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The use of biochemical signaling to derive smooth muscle cells (SMCs) from mesenchymal stem cells (MSCs) has been explored, but the induction of a fully functional SMC phenotype remains to be a major challenge. Cell morphology has been shown to regulate MSC differentiation into various lineages, including SMCs. We engineered substrates with microgrooves to induce cell elongation to study the mechanism underlying the MSC shape modulation in SMC differentiation. In comparison to those on flat substrates, MSCs cultured on engineered substrates were elongated with increased aspect ratios for both cell body and nucleus, as well as augmented cytoskeletal tensions. Biochemical studies indicated that the microgroove-elongated cells expressed significantly higher levels of SMC markers. MicroRNA analyses showed that up-regulation of miR-145 and the consequent repression of KLF4 in these elongated cells promoted MSC-to-SMC differentiation. Rho/ROCK inhibitions, which impair cytoskeletal tension, attenuated cell and nuclear elongations and disrupted the miR-145/KLF4 regulation for SMC differentiation. Furthermore, cell traction force measurements showed that miR-145 is essential for the functional contractility in the microgroove-induced SMC differentiation. Collectively, our findings demonstrate that, through a Rho-ROCK/miR-145/KLF4 pathway, the elongated cell shape serves as a decisive geometric cue to direct MSC differentiation into functional SMCs.

Leukocyte transmigration across vessel walls is a critical step in the innate immune response. Upon their activation and firm adhesion to vascular endothelial cells (VECs), leukocytes preferentially extravasate across junctional gaps in the endothelial monolayer (paracellular diapedesis). It has been hypothesized that VECs facilitate paracellular diapedesis by opening their cell-cell junctions in response to the presence of an adhering leukocyte. However, it is unclear how leukocytes interact mechanically with VECs to open the VEC junctions and migrate across the endothelium. In this study, we measured the spatial and temporal evolution of the 3D traction stresses generated by the leukocytes and VECs to elucidate the sequence of mechanical events involved in paracellular diapedesis. Our measurements suggest that the contractile stresses exerted by the leukocytes and the VECs can separately perturb the junctional tensions of VECs to result in the opening of gaps before the initiation of leukocyte transmigration. Decoupling the stresses exerted by the transmigrating leukocytes and the VECs reveals that the leukocytes actively contract the VECs to open a junctional gap and then push themselves across the gap by generating strong stresses that push into the matrix. In addition, we found that diapedesis is facilitated when the tension fluctuations in the VEC monolayer were increased by proinflammatory thrombin treatment. Our findings demonstrate that diapedesis can be mechanically regulated by the transmigrating leukocytes and by proinflammatory signals that increase VEC contractility.

Cells employing amoeboid motility exhibit repetitive cycles of rapid expansion and contraction and apply coordinated traction forces to their environment. Although aspects of this process are well studied, it is unclear how the cell controls the coordination of cell length changes with adhesion to the surface. Here, we develop a simple model to mechanistically explain the emergence of periodic changes in length and spatiotemporal dynamics of traction forces measured in chemotaxing unicellular amoeba, Dictyostelium discoideum. In contrast to the biochemical mechanisms that have been implicated in the coordination of some cellular processes, we show that many features of amoeboid locomotion emerge from a simple mechanochemical model. The mechanism for interaction with the environment in Dictyostelium is unknown and thus, we explore different cell-environment interaction models to reveal that mechanosensitive adhesions are necessary to reproduce the spatiotemporal adhesion patterns. In this modeling framework, we find that the other motility modes, such as smooth gliding, arise naturally with variations in the physical properties of the surface. Thus, our work highlights the prominent role of biomechanics in determining the emergent features of amoeboid locomotion.

Tyrosine kinase inhibitors (TKIs), despite their efficacy as anticancer therapeutics, are associated with cardiovascular side effects ranging from induced arrhythmias to heart failure. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), generated from 11 healthy individuals and 2 patients receiving cancer treatment, to screen U.S. Food and Drug Administration-approved TKIs for cardiotoxicities by measuring alterations in cardiomyocyte viability, contractility, electrophysiology, calcium handling, and signaling. With these data, we generated a "cardiac safety index" to reflect the cardiotoxicities of existing TKIs. TKIs with low cardiac safety indices exhibit cardiotoxicity in patients. We also derived endothelial cells (hiPSC-ECs) and cardiac fibroblasts (hiPSC-CFs) to examine cell type-specific cardiotoxicities. Using high-throughput screening, we determined that vascular endothelial growth factor receptor 2 (VEGFR2)/platelet-derived growth factor receptor (PDGFR)-inhibiting TKIs caused cardiotoxicity in hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs. With phosphoprotein analysis, we determined that VEGFR2/PDGFR-inhibiting TKIs led to a compensatory increase in cardioprotective insulin and insulin-like growth factor (IGF) signaling in hiPSC-CMs. Up-regulating cardioprotective signaling with exogenous insulin or IGF1 improved hiPSC-CM viability during cotreatment with cardiotoxic VEGFR2/PDGFR-inhibiting TKIs. Thus, hiPSC-CMs can be used to screen for cardiovascular toxicities associated with anticancer TKIs, and the results correlate with clinical phenotypes. This approach provides unexpected insights, as illustrated by our finding that toxicity can be alleviated via cardioprotective insulin/IGF signaling.

Cellular traction force microscopy (TFM) requires knowledge of the mechanical properties of the substratum where the cells adhere to calculate cell-generated forces from measurements of substratum deformation. Polymer-based hydrogels are broadly used for TFM due to their linearly elastic behavior in the range of measured deformations. However, the calculated stresses, particularly their spatial patterns, can be highly sensitive to the substratum's Poisson's ratio. We present two-layer elastographic TFM (2LETFM), a method that allows for simultaneously measuring the Poisson's ratio of the substratum while also determining the cell-generated forces. The new method exploits the analytical solution of the elastostatic equation and deformation measurements from two layers of the substratum. We perform an in silico analysis of 2LETFM concluding that this technique is robust with respect to TFM experimental parameters, and remains accurate even for noisy measurement data. We also provide experimental proof of principle of 2LETFM by simultaneously measuring the stresses exerted by migrating Physarum amoeboae on the surface of polyacrylamide substrata, and the Poisson's ratio of the substrata. The 2LETFM method could be generalized to concurrently determine the mechanical properties and cell-generated forces in more physiologically relevant extracellular environments, opening new possibilities to study cell-matrix interactions.

Spotted fever group (SFG) rickettsiae are human pathogens that infect cells in the vasculature. They disseminate through host tissues by a process of cell-to-cell spread that involves protrusion formation, engulfment, and vacuolar escape. Other bacterial pathogens rely on actin-based motility to provide a physical force for spread. Here, we show that SFG species Rickettsia parkeri typically lack actin tails during spread and instead manipulate host intercellular tension and mechanotransduction to promote spread. Using transposon mutagenesis, we identified surface cell antigen 4 (Sca4) as a secreted effector of spread that specifically promotes protrusion engulfment. Sca4 interacts with the cell-adhesion protein vinculin and blocks association with vinculin's binding partner, α-catenin. Using traction and monolayer stress microscopy, we show that Sca4 reduces vinculin-dependent mechanotransduction at cell-cell junctions. Our results suggest that Sca4 relieves intercellular tension to promote protrusion engulfment, which represents a distinctive strategy for manipulating cytoskeletal force generation to enable spread.

Cellular biophysical properties are novel biomarkers of cell phenotypes which may reflect the status of differentiating stem cells. Accurate characterizations of cellular biophysical properties, in conjunction with the corresponding biochemical properties could help to distinguish stem cells from primary cells, cancer cells, and differentiated cells. However, the correlated evolution of these properties in the course of directed stem cells differentiation has not been well characterized. In this study, we applied video particle tracking microrheology (VPTM) to measure intracellular viscoelasticity of differentiating human mesenchymal stromal/stem cells (hMSCs). Our results showed that osteogenesis not only increased both elastic and viscous moduli, but also converted the intracellular viscoelasticity of differentiating hMSCs from viscous-like to elastic-like. In contrast, adipogenesis decreased both elastic and viscous moduli while hMSCs remained viscous-like during the differentiation. In conjunction with bio- chemical and physical parameters, such as gene expression profiles, cell morphology, and cytoskeleton arrangement, we demonstrated that VPTM is a unique approach to quantify, with high data throughput, the maturation level of differentiating hMSCs and to anticipate their fate decisions. This approach is well suited for time-lapsed study of the mechanobiology of differentiating stem cells especially in three dimensional physico-chemical biomimetic environments including porous scaffolds.

Streams of migratory cells are initiated by the formation of tandem pairs of cells connected head to tail to which other cells subsequently adhere. The mechanisms regulating the transition from single to streaming cell migration remain elusive, although several molecules have been suggested to be involved. In this work, we investigate the mechanics of the locomotion ofDictyosteliumtandem pairs by analyzing the spatiotemporal evolution of their traction adhesions (TAs). We find that in migrating wild-type tandem pairs, each cell exerts traction forces on stationary sites (∼80% of the time), and the trailing cell reuses the location of the TAs of the leading cell. Both leading and trailing cells form contractile dipoles and synchronize the formation of new frontal TAs with ∼54-s time delay. Cells not expressing the lectin discoidin I or moving on discoidin I-coated substrata form fewer tandems, but the trailing cell still reuses the locations of the TAs of the leading cell, suggesting that discoidin I is not responsible for a possible chemically driven synchronization process. The migration dynamics of the tandems indicate that their TAs' reuse results from the mechanical synchronization of the leading and trailing cells' protrusions and retractions (motility cycles) aided by the cell-cell adhesions.

This work examines the relationship between spatio-temporal coordination of intracellular flow and traction stress and the speed of amoeboid locomotion of microplasmodia of Physarum polycephalum. We simultaneously perform particle image velocimetry and traction stress microscopy to measure the velocity of cytoplasmic flow and the stresses applied to the substrate by migrating Physarum microamoebae. In parallel, we develop a mathematical model of a motile cell which includes forces from the viscous cytosol, a poro-elastic, contractile cytoskeleton and adhesive interactions with the substrate. Our experiments show that flow and traction stress exhibit back-to-front-directed waves with a distinct phase difference. The model demonstrates that the direction and speed of locomotion are determined by this coordination between contraction, flow and adhesion. Using the model, we identify forms of coordination that generate model predictions consistent with experiments. We demonstrate that this coordination produces near optimal migration speed and is insensitive to heterogeneity in substrate adhesiveness. While it is generally thought that amoeboid motility is robust to changes in extracellular geometry and the nature of extracellular adhesion, our results demonstrate that coordination of adhesive forces is essential to producing robust migration.

Perturbed biomechanical stimuli are thought to be critical for the pathogenesis of a number of congenital heart defects, including Hypoplastic Left Heart Syndrome (HLHS). While embryonic cardiomyocytes experience biomechanical stretch every heart beat, their molecular responses to biomechanical stimuli during heart development are poorly understood. We hypothesized that biomechanical stimuli activate specific signaling pathways that impact proliferation, gene expression and myocyte contraction. The objective of this study was to expose embryonic mouse cardiomyocytes (EMCM) to cyclic stretch and examine key molecular and phenotypic responses. Analysis of RNA-Sequencing data demonstrated that gene ontology groups associated with myofibril and cardiac development were significantly modulated. Stretch increased EMCM proliferation, size, cardiac gene expression, and myofibril protein levels. Stretch also repressed several components belonging to the Transforming Growth Factor-β (Tgf-β) signaling pathway. EMCMs undergoing cyclic stretch had decreased Tgf-β expression, protein levels, and signaling. Furthermore, treatment of EMCMs with a Tgf-β inhibitor resulted in increased EMCM size. Functionally, Tgf-β signaling repressed EMCM proliferation and contractile function, as assayed via dynamic monolayer force microscopy (DMFM). Taken together, these data support the hypothesis that biomechanical stimuli play a vital role in normal cardiac development and for cardiac pathology, including HLHS.

Human mesenchymal stem cell (hMSC) proliferation, migration, and differentiation have all been linked to extracellular matrix stiffness, yet the signaling pathway(s) that are necessary for mechanotransduction remain unproven. Vinculin has been implicated as a mechanosensor in vitro, but here we demonstrate its ability to also regulate stem cell behavior, including hMSC differentiation. RNA interference-mediated vinculin knockdown significantly decreased stiffness-induced MyoD, a muscle transcription factor, but not Runx2, an osteoblast transcription factor, and impaired stiffness-mediated migration. A kinase binding accessibility screen predicted a cryptic MAPK1 signaling site in vinculin which could regulate these behaviors. Indeed, reintroduction of vinculin domains into knocked down cells indicated that MAPK1 binding site-containing vinculin constructs were necessary for hMSC expression of MyoD. Vinculin knockdown does not appear to interfere with focal adhesion assembly, significantly alter adhesive properties, or diminish cell traction force generation, indicating that its knockdown only adversely affected MAPK1 signaling. These data provide some of the first evidence that a force-sensitive adhesion protein can regulate stem cell fate.

Microscale and nanoscale surface topography changes can influence cell functions, including morphology. Although in vitro responses to static topography are novel, cells in vivo constantly remodel topography. To better understand how cells respond to changes in topography over time, we developed a soft polyacrylamide hydrogel with magnetic nickel microwires randomly oriented in the surface of the material. Varying the magnetic field around the microwires reversibly induced their alignment with the direction of the field, causing the smooth hydrogel surface to develop small wrinkles; changes in surface roughness, ΔRRMS , ranged from 0.05 to 0.70 μm and could be oscillated without hydrogel creep. Vascular smooth muscle cell morphology was assessed when exposed to acute and dynamic topography changes. Area and shape changes occurred when an acute topographical change was imposed for substrates exceeding roughness of 0.2 μm, but longer-term oscillating topography did not produce significant changes in morphology irrespective of wire stiffness. These data imply that cells may be able to use topography changes to transmit signals as they respond immediately to changes in roughness.

Atrial fibrillation (AF) is an increasingly prevalent and significant worldwide health problem. Manifested as an irregular atrial electrophysiological activation, it is associated with many serious health complications. AF affects the biomechanical function of the heart as contraction follows the electrical activation, subsequently leading to reduced blood flow. The underlying mechanisms behind AF are not fully understood, but it is known that AF is highly correlated with the presence of atrial fibrosis, and with a manifold increase in risk of stroke. In this review, we focus on biomechanical aspects in atrial fibrillation, current and emerging use of clinical images, and personalized computational models. We also discuss how these can be used to provide patient-specific care. Understanding the connection betweenatrial fibrillation and atrial remodeling might lead to valuable understanding of stroke and heart failure pathophysiology. Established and emerging imaging modalities can bring us closer to this understanding, especially with continued advancements in processing accuracy, reproducibility, and clinical relevance of the associated technologies. Computational models of cardiac electromechanics can be used to glean additional insights on the roles of AF and remodeling in heart function. People with atrial fibrillation (AF) experience a fast, chaotic heartbeat. AF greatly increases the risk of stroke. The hearts of AF patients often have an accumulation of fibrous tissue (fibrosis). Fibrosis patterns can be detected via medical imaging scans, like MRI. These images can be used to build patient-specific digital representations. These models can be used to explore how fibrosis might cause AF, stroke, and other health risks. Insights from imaging and modeling are becoming more and more useful as tools for personalizing AF treatment. [ABSTRACT FROM AUTHOR]