Determinants of electrical propagation and propagation block in Arrhythmogenic Cardiomyopathy.

Gap junction and ion channel remodeling occur early in Arrhythmogenic Cardiomyopathy (ACM), but their pathogenic consequences have not been elucidated. Here, we identified the arrhythmogenic substrate, consisting of propagation slowing and conduction block, in ACM models expressing two different desmosomal gene variants. Neonatal rat ventricular myocytes were transduced to express variants in genes encoding desmosomal proteins plakoglobin or plakophilin-2. Studies were performed in engineered cells and anisotropic tissues to quantify changes in conduction velocity, formation of unidirectional propagation, cell-cell electrical coupling, and ion currents. Conduction velocity decreased by 71% and 63% in the two ACM models. SB216763, an inhibitor of glycogen synthase kinase-3 beta, restored conduction velocity to near normal levels. Compared to control, both ACM models showed greater propensity for unidirectional conduction block, which increased further at greater stimulation frequencies. Cell-cell electrical conductance measured in cell pairs was reduced by 86% and 87% in the two ACM models. Computer modeling showed close correspondence between simulated and experimentally determined changes in conduction velocity. The simulation identified that reduced cell-cell electrical coupling was the dominant factor leading to slow conduction, while the combination of reduced cell-cell electrical coupling, reduced sodium current and inward rectifier potassium current explained the development of unidirectional block. Expression of two different ACM variants markedly reduced cell-cell electrical coupling and conduction velocity, and greatly increased the likelihood of developing unidirectional block - both key features of arrhythmogenesis. This study provides the first quantitative analysis of cellular electrophysiological changes leading to the substrate of reentrant arrhythmias in early stage ACM.

Self-organizing behaviors of cardiovascular cells on synthetic nanofiber scaffolds.

In tissues and organs, the extracellular matrix (ECM) helps maintain inter- and intracellular architectures that sustain the structure-function relationships defining physiological homeostasis. Combining fiber scaffolds and cells to form engineered tissues is a means of replicating these relationships. Engineered tissues' fiber scaffolds are designed to mimic the topology and chemical composition of the ECM network. Here, we asked how cells found in the heart compare in their propensity to align their cytoskeleton and self-organize in response to topological cues in fibrous scaffolds. We studied cardiomyocytes, valvular interstitial cells, and vascular endothelial cells as they adapted their inter- and intracellular architectures to the extracellular space. We used focused rotary jet spinning to manufacture aligned fibrous scaffolds to mimic the length scale and three-dimensional (3D) nature of the native ECM in the muscular, valvular, and vascular tissues of the heart. The representative cardiovascular cell types were seeded onto fiber scaffolds and infiltrated the fibrous network. We measured different cell types' propensity for cytoskeletal alignment in response to fiber scaffolds with differing levels of anisotropy. The results indicated that valvular interstitial cells on moderately anisotropic substrates have a higher propensity for cytoskeletal alignment than cardiomyocytes and vascular endothelial cells. However, all cell types displayed similar levels of alignment on more extreme (isotropic and highly anisotropic) fiber scaffold organizations. These data suggest that in the hierarchy of signals that dictate the spatiotemporal organization of a tissue, geometric cues within the ECM and cellular networks may homogenize behaviors across cell populations and demographics.

Spatiotemporal cell junction assembly in human iPSC-CM models of arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac disorder that causes life-threatening arrhythmias and myocardial dysfunction. Pathogenic variants in Plakophilin-2 (PKP2), a desmosome component within specialized cardiac cell junctions, cause the majority of ACM cases. However, the molecular mechanisms by which PKP2 variants induce disease phenotypes remain unclear. Here we built bioengineered platforms using genetically modified human induced pluripotent stem cell-derived cardiomyocytes to model the early spatiotemporal process of cardiomyocyte junction assembly in vitro. Heterozygosity for truncating variant PKP2R413X reduced Wnt/ß-catenin signaling, impaired myofibrillogenesis, delayed mechanical coupling, and reduced calcium wave velocity in engineered tissues. These abnormalities were ameliorated by SB216763, which activated Wnt/ß-catenin signaling, improved cytoskeletal organization, restored cell junction integrity in cell pairs, and improved calcium wave velocity in engineered tissues. Together, these findings highlight the therapeutic potential of modulating Wnt/ß-catenin signaling in a human model of ACM.

Fibre-infused gel scaffolds guide cardiomyocyte alignment in 3D-printed ventricles.

Hydrogels are attractive materials for tissue engineering, but efforts to date have shown limited ability to produce the microstructural features necessary to promote cellular self-organization into hierarchical three-dimensional (3D) organ models. Here we develop a hydrogel ink containing prefabricated gelatin fibres to print 3D organ-level scaffolds that recapitulate the intra- and intercellular organization of the heart. The addition of prefabricated gelatin fibres to hydrogels enables the tailoring of the ink rheology, allowing for a controlled sol-gel transition to achieve precise printing of free-standing 3D structures without additional supporting materials. Shear-induced alignment of fibres during ink extrusion provides microscale geometric cues that promote the self-organization of cultured human cardiomyocytes into anisotropic muscular tissues in vitro. The resulting 3D-printed ventricle in vitro model exhibited biomimetic anisotropic electrophysiological and contractile properties.

Recreating the heart's helical structure-function relationship with focused rotary jet spinning.

Helical alignments within the heart's musculature have been speculated to be important in achieving physiological pumping efficiencies. Testing this possibility is difficult, however, because it is challenging to reproduce the fine spatial features and complex structures of the heart's musculature using current techniques. Here we report focused rotary jet spinning (FRJS), an additive manufacturing approach that enables rapid fabrication of micro/nanofiber scaffolds with programmable alignments in three-dimensional geometries. Seeding these scaffolds with cardiomyocytes enabled the biofabrication of tissue-engineered ventricles, with helically aligned models displaying more uniform deformations, greater apical shortening, and increased ejection fractions compared with circumferential alignments. The ability of FRJS to control fiber arrangements in three dimensions offers a streamlined approach to fabricating tissues and organs, with this work demonstrating how helical architectures contribute to cardiac performance.

Differential modulation of endothelial cytoplasmic protrusions after exposure to graphene-family nanomaterials.

Engineered nanomaterials offer the benefit of having systematically tunable physicochemical characteristics (e.g., size, dimensionality, and surface chemistry) that highly dictate the biological activity of a material. Among the most promising engineered nanomaterials to date are graphene-family nanomaterials (GFNs), which are 2-D nanomaterials (2DNMs) with unique electrical and mechanical properties. Beyond engineering new nanomaterial properties, employing safety-by-design through considering the consequences of cell-material interactions is essential for exploring their applicability in the biomedical realm. In this study, we asked the effect of GFNs on the endothelial barrier function and cellular architecture of vascular endothelial cells. Using micropatterned cell pairs as a reductionist in vitro model of the endothelium, the progression of cytoskeletal reorganization as a function of GFN surface chemistry and time was quantitatively monitored. Here, we show that the surface oxidation of GFNs (graphene, reduced graphene oxide, partially reduced graphene oxide, and graphene oxide) differentially affect the endothelial barrier at multiple scales; from the biochemical pathways that influence the development of cellular protrusions to endothelial barrier integrity. More oxidized GFNs induce higher endothelial permeability and the increased formation of cytoplasmic protrusions such as filopodia. We found that these changes in cytoskeletal organization, along with barrier function, can be potentiated by the effect of GFNs on the Rho/Rho-associated kinase (ROCK) pathway. Specifically, GFNs with higher surface oxidation elicit stronger ROCK2 inhibitory behavior as compared to pristine graphene sheets. Overall, findings from these studies offer a new perspective towards systematically controlling the surface-dependent effects of GFNs on cytoskeletal organization via ROCK2 inhibition, providing insight for implementing safety-by-design principles in GFN manufacturing towards their targeted biomedical applications.

An autonomously swimming biohybrid fish designed with human cardiac biophysics.

Biohybrid systems have been developed to better understand the design principles and coordination mechanisms of biological systems. We consider whether two functional regulatory features of the heart-mechanoelectrical signaling and automaticity-could be transferred to a synthetic analog of another fluid transport system: a swimming fish. By leveraging cardiac mechanoelectrical signaling, we recreated reciprocal contraction and relaxation in a muscular bilayer construct where each contraction occurs automatically as a response to the stretching of an antagonistic muscle pair. Further, to entrain this closed-loop actuation cycle, we engineered an electrically autonomous pacing node, which enhanced spontaneous contraction. The biohybrid fish equipped with intrinsic control strategies demonstrated self-sustained body-caudal fin swimming, highlighting the role of feedback mechanisms in muscular pumps such as the heart and muscles.

High-throughput coating with biodegradable antimicrobial pullulan fibres extends shelf life and reduces weight loss in an avocado model.

Food waste and food safety motivate the need for improved food packaging solutions. However, current films/coatings addressing these issues are often limited by inefficient release dynamics that require large quantities of active ingredients. Here we developed antimicrobial pullulan fibre (APF)-based packaging that is biodegradable and capable of wrapping food substrates, increasing their longevity and enhancing their safety. APFs were spun using a high-throughput system, termed focused rotary jet spinning, with water as the only solvent, allowing the incorporation of naturally derived antimicrobial agents. Using avocados as a representative example, we demonstrate that APF-coated samples had their shelf life extended by inhibited proliferation of natural microflora, and lost less weight than uncoated control samples. This work offers a promising technique to produce scalable, low-cost and environmentally friendly biodegradable antimicrobial packaging systems.

Prednisolone rescues Duchenne muscular dystrophy phenotypes in human pluripotent stem cell-derived skeletal muscle in vitro.

Duchenne muscular dystrophy (DMD) is a devastating genetic disease leading to degeneration of skeletal muscles and premature death. How dystrophin absence leads to muscle wasting remains unclear. Here, we describe an optimized protocol to differentiate human induced pluripotent stem cells (iPSC) to a late myogenic stage. This allows us to recapitulate classical DMD phenotypes (mislocalization of proteins of the dystrophin-associated glycoprotein complex, increased fusion, myofiber branching, force contraction defects, and calcium hyperactivation) in isogenic DMD-mutant iPSC lines in vitro. Treatment of the myogenic cultures with prednisolone (the standard of care for DMD) can dramatically rescue force contraction, fusion, and branching defects in DMD iPSC lines. This argues that prednisolone acts directly on myofibers, challenging the largely prevalent view that its beneficial effects are caused by antiinflammatory properties. Our work introduces a human in vitro model to study the onset of DMD pathology and test novel therapeutic approaches.

Mapping 2D- and 3D-distributions of metal/metal oxide nanoparticles within cleared human ex vivo skin tissues.

An increasing number of commercial skincare products are being manufactured with engineered nanomaterials (ENMs), prompting a need to fully understand how ENMs interact with the dermal barrier as a major biodistribution entry route. Although animal studies show that certain nanomaterials can cross the skin barrier, physiological differences between human and animal skin, such as the lack of sweat glands, limit the translational validity of these results. Current optical microscopy methods have limited capabilities to visualize ENMs within human skin tissues due to the high amount of background light scattering caused by the dense, ubiquitous extracellular matrix (ECM) of the skin. Here, we hypothesized that organic solvent-based tissue clearing ("immunolabeling-enabled three-dimensional imaging of solvent-cleared organs", or "iDISCO") would reduce background light scattering from the extracellular matrix of the skin to sufficiently improve imaging contrast for both 2D mapping of unlabeled metal oxide ENMs and 3D mapping of fluorescent nanoparticles. We successfully mapped the 2D distribution of label-free TiO2 and ZnO nanoparticles in cleared skin sections using correlated signals from darkfield, brightfield, and confocal microscopy, as well as micro-spectroscopy. Specifically, hyperspectral microscopy and Raman spectroscopy confirmed the identity of label-free ENMs which we mapped within human skin sections. We also measured the 3D distribution of fluorescently labeled Ag nanoparticles in cleared skin biopsies with wounded epidermal layers using light sheet fluorescence microscopy. Overall, this study explores a novel strategy for quantitatively mapping ENM distributions in cleared ex vivo human skin tissue models using multiple imaging modalities. By improving the imaging contrast, we present label-free 2D ENM tracking and 3D ENM mapping as promising capabilities for nanotoxicology investigations.

Human brain microvascular endothelial cell pairs model tissue-level blood-brain barrier function.

The blood-brain barrier plays a critical role in delivering oxygen and nutrients to the brain while preventing the transport of neurotoxins. Predicting the ability of potential therapeutics and neurotoxicants to modulate brain barrier function remains a challenge due to limited spatial resolution and geometric constraints offered by existing in vitro models. Using soft lithography to control the shape of microvascular tissues, we predicted blood-brain barrier permeability states based on structural changes in human brain endothelial cells. We quantified morphological differences in nuclear, junction, and cytoskeletal proteins that influence, or indicate, barrier permeability. We established a correlation between brain endothelial cell pair structure and permeability by treating cell pairs and tissues with known cytoskeleton-modulating agents, including a Rho activator, a Rho inhibitor, and a cyclic adenosine monophosphate analog. Using this approach, we found that high-permeability cell pairs showed nuclear elongation, loss of junction proteins, and increased actin stress fiber formation, which were indicative of increased contractility. We measured traction forces generated by high- and low-permeability pairs, finding that higher stress at the intercellular junction contributes to barrier leakiness. We further tested the applicability of this platform to predict modulations in brain endothelial permeability by exposing cell pairs to engineered nanomaterials, including gold, silver-silica, and cerium oxide nanoparticles, thereby uncovering new insights into the mechanism of nanoparticle-mediated barrier disruption. Overall, we confirm the utility of this platform to assess the multiscale impact of pharmacological agents or environmental toxicants on blood-brain barrier integrity.

Inhibition of mTOR Signaling Enhances Maturation of Cardiomyocytes Derived From Human-Induced Pluripotent Stem Cells via p53-Induced Quiescence.

BACKGROUND: Current differentiation protocols to produce cardiomyocytes from human induced pluripotent stem cells (iPSCs) are capable of generating highly pure cardiomyocyte populations as determined by expression of cardiac troponin T. However, these cardiomyocytes remain immature, more closely resembling the fetal state, with a lower maximum contractile force, slower upstroke velocity, and immature mitochondrial function compared with adult cardiomyocytes. Immaturity of iPSC-derived cardiomyocytes may be a significant barrier to clinical translation of cardiomyocyte cell therapies for heart disease. During development, cardiomyocytes undergo a shift from a proliferative state in the fetus to a more mature but quiescent state after birth. The mechanistic target of rapamycin (mTOR)-signaling pathway plays a key role in nutrient sensing and growth. We hypothesized that transient inhibition of the mTOR-signaling pathway could lead cardiomyocytes to a quiescent state and enhance cardiomyocyte maturation. METHODS: Cardiomyocytes were differentiated from 3 human iPSC lines using small molecules to modulate the Wnt pathway. Torin1 (0 to 200 nmol/L) was used to inhibit the mTOR pathway at various time points. We quantified contractile, metabolic, and electrophysiological properties of matured iPSC-derived cardiomyocytes. We utilized the small molecule inhibitor, pifithrin-α, to inhibit p53 signaling, and nutlin-3a, a small molecule inhibitor of MDM2 (mouse double minute 2 homolog) to upregulate and increase activation of p53. RESULTS: Torin1 (200 nmol/L) increased the percentage of quiescent cells (G0 phase) from 24% to 48% compared with vehicle control (P<0.05). Torin1 significantly increased expression of selected sarcomere proteins (including TNNI3 [troponin I, cardiac muscle]) and ion channels (including Kir2.1) in a dose-dependent manner when Torin1 was initiated after onset of cardiomyocyte beating. Torin1-treated cells had an increased relative maximum force of contraction, increased maximum oxygen consumption rate, decreased peak rise time, and increased downstroke velocity. Torin1 treatment increased protein expression of p53, and these effects were inhibited by pifithrin-α. In contrast, nutlin-3a independently upregulated p53, led to an increase in TNNI3 expression and worked synergistically with Torin1 to further increase expression of both p53 and TNNI3. CONCLUSIONS: Transient treatment of human iPSC-derived cardiomyocytes with Torin1 shifts cells to a quiescent state and enhances cardiomyocyte maturity.

Muscle tissue engineering in fibrous gelatin: implications for meat analogs.

Bioprocessing applications that derive meat products from animal cell cultures require food-safe culture substrates that support volumetric expansion and maturation of adherent muscle cells. Here we demonstrate scalable production of microfibrous gelatin that supports cultured adherent muscle cells derived from cow and rabbit. As gelatin is a natural component of meat, resulting from collagen denaturation during processing and cooking, our extruded gelatin microfibers recapitulated structural and biochemical features of natural muscle tissues. Using immersion rotary jet spinning, a dry-jet wet-spinning process, we produced gelatin fibers at high rates (~ 100 g/h, dry weight) and, depending on process conditions, we tuned fiber diameters between ~ 1.3 ± 0.1 µm (mean ± SEM) and 8.7 ± 1.4 µm (mean ± SEM), which are comparable to natural collagen fibers. To inhibit fiber degradation during cell culture, we crosslinked them either chemically or by co-spinning gelatin with a microbial crosslinking enzyme. To produce meat analogs, we cultured bovine aortic smooth muscle cells and rabbit skeletal muscle myoblasts in gelatin fiber scaffolds, then used immunohistochemical staining to verify that both cell types attached to gelatin fibers and proliferated in scaffold volumes. Short-length gelatin fibers promoted cell aggregation, whereas long fibers promoted aligned muscle tissue formation. Histology, scanning electron microscopy, and mechanical testing demonstrated that cultured muscle lacked the mature contractile architecture observed in natural muscle but recapitulated some of the structural and mechanical features measured in meat products.

Quantifying the effects of engineered nanomaterials on endothelial cell architecture and vascular barrier integrity using a cell pair model.

Engineered nanomaterials (ENMs) are increasingly used in consumer products due to their unique physicochemical properties, but the specific hazards they pose to the structural and functional integrity of endothelial barriers remain elusive. When assessing the effects of ENMs on vascular barrier function, endothelial cell monolayers are commonly used as in vitro models. Monolayer models, however, do not offer a granular understanding of how the structure-function relationships between endothelial cells and tissues are disrupted due to ENM exposure. To address this issue, we developed a micropatterned endothelial cell pair model to quantitatively evaluate the effects of 10 ENMs (8 metal/metal oxides and 2 organic ENMs) on multiple cellular parameters and determine how these parameters correlate to changes in vascular barrier function. This minimalistic approach showed concerted changes in endothelial cell morphology, intercellular junction formation, and cytoskeletal organization due to ENM exposure, which were then quantified and compared to unexposed pairs using a "similarity scoring" method. Using the cell pair model, this study revealed dose-dependent changes in actin organization and adherens junction formation following exposure to representative ENMs (Ag, TiO2 and cellulose nanocrystals), which exhibited trends that correlate with changes in tissue permeability measured using an endothelial monolayer assay. Together, these results demonstrate that we can quantitatively evaluate changes in endothelial architecture emergent from nucleo-cytoskeletal network remodeling using micropatterned cell pairs. The endothelial pair model therefore presents potential applicability as a standardized assay for systematically screening ENMs and other test agents for their cellular-level structural effects on vascular barriers.

Synchronized stimulation and continuous insulin sensing in a microfluidic human Islet on a Chip designed for scalable manufacturing.

Pancreatic ß cell function is compromised in diabetes and is typically assessed by measuring insulin secretion during glucose stimulation. Traditionally, measurement of glucose-stimulated insulin secretion involves manual liquid handling, heterogeneous stimulus delivery, and enzyme-linked immunosorbent assays that require large numbers of islets and processing time. Though microfluidic devices have been developed to address some of these limitations, traditional methods for islet testing remain the most common due to the learning curve for adopting microfluidic devices and the incompatibility of most device materials with large-scale manufacturing. We designed and built a thermoplastic, microfluidic-based Islet on a Chip compatible with commercial fabrication methods, that automates islet loading, stimulation, and insulin sensing. Inspired by the perfusion of native islets by designated arterioles and capillaries, the chip delivers synchronized glucose pulses to islets positioned in parallel channels. By flowing suspensions of human cadaveric islets onto the chip, we confirmed automatic capture of islets. Fluorescent glucose tracking demonstrated that stimulus delivery was synchronized within a two-minute window independent of the presence or size of captured islets. Insulin secretion was continuously sensed by an automated, on-chip immunoassay and quantified by fluorescence anisotropy. By integrating scalable manufacturing materials, on-line, continuous insulin measurement, and precise spatiotemporal stimulation into an easy-to-use design, the Islet on a Chip should accelerate efforts to study and develop effective treatments for diabetes.

Scatter Enhanced Phase Contrast Microscopy for Discriminating Mechanisms of Active Nanoparticle Transport in Living Cells.

Understanding the uptake and transport dynamics of engineered nanomaterials (ENMs) by mammalian cells is an important step in designing next-generation drug delivery systems. However, to track these materials and their cellular interactions, current studies often depend on surface-bound fluorescent labels, which have the potential to alter native cellular recognition events. As a result, there is still a need to develop methods capable of monitoring ENM-cell interactions independent of surface modification. Addressing these concerns, here we show how scatter enhanced phase contrast (SEPC) microscopy can be extended to work as a generalized label-free approach for monitoring nanoparticle uptake and transport dynamics. To determine which materials can be studied using SEPC, we turn to Lorenz-Mie theory, which predicts that individual particles down to ∼35 nm can be observed. We confirm this experimentally, demonstrating that SEPC works for a variety of metal and metal oxides, including Au, Ag, TiO2, CeO2, Al2O3, and Fe2O3 nanoparticles. We then demonstrate that SEPC microscopy can be used in a quantitative, time-dependent fashion to discriminate between distinct modes of active cellular transport, including intracellular transport and membrane-assisted transport. Finally, we combine this technique with microcontact printing to normalize transport dynamics across multiple cells, allowing for a careful study of ensemble TiO2 nanoparticle uptake. This revealed three distinct regions of particle transport across the cell, indicating that membrane dynamics play an important role in regulating particle flow. By avoiding fluorescent labels, SEPC allows for a rational exploration of the surface properties of nanomaterials in their native state and their role in endocytosis and cellular transport.

Mussel-inspired 3D fiber scaffolds for heart-on-a-chip toxicity studies of engineered nanomaterials.

Due to the unique physicochemical properties exhibited by materials with nanoscale dimensions, there is currently a continuous increase in the number of engineered nanomaterials (ENMs) used in consumer goods. However, several reports associate ENM exposure to negative health outcomes such as cardiovascular diseases. Therefore, understanding the pathological consequences of ENM exposure represents an important challenge, requiring model systems that can provide mechanistic insights across different levels of ENM-based toxicity. To achieve this, we developed a mussel-inspired 3D microphysiological system (MPS) to measure cardiac contractility in the presence of ENMs. While multiple cardiac MPS have been reported as alternatives to in vivo testing, most systems only partially recapitulate the native extracellular matrix (ECM) structure. Here, we show how adhesive and aligned polydopamine (PDA)/polycaprolactone (PCL) nanofiber can be used to emulate the 3D native ECM environment of the myocardium. Such nanofiber scaffolds can support the formation of anisotropic and contractile muscular tissues. By integrating these fibers in a cardiac MPS, we assessed the effects of TiO2 and Ag nanoparticles on the contractile function of cardiac tissues. We found that these ENMs decrease the contractile function of cardiac tissues through structural damage to tissue architecture. Furthermore, the MPS with embedded sensors herein presents a way to non-invasively monitor the effects of ENM on cardiac tissue contractility at different time points. These results demonstrate the utility of our MPS as an analytical platform for understanding the functional impacts of ENMs while providing a biomimetic microenvironment to in vitro cardiac tissue samples. Graphical Abstract Heart-on-a-chip integrated with mussel-inspired fiber scaffolds for a high-throughput toxicological assessment of engineered nanomaterials.

Nongenetic Optical Methods for Measuring and Modulating Neuronal Response.

The ability to probe and modulate electrical signals sensitively at cellular length scales is a key challenge in the field of electrophysiology. Electrical signals play integral roles in regulating cellular behavior and in controlling biological function. From cardiac arrhythmias to neurodegenerative disorders, maladaptive phenotypes in electrophysiology can result in serious and potentially deadly medical conditions. Understanding how to monitor and to control these behaviors precisely and noninvasively represents an important step in developing next-generation therapeutic devices. As we develop a deeper understanding of neural network formation, electrophysiology has the potential to offer fundamental insights into the inner working of the brain. In this Perspective, we explore traditional methods for examining neural function, discuss recent genetic advances in electrophysiology, and then focus on the latest innovations in optical sensing and stimulation of action potentials in neurons. We emphasize nongenetic optical methods, as these provide high spatiotemporal resolution and can be achieved with minimal invasiveness.