression in hESC-VCMs shortened the decay of Ca2+ transient at low frequencies (0.5 Hz) without affecting the amplitude, SR Ca2+ content and Ca2+ baseline. Interestingly, short hairpin RNA-based NCX suppression did not prolong the transient decay, indicating a compensatory response for Ca2+ removal. Although hESC-VCMs and their derived microtissues exhibited negative frequency-transient/force responses, SERCA overexpression rendered them less negative at high frequencies (>2 Hz) by accelerating Ca2+ sequestration. We conclude that for hESC-VCMs and their microtissues, SERCA, rather than NCX, is the main Ca2+ remover during diastole; poor SERCA expression is the leading cause for immature negative-frequency/force responses, which can be partially reverted by forced expression. Combinatorial approach to mature calcium handling in hESC-VCMs may help shed further mechanistic insights. NEW & NOTEWORTHY In this study of human pluripotent stem cell-derived cardiomyocytes, we studied the role of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and Na+-Ca2+ exchanger (NCX) in Ca2+ handling. Our data support the notion that SERCA is more effective in cytosolic calcium removal than the NCX.

Background and aims Chronic cholestatic liver diseases, such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), are frequently associated with damage to the barrier function of the biliary epithelium. Here, we report on a bile duct-on-a-chip that phenocopies not only the tubular architecture of the bile duct in three dimensions, but also its barrier functions. Approach and results We showed that mouse cholangiocytes in the channel of the device became polarized and formed mature tight junctions, that the permeability of the cholangiocyte monolayer was comparable to ex vivo measurements, and that cholangiocytes in the device were mechanosensitive (as demonstrated by changes in calcium flux under applied luminal flow). Permeability decreased significantly when cells formed a compact monolayer with cell densities comparable to those observed in vivo. This device enabled independent access to the apical and basolateral surfaces of the cholangiocyte channel, allowing proof-of-concept toxicity studies with the biliary toxin, biliatresone, and the bile acid, glycochenodeoxycholic acid. The cholangiocyte basolateral side was more vulnerable than the apical side to treatment with either agent, suggesting a protective adaptation of the apical surface that is normally exposed to bile. Further studies revealed a protective role of the cholangiocyte apical glycocalyx, wherein disruption of the glycocalyx with neuraminidase increased the permeability of the cholangiocyte monolayer after treatment with glycochenodeoxycholic acid. Conclusions This bile duct-on-a-chip captured essential features of a simplified bile duct in structure and organ-level functions and represents an in vitro platform to study the pathophysiology of the bile duct using cholangiocytes from a variety of sources.

Supplemental Digital Content is available in the text., Rationale: Human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) in combination with CRISPR/Cas9 genome editing provide unparalleled opportunities to study cardiac biology and disease. However, sarcomeres, the fundamental units of myocyte contraction, are immature and nonlinear in hiPSC-CMs, which technically challenge accurate functional interrogation of contractile parameters in beating cells. Furthermore, existing analysis methods are relatively low-throughput, indirectly assess contractility, or only assess well-aligned sarcomeres found in mature cardiac tissues. Objective: We aimed to develop an analysis platform that directly, rapidly, and automatically tracks sarcomeres in beating cardiomyocytes. The platform should assess sarcomere content, contraction and relaxation parameters, and beat rate. Methods and Results: We developed SarcTrack, a MatLab software that monitors fluorescently tagged sarcomeres in hiPSC-CMs. The algorithm determines sarcomere content, sarcomere length, and returns rates of sarcomere contraction and relaxation. By rapid measurement of hundreds of sarcomeres in each hiPSC-CM, SarcTrack provides large data sets for robust statistical analyses of multiple contractile parameters. We validated SarcTrack by analyzing drug-treated hiPSC-CMs, confirming the contractility effects of compounds that directly activate (CK-1827452) or inhibit (MYK-461) myosin molecules or indirectly alter contractility (verapamil and propranolol). SarcTrack analysis of hiPSC-CMs carrying a heterozygous truncation variant in the myosin-binding protein C (MYBPC3) gene, which causes hypertrophic cardiomyopathy, recapitulated seminal disease phenotypes including cardiac hypercontractility and diminished relaxation, abnormalities that normalized with MYK-461 treatment. Conclusions: SarcTrack provides a direct and efficient method to quantitatively assess sarcomere function. By improving existing contractility analysis methods and overcoming technical challenges associated with functional evaluation of hiPSC-CMs, SarcTrack enhances translational prospects for sarcomere-regulating therapeutics and accelerates interrogation of human cardiac genetic variants.

SummaryEpithelial to mesenchymal transition (EMT) is a complex cellular program proceeding through a hybrid E/M state linked to cancer-associated stemness, migration and chemoresistance. Deeper molecular understanding of this dynamic physiological landscape is needed to define events which regulate the transition and entry into and exit from the E/M state. Here, we quantified >60,000 molecules across ten time points and twelve omic layers in human mammary epithelial cells undergoing TGFβ-induced EMT. Deep proteomic profiles of whole cells, nuclei, extracellular vesicles, secretome, membrane and phosphoproteome defined state-specific signatures and major transition points. Parallel metabolomics showed metabolic reprogramming preceded changes in other layers, while single-cell RNA sequencing identified transcription factors controlling entry into E/M. Covariance analysis exposed unexpected discordance between the molecular layers. Integrative causal modeling revealed co-dependencies governing entry into E/M that were verified experimentally using combinatorial inhibition. Overall, this dataset provides an unprecedented resource on TGFβ signaling, EMT and cancer.

We have designed and fabricated a microfluidic-based platform for sensing mechanical forces generated by cardiac microtissues in a highly-controlled microenvironment. Our fabrication approach combines Direct Laser Writing (DLW) lithography with soft lithography. At the center of our platform is a cylindrical volume, divided into two chambers by a cylindrical polydimethylsiloxane (PDMS) shell. Cells are seeded into the inner chamber from a top opening, and the microtissue assembles onto tailor-made attachment sites on the inner walls of the cylindrical shell. The outer chamber is electrically and fluidically isolated from the inner one by the cylindrical shell and is designed for actuation and sensing purposes. Externally applied pressure waves to the outer chamber deform parts of the cylindrical shell and thus allow us to exert time-dependent forces on the microtissue. Oscillatory forces generated by the microtissue similarly deform the cylindrical shell and change the volume of the outer chamber, resulting in measurable electrical conductance changes. We have used this platform to study the response of cardiac microtissues derived from human induced pluripotent stem cells (hiPSC) under prescribed mechanical loading and pacing., Main article 15 pages, 6 figures, 1 tables; supplementary 11 pages, 7 figures, 1 table, 6 movies

Interactions between cardiac myofibroblasts and myocytes may slow conduction and generate spontaneous beating in fibrosis, increasing the chance of life-threatening arrhythmia. While co-culture studies have shown that myofibroblasts can affect cardiomyocyte electrophysiology in vitro, the extent of myofibroblast-myocyte electrical conductance in a syncytium is unknown. In this neonatal rat study, cardiac myofibroblasts were transduced with Channelrhodopsin-2, which allowed acute and selective increase of myofibroblast current, and plated on top of cardiomyocytes. Optical mapping revealed significantly decreased conduction velocity (− 27 ± 6%, p –3), upstroke rate (− 13 ± 4%, p = 0.002), and action potential duration (− 14 ± 7%, p = 0.004) in co-cultures when 0.017 mW/mm2 light was applied, as well as focal spontaneous beating in 6/7 samples and a decreased cycle length (− 36 ± 18%, p = 0.002) at 0.057 mW/mm2 light. In silico modeling of the experiments reproduced the experimental findings and suggested the light levels used in experiments produced excess current similar in magnitude to endogenous myofibroblast current. Fitting the model to experimental data predicted a tissue-level electrical conductance across the 3-D interface between myofibroblasts and cardiomyocytes of ~ 5 nS/cardiomyocyte, and showed how increased myofibroblast-myocyte conductance, increased myofibroblast/myocyte capacitance ratio, and increased myofibroblast current, which occur in fibrosis, can work in tandem to produce pro-arrhythmic increases in conduction and spontaneous beating.

Rationale: NAA15 (N-alpha-acetyltransferase 15) is a component of the NatA (N-terminal acetyltransferase complex). The mechanism by which NAA15 haploinsufficiency causes congenital heart disease remains unknown. To better understand molecular processes by which NAA15 haploinsufficiency perturbs cardiac development, we introduced NAA15 variants into human induced pluripotent stem cells (iPSCs) and assessed the consequences of these mutations on RNA and protein expression. Objective: We aim to understand the role of NAA15 haploinsufficiency in cardiac development by investigating proteomic effects on NatA complex activity and identifying proteins dependent upon a full amount of NAA15. Methods and Results: We introduced heterozygous loss of function, compound heterozygous, and missense residues (R276W) in iPSCs using CRISPR/Cas9. Haploinsufficient NAA15 iPSCs differentiate into cardiomyocytes, unlike NAA15 -null iPSCs, presumably due to altered composition of NatA. Mass spectrometry analyses reveal ≈80% of identified iPSC NatA targeted proteins displayed partial or complete N-terminal acetylation. Between null and haploinsufficient NAA15 cells, N-terminal acetylation levels of 32 and 9 NatA-specific targeted proteins were reduced, respectively. Similar acetylation loss in few proteins occurred in NAA15 R276W induced pluripotent stem cells. In addition, steady-state protein levels of 562 proteins were altered in both null and haploinsufficient NAA15 cells; 18 were ribosomal-associated proteins. At least 4 proteins were encoded by genes known to cause autosomal dominant congenital heart disease. Conclusions: These studies define a set of human proteins that requires a full NAA15 complement for normal synthesis and development. A 50% reduction in the amount of NAA15 alters levels of at least 562 proteins and N-terminal acetylation of only 9 proteins. One or more modulated proteins are likely responsible for NAA15-haploinsufficiency mediated congenital heart disease. Additionally, genetically engineered induced pluripotent stem cells provide a platform for evaluating the consequences of amino acid sequence variants of unknown significance on NAA15 function.

A better fundamental understanding of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has the potential to advance applications ranging from drug discovery to cardiac repair. Automated quantitative analysis of beating hiPSC-CMs is an important and fast developing component of the hiPSC-CM research pipeline. Here we introduce “Sarc-Graph,” a computational framework to segment, track, and analyze sarcomeres in fluorescently tagged hiPSC-CMs. Our framework includes functions to segment z-discs and sarcomeres, track z-discs and sarcomeres in beating cells, and perform automated spatiotemporal analysis and data visualization. In addition to reporting good performance for sarcomere segmentation and tracking with little to no parameter tuning and a short runtime, we introduce two novel analysis approaches. First, we construct spatial graphs where z-discs correspond to nodes and sarcomeres correspond to edges. This makes measuring the network distance between each sarcomere (i.e., the number of connecting sarcomeres separating each sarcomere pair) straightforward. Second, we treat tracked and segmented components as fiducial markers and use them to compute the approximate deformation gradient of the entire tracked population. This represents a new quantitative descriptor of hiPSC-CM function. We showcase and validate our approach with both synthetic and experimental movies of beating hiPSC-CMs. By publishing Sarc-Graph, we aim to make automated quantitative analysis of hiPSC-CM behavior more accessible to the broader research community., Author summary Heart disease is the leading cause of death worldwide. Because of this, many researchers are studying heart cells in the lab and trying to create artificial heart tissue. Recently, there has been a growing focus on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). These are cells that are safely sampled from living humans, for example from the blood or skin, that are then transformed into human heart muscle cells. One active research goal is to use these cells to repair the damaged heart. Another active research goal is to test new drugs on these cells before testing them in animals and humans. However, one major challenge is that hiPSC-CMs often have an irregular internal structure that is difficult to analyze. At present, their behavior is far from fully understood. To address this, we have created software to automatically analyze movies of beating hiPSC-CMs. With our software, it is possible to quantify properties such as the amount and direction of beating cell contraction, and the variation in behavior across different parts of each cell. These tools will enable further quantitative analysis of hiPSC-CMs. With these tools, it will be easier to understand, control, and optimize artificial heart tissue created with hiPSC-CMs, and quantify the effects of drugs on hiPSC-CM behavior.

The inherent constraints on resolution, speed and field of view have hindered the development of high-speed, three-dimensional microscopy techniques over large scales. Here, we present a multiplane line-scan imaging strategy, which uses a series of axially distributed reflecting slits to probe different depths within a sample volume. Our technique enables the simultaneous imaging of an optically sectioned image stack with a single camera at frame rates of hundreds of hertz, without the need for axial scanning. We demonstrate the applicability of our system to monitor fast dynamics in biological samples by performing calcium imaging of neuronal activity in mouse brains and voltage imaging of cardiomyocytes in cardiac samples.

How adhesive forces are transduced and integrated into biochemical signals at focal adhesions (FAs) is poorly understood. Using cells adhering to deformable micropillar arrays, we demonstrate that traction force and FAK localization as well as traction force and Y397-FAK phosphorylation are linearly coupled at individual FAs on stiff, but not soft, substrates. Similarly, FAK phosphorylation increases linearly with external forces applied to FAs using magnetic beads. This mechanosignaling coupling requires actomyosin contractility, talin-FAK binding, and full-length vinculin that binds talin and actin. Using an in vitro 3D biomimetic wound healing model, we show that force-FAK signaling coupling coordinates cell migration and tissue-scale forces to promote microtissue repair. A simple kinetic binding model of talin-FAK interactions under force can recapitulate the experimental observations. This study provides insights on how talin and vinculin convert forces into FAK signaling events regulating cell migration and tissue repair., How adhesive forces are transduced and integrated into biochemical signals at focal adhesions (FAs) is poorly understood. Here authors show that force- FAK signaling coupling coordinates cell migration and tissue-scale forces to promote microtissue repair.

SARS-CoV-2 can infect multiple organs, including lung, intestine, kidney, heart, liver, and brain. The molecular details of how the virus navigates through diverse cellular environments and establishes replication are poorly defined. Here, we performed global proteomic analysis of the virus-host interface in a newly established panel of phenotypically diverse, SARS-CoV-2-infectable human cell lines representing different body organs. This revealed universal inhibition of interferon signaling across cell types following SARS-CoV-2 infection. We performed systematic analyses of the JAK-STAT pathway in a broad range of cellular systems, including immortalized cell lines and primary-like cardiomyocytes, and found that several pathway components were targeted by SARS-CoV-2 leading to cellular desensitization to interferon. These findings indicate that the suppression of interferon signaling is a mechanism widely used by SARS-CoV-2 in diverse tissues to evade antiviral innate immunity, and that targeting the viral mediators of immune evasion may help block virus replication in patients with COVID-19.

The extracellular matrix (ECM) is a complex mixture composed of fibrillar collagens as well as additional protein and carbohydrate components. Proteoglycans (PGs) contribute to the heterogeneity of the ECM and play an important role in its structure and function. While the small leucine rich proteoglycans (SLRPs), including decorin and lumican, have been studied extensively as mediators of collagen fibrillogenesis and organization, the function of large matrix PGs in collagen matrices is less well known. In this study, we showed that different matrix PGs have distinct roles in regulating collagen behaviors. We found that versican, a large chondroitin sulfate PG, promotes collagen fibrillogenesis in a turbidity assay and upregulates cell-mediated collagen compaction and reorganization, whereas aggrecan, a structurally-similar large PG, has different and often opposing effects on collagen. Compared to versican, decorin and lumican also have distinct functions in regulating collagen behaviors. The different ways in which matrix PGs interact with collagen have important implications for understanding the role of the ECM in diseases such as fibrosis and cancer, and suggest that matrix PGs are potential therapeutic targets.

Summary A primary challenge in tissue engineering is to recapitulate both the structural and functional features of whole tissues and organs. In vivo, patterning of the body plan and constituent tissues emerges from the carefully orchestrated interactions between the transcriptional programs that give rise to cell types and the mechanical forces that drive the bending, twisting, and extensions critical to morphogenesis. Substantial recent progress in mechanobiology—understanding how mechanics regulate cell behaviors and what cellular machineries are responsible—raises the possibility that one can begin to use these insights to help guide the strategy and design of functional engineered tissues. In this perspective, we review and propose the development of different approaches, from providing appropriate extracellular mechanical cues to interfering with cellular mechanosensing machinery, to aid in controlling cell and tissue structure and function.

The structural and functional maturation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is essential for application to pharmaceutical testing, disease modeling, and ultimately therapeutic use. Multicellular 3D-tissue platforms have improved functional maturation of hiPSC-CMs, but probing cardiac contractile properties remains challenging in a 3D environment, especially at depth and in live tissues. Using small angle X-ray scattering (SAXS) images, we show that hiPSC-CMs, matured and examined in a 3D environment, exhibit periodic spatial arrangement of the myofilament lattice, which has not been previously detected in hiPSC-CMs. Contractile force is found to correlate with both scattering intensity (R2=0.44) and lattice spacing (R2=0.46). Scattering intensity also correlates with lattice spacing (R2=0.81), suggestive of lower noise in our structural measurement relative to the functional measurement. Notably, we observe decreased myofilament ordering in tissues with a myofilament mutation known to lead to hypertrophic cardiomyopathy (HCM). Our results highlight the progress of human cardiac tissue engineering and enable unprecedented study of structural maturation in hiPSC-CMs., Comment: 12 pages, 6 figures, supplementary material available upon request to the lead author

The mammary gland is a highly vascularized tissue capable of expansion and regression during development and disease. To enable mechanistic insight into the coordinated morphogenic crosstalk between the epithelium and vasculature, we introduce a 3D microfluidic platform that juxtaposes a human mammary duct in proximity to a perfused endothelial vessel. Both compartments recapitulate stable architectural features of native tissue and the ability to undergo distinct forms of branching morphogenesis. Modeling HER2/ERBB2 amplification or activating PIK3CA(H1047R) mutation each produces ductal changes observed in invasive progression, yet with striking morphogenic and behavioral differences. Interestingly, PI3KαH1047R ducts also elicit increased permeability and structural disorganization of the endothelium, and we identify the distinct secretion of IL-6 as the paracrine cause of PI3KαH1047R-associated vascular dysfunction. These results demonstrate the functionality of a model system that facilitates the dissection of 3D morphogenic behaviors and bidirectional signaling between mammary epithelium and endothelium during homeostasis and pathogenesis., In vitro models of the human mammary gland have struggled to mimic the 3D morphogenic processes that occur in vivo. Here the authors develop a 3D microfluidic platform of a vascularized human mammary duct that simulates diverse morphogenic transitions and paracrine crosstalk.

Interactions between cardiac myofibroblasts and myocytes may slow conduction after cardiac injury, increasing the chance of life-threatening arrhythmia. While co-culture studies have shown that myofibroblasts can affect cardiomyocyte electrophysiology in vitro, the mechanism(s) remain debatable. In this study, primary neonatal rat cardiac myofibroblasts were transduced with the light-activated ion channel Channelrhodopsin-2, which allowed acute and selective modulation of myofibroblast currents in co-cultures with cardiomyocytes. Optical mapping revealed that myofibroblast-specific optogenetically induced inward currents decreased conduction velocity in the co-cultures by 27±6% (baseline = 17.7±5.3 cm/s), and shortened the cardiac action potential duration by 14±7% (baseline = 161±11 ms) when 0.017 mW/mm2 light was applied. When light irradiance was increased to 0.057 mW/mm2, the myofibroblast currents led to spontaneous beating in 6/7 co-cultures. Experiments showed that optogenetic perturbation did not lead to changes in myofibroblast strain and force generation, suggesting purely electrical effects in this model. In silico modeling of optogenetically modified myofibroblast-cardiomyocyte co-cultures largely reproduced these results and enabled a comprehensive study of relevant parameters. These results clearly demonstrate that myofibroblasts are sufficiently electrically connected to cardiomyocytes to effectively alter macroscopic electrophysiological properties in this model of cardiac tissue.

Dendritic cell (DC) migration is required for efficient presentation of antigen to T cells and the initiation of an adaptive immune response. In spite of its importance, many aspects of DC migration have not been characterized. DCs encounter a variety of environments with different stiffness and geometry, but the effect of these parameters on DC migration has not yet been determined. We addressed this question by comparing DC motility on standard migration surfaces (polydimethylsiloxane (PDMS)-coated coverslips) and micropost array detectors (mPADs). These two surfaces differ in both stiffness and geometry. We found that DC migration was affected by substrate type, with significant increases in speed and significant decreases in persistence time on mPADs made of PDMS as compared to spin-coated PDMS coverslips. To determine whether the geometry or compliance of the post arrays was responsible for these changes in DC migration, we quantified DC motility on mPADs of identical geometry but different stiffness. Migration was indistinguishable on these mPADs, suggesting that DCs are responsive to geometry of ligand presentation and not stiffness. Further, by micropatterning ligands on flat PDMS surfaces in similar geometries to the mPAD arrays, we determined that DCs respond to the geometry of printed ligand. Finally, we used a variety of small molecule inhibitors to identify pathways involved in geometry sensing. We saw a significant role for myosin contractility and α(5)β(1) integrin engagement. We also noted significant reorganization of the actin cytoskeleton into dynamic actin rings when DCs were motile on posts. From these experiments, we conclude that DCs are insensitive to substrate compliance in the range tested but respond to changes in geometry via a mechanism that involves integrin function, myosin contractility, and remodeling of the actin cytoskeleton. As a possible explanation, we postulate a consistent role for filopodial extension and contraction as the driver of DC motility.

Biomaterials engineered with specific bioactive ligands, tunable mechanical properties, and complex architectural features have emerged as powerful tools to probe how cells sense and respond to the physical properties of their material surroundings, and ultimately provide designer approaches to control cell function.

The vascular barrier that separates blood from tissues is actively regulated by the endothelium and is essential for transport, inflammation, and haemostasis. Haemodynamic shear stress plays a critical role in maintaining endothelial barrier function, but how this occurs remains unknown. Here we use an engineered organotypic model of perfused microvessels to show that activation of the transmembrane receptor NOTCH1 directly regulates vascular barrier function through a non-canonical, transcription-independent signalling mechanism that drives assembly of adherens junctions, and confirm these findings in mouse models. Shear stress triggers DLL4-dependent proteolytic activation of NOTCH1 to expose the transmembrane domain of NOTCH1. This domain mediates establishment of the endothelial barrier; expression of the transmembrane domain of NOTCH1 is sufficient to rescue defects in barrier function induced by knockout of NOTCH1. The transmembrane domain restores barrier function by catalysing the formation of a receptor complex in the plasma membrane consisting of vascular endothelial cadherin, the transmembrane protein tyrosine phosphatase LAR, and the RAC1 guanidine-exchange factor TRIO. This complex activates RAC1 to drive assembly of adherens junctions and establish barrier function. Canonical transcriptional signalling via Notch is highly conserved in metazoans and is required for many processes in vascular development, including arterial-venous differentiation, angiogenesis and remodelling. We establish the existence of a non-canonical cortical NOTCH1 signalling pathway that regulates vascular barrier function, and thus provide a mechanism by which a single receptor might link transcriptional programs with adhesive and cytoskeletal remodelling.

Loss of the microvascular (MV) network results in tissue ischemia, loss of tissue function, and is a hallmark of chronic diseases. The incorporation of a functional vascular network with that of the host remains a challenge to utilizing engineered tissues in clinically relevant therapies. We showed that vascular-bed-specific endothelial cells (ECs) exhibit differing angiogenic capacities, with kidney microvascular endothelial cells (MVECs) being the most deficient, and sought to explore the underlying mechanism. Constitutive activation of the phosphatase PTEN in kidney MVECs resulted in impaired PI3K/AKT activity in response to vascular endothelial growth factor (VEGF). Suppression of PTEN in vivo resulted in microvascular regeneration, but was insufficient to improve tissue function. Promoter analysis of the differentially regulated genes in KMVECs suggests that the transcription factor FOXO1 is highly active and RNAseq analysis revealed that hyperactive FOXO1 inhibits VEGF-Notch-dependent tip-cell formation by direct and indirect inhibition of DLL4 expression in response to VEGF. Inhibition of FOXO1 enhanced angiogenesis in human bio-engineered capillaries, and resulted in microvascular regeneration and improved function in mouse models of injury-repair.

Cell-generated tractions play an important role in various physiological and pathological processes such as stem-cell differentiation, cell migration, wound healing, and cancer metastasis. Traction force microscopy (TFM) is a technique for quantifying cellular tractions during cell–matrix interactions. Most applications of this technique have heretofore assumed that the matrix surrounding the cells is linear elastic and undergoes infinitesimal strains, but recent experiments have shown that the traction-induced strains can be large (e.g., more than 50%). In this paper, we propose a novel three-dimensional (3D) TFM approach that consistently accounts for both the geometric nonlinearity introduced by large strains in the matrix, and the material nonlinearity due to strain-stiffening of the matrix. In particular, we pose the TFM problem as a nonlinear inverse hyperelasticity problem in the stressed configuration of the matrix, with the objective of determining the cellular tractions that are consistent with the measured displacement field in the matrix. We formulate the inverse problem as a constrained minimization problem and develop an efficient adjoint-based minimization procedure to solve it. We first validate our approach using simulated data, and quantify its sensitivity to noise. We then employ the new approach to recover tractions exerted by NIH 3T3 cells fully encapsulated in hydrogel matrices of varying stiffness. We find that neglecting nonlinear effects can induce significant errors in traction reconstructions. We also find that cellular tractions roughly increase with gel stiffness, while the strain energy appears to saturate.

This work presents a microscale tissue testbed with closed loop mechanical control. The platform leverages a non-contact technique capable of simultaneous actuation and detection, both derived from magnetic fields. We demonstrate cyclic tension and compression of engineered microtissue as well as long-term monitoring of spontaneous beating inside an incubator. The device is capable of positional feedback with high spatial and temporal resolution, while maintaining optical access from a standard microscope. Such a platform will enable experimental design of arbitrary mechanical environments for tissue conditioning, maturation, and monitoring.