Proteomics of novel induced pluripotent stem cell-derived vascular endothelial cells reveal extensive similarity with an immortalized human endothelial cell line.

The vascular endothelium constitutes the inner lining of the blood vessel, and malfunction and injuries of the endothelium can cause cardiovascular diseases as well as other diseases including stroke, tumor growth, and chronic kidney failure. Generation of effective sources to replace injured endothelial cells (ECs) could have significant clinical impact, and somatic cell sources like peripheral or cord blood cannot credibly supply enough endothelial cell progenitors for multitude of treatments. Pluripotent stem cells are a promising source for a reliable EC supply, which have the potential to restore tissue function and treat vascular diseases. We have developed methods to differentiate induced pluripotent stem cells (iPSCs) efficiently and robustly across multiple iPSC lines into nontissue-specific pan vascular ECs (iECs) with high purity. These iECs present with canonical endothelial cell markers and exhibit measures of endothelial cell functionality with the uptake of Dil fluorescent dye-labeled acetylated low-density lipoprotein (Dil-Ac-LDL) and tube formation. Using proteomic analysis, we revealed that the iECs are more proteomically similar to established human umbilical vein ECs (HUVECs) than to iPSCs. Posttranslational modifications (PTMs) were most shared between HUVECs and iECs, and potential targets for increasing the proteomic similarity of iECs to HUVECs were identified. Here we demonstrate an efficient robust method to differentiate iPSCs into functional ECs, and for the first time provide a comprehensive protein expression profile of iECs, which indicates their similarities with a widely used immortalized HUVECs, allowing for further mechanistic studies of EC development, signaling, and metabolism for future regenerative applications.NEW & NOTEWORTHY We have developed methods to differentiate induced pluripotent stem cells (iPSCs) across multiple iPSC lines into nontissue-specific pan vascular ECs (iECs) and demonstrated the proteomic similarity of these cells to a widely used endothelial cell line (HUVECs). We also identified posttranslational modifications and targets for increasing the proteomic similarity of iECs to HUVECs. In the future, iECs can be used to study EC development, signaling, and metabolism for future regenerative applications.

BIRCH: An Automated Workflow for Evaluation, Correction, and Visualization of Batch Effect in Bottom-Up Mass Spectrometry-Based Proteomics Data.

Recent surges in large-scale mass spectrometry (MS)-based proteomics studies demand a concurrent rise in methods to facilitate reliable and reproducible data analysis. Quantification of proteins in MS analysis can be affected by variations in technical factors such as sample preparation and data acquisition conditions leading to batch effects, which adds to noise in the data set. This may in turn affect the effectiveness of any biological conclusions derived from the data. Here we present Batch-effect Identification, Representation, and Correction of Heterogeneous data (BIRCH), a workflow for analysis and correction of batch effect through an automated, versatile, and easy to use web-based tool with the goal of eliminating technical variation. BIRCH also supports diagnosis of the data to check for the presence of batch effects, feasibility of batch correction, and imputation to deal with missing values in the data set. To illustrate the relevance of the tool, we explore two case studies, including an iPSC-derived cell study and a Covid vaccine study to show different context-specific use cases. Ultimately this tool can be used as an extremely powerful approach for eliminating technical bias while retaining biological bias, toward understanding disease mechanisms and potential therapeutics.

Optical Clearing of Skeletal Muscle Bundles Engineered in 3-D Printed Templates.

Many techniques for engineering and interrogating three-dimensional (3-D) muscle bundles from animal- or patient-derived myoblasts have recently been developed to overcome the limitations of existing in vitro and in vivo model systems. However, many approaches for engineering 3-D muscle bundles rely on specialized and time-consuming techniques, such as photolithography for fabrication and cryosectioning for histology. Cryosectioning also limits visualization to a single plane instead of the entire 3-D structure. To address these challenges, we first implemented a consumer-grade 3-D-printer to rapidly prototype multiple templates for engineering muscle bundles. We then employed our templates to engineer 3D muscle bundles and identify template geometries that promoted bundle survival over three weeks. Subsequently, we implemented tissue clearing, immunostaining, and confocal imaging to acquire z-stacks of intact muscle bundles labelled for myogenic markers. With this approach, we could select the imaging plane on-demand and visualize the intact 3-D structure of bundles. However, tissue clearing did cause some tissue degradation that should be considered. Together, these advances in muscle tissue engineering and imaging will accelerate the use of these 3-D tissue platforms for disease modeling and therapeutic discovery.

Mitochondrial architecture in cardiac myocytes depends on cell shape and matrix rigidity.

Contraction of cardiac myocytes depends on energy generated by the mitochondria. During cardiac development and disease, the structure and function of the mitochondrial network in cardiac myocytes is known to remodel in concert with many other factors, including changes in nutrient availability, hemodynamic load, extracellular matrix (ECM) rigidity, cell shape, and maturation of other intracellular structures. However, the independent role of each of these factors on mitochondrial network architecture is poorly understood. In this study, we tested the hypothesis that cell aspect ratio (AR) and ECM rigidity regulate the architecture of the mitochondrial network in cardiac myocytes. To do this, we spin-coated glass coverslips with a soft, moderate, or stiff polymer. Next, we microcontact printed cell-sized rectangles of fibronectin with AR matching cardiac myocytes at various developmental or disease states onto the polymer surface. We then cultured neonatal rat ventricular myocytes on the patterned surfaces and used confocal microscopy and image processing techniques to quantify sarcomeric α-actinin volume, nucleus volume, and mitochondrial volume, surface area, and size distribution. On some substrates, α-actinin volume increased with cell AR but was not affected by ECM rigidity. Nucleus volume was mostly uniform across all conditions. In contrast, mitochondrial volume increased with cell AR on all substrates. Furthermore, mitochondrial surface area to volume ratio decreased as AR increased on all substrates. Large mitochondria were also more prevalent in cardiac myocytes with higher AR. For select AR, mitochondria were also smaller as ECM rigidity increased. Collectively, these results suggest that mitochondrial architecture in cardiac myocytes is strongly influenced by cell shape and moderately influenced by ECM rigidity. These data have important implications for understanding the factors that impact metabolic performance during heart development and disease.

Engineering Shape-Controlled Microtissues on Compliant Hydrogels with Tunable Rigidity and Extracellular Matrix Ligands.

In vitro models that recapitulate key aspects of native tissue architecture and the physical microenvironment are emerging systems for modeling development and disease. For example, the myocardium consists of layers of aligned and coupled cardiac myocytes that are interspersed with supporting cells and embedded in a compliant extracellular matrix (ECM). These cell-cell and cell-matrix interactions are known to be important regulators of tissue physiology and pathophysiology. In this protocol, we describe a method for mimicking the alignment, cell-cell interactions, and rigidity of the myocardium by engineering an array of square, aligned cardiac microtissues on polyacrylamide hydrogels. This entails three key methods: (1) fabricating elastomer stamps with a microtissue pattern; (2) preparing polyacrylamide hydrogel culture substrates with tunable elastic moduli; and (3) transferring ECM proteins onto the surface of the hydrogels using microcontact printing. These hydrogels can then be seeded with cardiac myocytes or mixtures of cardiac myocytes and fibroblasts to adjust cell-cell interactions. Overall, this approach is advantageous because shape-controlled microtissues encompass both cell-cell and cell-matrix adhesions in a form factor that is relatively reproducible and scalable. Furthermore, polyacrylamide hydrogels are compatible with the traction force microscopy assay for quantifying contractility, a critical function of the myocardium. Although cardiac microtissues are the example presented in this protocol, the techniques are relatively versatile and could have many applications in modeling other tissue systems.

Regulation of calcium dynamics and propagation velocity by tissue microstructure in engineered strands of cardiac tissue.

Disruptions to cardiac tissue microstructure are common in diseased or injured myocardium and are known substrates for arrhythmias. However, we have a relatively coarse understanding of the relationships between myocardial tissue microstructure, propagation velocity and calcium cycling, due largely to the limitations of conventional experimental tools. To address this, we used microcontact printing to engineer strands of cardiac tissue with eight different widths, quantified several structural and functional parameters and established correlation coefficients. As strand width increased, actin alignment, nuclei density, sarcomere index and cell aspect ratio decreased with unique trends. The propagation velocity of calcium waves decreased and the rise time of calcium transients increased with increasing strand width. The decay time constant of calcium transients decreased and then slightly increased with increasing strand width. Based on correlation coefficients, actin alignment was the strongest predictor of propagation velocity and calcium transient rise time. Sarcomere index and cell aspect ratio were also strongly correlated with propagation velocity. Actin alignment, sarcomere index and cell aspect ratio were all weak predictors of the calcium transient decay time constant. We also measured the expression of several genes relevant to propagation and calcium cycling and found higher expression of the genes that encode for connexin 43 (Cx43) and a subunit of L-type calcium channels in thin strands compared to isotropic tissues. Together, these results suggest that thinner strands have higher values of propagation velocity and calcium transient rise time due to a combination of favorable tissue microstructure and enhanced expression of genes for Cx43 and L-type calcium channels. These data are important for defining how microstructural features regulate intercellular and intracellular calcium handling, which is needed to understand mechanisms of propagation in physiological situations and arrhythmogenesis in pathological situations.

Matrix-guided control of mitochondrial function in cardiac myocytes.

In ventricular myocardium, extracellular matrix (ECM) remodeling is a hallmark of physiological and pathological growth, coincident with metabolic rewiring of cardiac myocytes. However, the direct impact of the biochemical and mechanical properties of the ECM on the metabolic function of cardiac myocytes is mostly unknown. Furthermore, understanding the impact of distinct biomaterials on cardiac myocyte metabolism is critical for engineering physiologically-relevant models of healthy and diseased myocardium. For these reasons, we systematically measured morphological and metabolic responses of neonatal rat ventricular myocytes cultured on fibronectin- or gelatin-coated polydimethylsiloxane (PDMS) of three elastic moduli and gelatin hydrogels with four elastic moduli. On all substrates, total protein content, cell morphology, and the ratio of mitochondrial DNA to nuclear DNA were preserved. Cytotoxicity was low on all substrates, although slightly higher on PDMS compared to gelatin hydrogels. We also quantified oxygen consumption rates and extracellular acidification rates using a Seahorse extracellular flux analyzer. Our data indicate that several metrics associated with baseline glycolysis and baseline and maximum mitochondrial function are enhanced when cardiac myocytes are cultured on gelatin hydrogels compared to all PDMS substrates, irrespective of substrate rigidity. These results yield new insights into how mechanical and biochemical cues provided by the ECM impact mitochondrial function in cardiac myocytes. STATEMENT OF SIGNIFICANCE: Cardiac development and disease are associated with remodeling of the extracellular matrix coincident with metabolic rewiring of cardiac myocytes. However, little is known about the direct impact of the biochemical and mechanical properties of the extracellular matrix on the metabolic function of cardiac myocytes. In this study, oxygen consumption rates were measured in neonatal rat ventricular myocytes maintained on several commonly-used biomaterial substrates to reveal new relationships between the extracellular matrix and cardiac myocyte metabolism. Several mitochondrial parameters were enhanced on gelatin hydrogels compared to synthetic PDMS substrates. These data are important for comprehensively understanding matrix-regulation of cardiac myocyte physiology. Additionally, these data should be considered when selecting scaffolds for engineering in vitro cardiac tissue models.

Engineering cardiac microphysiological systems to model pathological extracellular matrix remodeling.

Many cardiovascular diseases are associated with pathological remodeling of the extracellular matrix (ECM) in the myocardium. ECM remodeling is a complex, multifactorial process that often contributes to declines in myocardial function and progression toward heart failure. However, the direct effects of the many forms of ECM remodeling on myocardial cell and tissue function remain elusive, in part because conventional model systems used to investigate these relationships lack robust experimental control over the ECM. To address these shortcomings, microphysiological systems are now being developed and implemented to establish direct relationships between distinct features in the ECM and myocardial function with unprecedented control and resolution in vitro. In this review, we will first highlight the most prominent characteristics of ECM remodeling in cardiovascular disease and describe how these features can be mimicked with synthetic and natural biomaterials that offer independent control over multiple ECM-related parameters, such as rigidity and composition. We will then detail innovative microfabrication techniques that enable precise regulation of cellular architecture in two and three dimensions. We will also describe new approaches for quantifying multiple aspects of myocardial function in vitro, such as contractility, action potential propagation, and metabolism. Together, these collective technologies implemented as cardiac microphysiological systems will continue to uncover important relationships between pathological ECM remodeling and myocardial cell and tissue function, leading to new fundamental insights into cardiovascular disease, improved human disease models, and novel therapeutic approaches.

Microenvironmental Modulation of Calcium Wave Propagation Velocity in Engineered Cardiac Tissues.

INTRODUCTION: In the myocardium, rapid propagation of action potentials and subsequent calcium waves is critical for synchronizing the contraction of cardiac myocytes and maximizing cardiac output. In many pathological settings, diverse remodeling of the tissue microenvironment is correlated with arrhythmias and decreased cardiac output, but the precise impact of tissue remodeling on propagation is not completely understood. Our objective was to delineate how multiple features within the cardiac tissue microenvironment modulate propagation velocity. METHODS: To recapitulate diverse myocardial tissue microenvironments, we engineered substrates with tunable elasticity, patterning, composition, and topography using two formulations of polydimethylsiloxane (PDMS) micropatterned with fibronectin and gelatin hydrogels with flat or micromolded features. We cultured neonatal rat ventricular myocytes on these substrates and quantified cell density, tissue alignment, and cell shape. We used a fluorescent calcium indicator, high-speed microscopy, and newly-developed analysis software to record and quantify calcium wave propagation velocity (CPV). RESULTS: For all substrates, tissue alignment and cell aspect ratio were higher in aligned compared to isotropic tissues. Isotropic CPV and longitudinal CPV were similar across conditions, but transverse CPV was lower on micromolded gelatin hydrogels compared to micropatterned soft and stiff PDMS. In aligned tissues, the anisotropy ratio of CPV (longitudinal CPV/transverse CPV) was lower on micropatterned soft PDMS compared to micropatterned stiff PDMS and micromolded gelatin hydrogels. CONCLUSION: Propagation velocity in engineered cardiac tissues is sensitive to features in the tissue microenvironment, such as alignment, matrix elasticity, and matrix topography, which may underlie arrhythmias in conditions with pathological tissue remodeling.

Engineering micromyocardium to delineate cellular and extracellular regulation of myocardial tissue contractility.

Cardiovascular diseases are a leading cause of death, in part due to limitations of existing models of the myocardium. Myocardium consists of aligned, contractile cardiac myocytes interspersed with fibroblasts that synthesize extracellular matrix (ECM). The cellular demographics and biochemical and mechanical properties of the ECM remodel in many different cardiac diseases. However, the impact of diverse cellular and extracellular remodeling on the contractile output of the myocardium are poorly understood. To address this, we micropatterned 13 kPa and 90 kPa polyacrylamide gels with aligned squares of fibronectin (FN) or laminin (LN). We seeded gels with two concentrations of primary neonatal rat ventricular myocytes, which naturally contain fibroblasts. Cells assembled into aligned "µMyocardia" with fibroblast : myocyte ratios dependent on initial seeding concentration. Using traction force microscopy (TFM), we found that the peak systolic longitudinal cross-sectional force was similar across conditions, but the peak systolic work was significantly lower on 90 kPa gels. This indicates that ECM elasticity dominates over ECM ligand and cell demographics in regulating contractile output. Because our platform provides independent control over cell-cell and cell-matrix interactions, it has many applications for cardiac disease modeling.

Mitochondrial function in engineered cardiac tissues is regulated by extracellular matrix elasticity and tissue alignment.

Mitochondria in cardiac myocytes are critical for generating ATP to meet the high metabolic demands associated with sarcomere shortening. Distinct remodeling of mitochondrial structure and function occur in cardiac myocytes in both developmental and pathological settings. However, the factors that underlie these changes are poorly understood. Because remodeling of tissue architecture and extracellular matrix (ECM) elasticity are also hallmarks of ventricular development and disease, we hypothesize that these environmental factors regulate mitochondrial function in cardiac myocytes. To test this, we developed a new procedure to transfer tunable polydimethylsiloxane disks microcontact-printed with fibronectin into cell culture microplates. We cultured Sprague-Dawley neonatal rat ventricular myocytes within the wells, which consistently formed tissues following the printed fibronectin, and measured oxygen consumption rate using a Seahorse extracellular flux analyzer. Our data indicate that parameters associated with baseline metabolism are predominantly regulated by ECM elasticity, whereas the ability of tissues to adapt to metabolic stress is regulated by both ECM elasticity and tissue alignment. Furthermore, bioenergetic health index, which reflects both the positive and negative aspects of oxygen consumption, was highest in aligned tissues on the most rigid substrate, suggesting that overall mitochondrial function is regulated by both ECM elasticity and tissue alignment. Our results demonstrate that mitochondrial function is regulated by both ECM elasticity and myofibril architecture in cardiac myocytes. This provides novel insight into how extracellular cues impact mitochondrial function in the context of cardiac development and disease.NEW & NOTEWORTHY A new methodology has been developed to measure O2 consumption rates in engineered cardiac tissues with independent control over tissue alignment and matrix elasticity. This led to the findings that matrix elasticity regulates basal mitochondrial function, whereas both matrix elasticity and tissue alignment regulate mitochondrial stress responses.