Combinatorial extracellular matrix cues with mechanical strain induce differential effects on myogenesis in vitro.

Skeletal muscle regeneration remains a clinical unmet need for volumetric muscle loss and atrophy where muscle function cannot be restored to prior capacity. Current experimental approaches do not account for the complex microenvironmental factors that modulate myogenesis. In this study we developed a biomimetic tissue chip platform to systematically study the combined effects of the extracellular matrix (ECM) microenvironment and mechanical strain on myogenesis of murine myoblasts. Using stretchable tissue chips composed of collagen I (C), fibronectin (F) and laminin (L), as well as their combinations thereof, we tested the addition of mechanical strain regimens on myogenesis at the transcriptomic and translational levels. Our results show that ECMs have a significant effect on myotube formation in C2C12 murine myoblasts. Under static conditions, laminin substrates induced the longest myotubes, whereas fibronectin produced the widest myotubes. Combinatorial ECMs showed non-intuitive effects on myotube formation. Genome-wide analysis revealed the upregulation in actin cytoskeletal related genes that are suggestive of myogenesis. When mechanical strain was introduced to C + F + L combinatorial ECM substrates in the form of constant or intermittent uniaxial strain at low (5%) and high (15%) levels, we observed synergistic enhancements in myotube width, along with transcriptomic upregulation in myosin heavy chain genes. Together, these studies highlight the complex role of microenvironmental factors such as ECM interactions and strain on myotube formation and the underlying signaling pathways.

Preclinical Drug Testing in Scalable 3D Engineered Muscle Tissues.

Accurately modeling healthy and disease conditions in vitro is vital for the development of new treatment strategies and therapeutics. For cardiac and skeletal muscle diseases, contractile force and kinetics constitute key metrics for assessing muscle function. New and improved methods for generating engineered muscle tissues (EMTs) from induced pluripotent stem cells have made in vitro disease modeling more reliable for contractile tissues; however, reproducibly fabricating tissues from suspended cell cultures and measuring their contractility is challenging. Such techniques are often plagued with high failure rates and require complex instrumentation and customized data analysis routines. A new platform and device that utilizes 3D EMTs in conjunction with a label-free, highly-parallel, and automation-friendly contractility assay circumvent many of these obstacles. The platform enables facile and reproducible fabrication of 3D EMTs using virtually any cell source. Tissue contractility is then measured via an instrument that simultaneously measures 24 tissues without the need for complex software analysis routines. The instrument can reliably measure micronewton changes in force, allowing for dose-dependent compound screening to measure the effect of a drug or therapeutic on contractile output. Engineered tissues made with this device are fully functional, generating twitch and tetanic contractions upon electrical stimulation, and can be analyzed longitudinally in culture over weeks or months. Here, we show data from cardiac muscle EMTs under acute and chronic dosing with known toxicants, including a drug (BMS-986094) that was pulled from clinical trials after patient fatalities due to unanticipated cardiotoxicity. Altered skeletal muscle function in engineered tissues in response to treatment with a myosin inhibitor is also presented. This platform enables the researcher to integrate complex, information-rich bioengineered model systems into their drug discovery workflow with minimal additional training or skills required.

Myoscaffolds reveal laminin scarring is detrimental for stem cell function while sarcospan induces compensatory fibrosis.

We developed an on-slide decellularization approach to generate acellular extracellular matrix (ECM) myoscaffolds that can be repopulated with various cell types to interrogate cell-ECM interactions. Using this platform, we investigated whether fibrotic ECM scarring affected human skeletal muscle progenitor cell (SMPC) functions that are essential for myoregeneration. SMPCs exhibited robust adhesion, motility, and differentiation on healthy muscle-derived myoscaffolds. All SPMC interactions with fibrotic myoscaffolds from dystrophic muscle were severely blunted including reduced motility rate and migration. Furthermore, SMPCs were unable to remodel laminin dense fibrotic scars within diseased myoscaffolds. Proteomics and structural analysis revealed that excessive collagen deposition alone is not pathological, and can be compensatory, as revealed by overexpression of sarcospan and its associated ECM receptors in dystrophic muscle. Our in vivo data also supported that ECM remodeling is important for SMPC engraftment and that fibrotic scars may represent one barrier to efficient cell therapy.

High-throughput, real-time monitoring of engineered skeletal muscle function using magnetic sensing.

Engineered muscle tissues represent powerful tools for examining tissue level contractile properties of skeletal muscle. However, limitations in the throughput associated with standard analysis methods limit their utility for longitudinal study, high throughput drug screens, and disease modeling. Here we present a method for integrating 3D engineered skeletal muscles with a magnetic sensing system to facilitate non-invasive, longitudinal analysis of developing contraction kinetics. Using this platform, we show that engineered skeletal muscle tissues derived from both induced pluripotent stem cell and primary sources undergo improvements in contractile output over time in culture. We demonstrate how magnetic sensing of contractility can be employed for simultaneous assessment of multiple tissues subjected to different doses of known skeletal muscle inotropes as well as the stratification of healthy versus diseased functional profiles in normal and dystrophic muscle cells. Based on these data, this combined culture system and magnet-based contractility platform greatly broadens the potential for 3D engineered skeletal muscle tissues to impact the translation of novel therapies from the lab to the clinic.

Cancer cells become less deformable and more invasive with activation of ß-adrenergic signaling.

Invasion by cancer cells is a crucial step in metastasis. An oversimplified view in the literature is that cancer cells become more deformable as they become more invasive. ß-adrenergic receptor (ßAR) signaling drives invasion and metastasis, but the effects on cell deformability are not known. Here, we show that activation of ß-adrenergic signaling by ßAR agonists reduces the deformability of highly metastatic human breast cancer cells, and that these stiffer cells are more invasive in vitro We find that ßAR activation also reduces the deformability of ovarian, prostate, melanoma and leukemia cells. Mechanistically, we show that ßAR-mediated cell stiffening depends on the actin cytoskeleton and myosin II activity. These changes in cell deformability can be prevented by pharmacological ß-blockade or genetic knockout of the ß2-adrenergic receptor. Our results identify a ß2-adrenergic-Ca2+-actin axis as a new regulator of cell deformability, and suggest that the relationship between cell mechanical properties and invasion might be dependent on context.

Stiffness of pancreatic cancer cells is associated with increased invasive potential.

Metastasis is a fundamentally physical process in which cells are required to deform through narrow gaps as they invade surrounding tissues and transit to distant sites. In many cancers, more invasive cells are more deformable than less invasive cells, but the extent to which mechanical phenotype, or mechanotype, can predict disease aggressiveness in pancreatic ductal adenocarcinoma (PDAC) remains unclear. Here we investigate the invasive potential and mechanical properties of immortalized PDAC cell lines derived from primary tumors and a secondary metastatic site, as well as noncancerous pancreatic ductal cells. To investigate how invasive behavior is associated with cell mechanotype, we flow cells through micron-scale pores using parallel microfiltration and microfluidic deformability cytometry; these results show that the ability of PDAC cells to passively transit through pores is only weakly correlated with their invasive potential. We also measure the Young's modulus of pancreatic ductal cells using atomic force microscopy, which reveals that there is a strong association between cell stiffness and invasive potential in PDAC cells. To determine the molecular origins of the variability in mechanotype across our PDAC cell lines, we analyze RNAseq data for genes that are known to regulate cell mechanotype. Our results show that vimentin, actin, and lamin A are among the most differentially expressed mechanoregulating genes across our panel of PDAC cell lines, as well as a cohort of 38 additional PDAC cell lines. We confirm levels of these proteins across our cell panel using immunoblotting, and find that levels of lamin A increase with both invasive potential and Young's modulus. Taken together, we find that stiffer PDAC cells are more invasive than more compliant cells, which challenges the paradigm that decreased cell stiffness is a hallmark of metastatic potential.

Prolonged Culture of Aligned Skeletal Myotubes on Micromolded Gelatin Hydrogels.

In vitro models of skeletal muscle are critically needed to elucidate disease mechanisms, identify therapeutic targets, and test drugs pre-clinically. However, culturing skeletal muscle has been challenging due to myotube delamination from synthetic culture substrates approximately one week after initiating differentiation from myoblasts. In this study, we successfully maintained aligned skeletal myotubes differentiated from C2C12 mouse skeletal myoblasts for three weeks by utilizing micromolded (µmolded) gelatin hydrogels as culture substrates, which we thoroughly characterized using atomic force microscopy (AFM). Compared to polydimethylsiloxane (PDMS) microcontact printed (µprinted) with fibronectin (FN), cell adhesion on gelatin hydrogel constructs was significantly higher one week and three weeks after initiating differentiation. Delamination from FN-µprinted PDMS precluded robust detection of myotubes. Compared to a softer blend of PDMS µprinted with FN, myogenic index, myotube width, and myotube length on µmolded gelatin hydrogels was similar one week after initiating differentiation. However, three weeks after initiating differentiation, these parameters were significantly higher on µmolded gelatin hydrogels compared to FN-µprinted soft PDMS constructs. Similar results were observed on isotropic versions of each substrate, suggesting that these findings are independent of substrate patterning. Our platform enables novel studies into skeletal muscle development and disease and chronic drug testing in vitro.

Myocyte shape regulates lateral registry of sarcomeres and contractility.

The heart actively remodels architecture in response to various physiological and pathological conditions. Gross structural change of the heart chambers is directly reflected at the cellular level by altering the morphological characteristics of individual cardiomyocytes. However, an understanding of the relationship between cardiomyocyte shape and the contractile function remains unclear. By using in vitro assays to analyze systolic stress of cardiomyocytes with controlled shape, we demonstrated that the characteristic morphological features of cardiomyocytes observed in a variety of pathophysiological conditions are correlated with mechanical performance. We found that cardiomyocyte contractility is optimized at the cell length/width ratio observed in normal hearts, and decreases in cardiomyocytes with morphological characteristics resembling those isolated from failing hearts. Quantitative analysis of sarcomeric architecture revealed that the change of contractility may arise from alteration of myofibrillar structure. Measurements of intracellular calcium in myocytes revealed unique characteristics of calcium metabolism as a function of myocyte shape. Our data suggest that cell shape is critical in determining contractile performance of single cardiomyocytes by regulating the intracellular structure and calcium handling ability.

Cell-to-cell coupling in engineered pairs of rat ventricular cardiomyocytes: relation between Cx43 immunofluorescence and intercellular electrical conductance.

Gap junctions are composed of connexin (Cx) proteins, which mediate intercellular communication. Cx43 is the dominant Cx in ventricular myocardium, and Cx45 is present in trace amounts. Cx43 immunosignal has been associated with cell-to-cell coupling and electrical propagation, but no studies have directly correlated Cx43 immunosignal to electrical cell-to-cell conductance, g(j), in ventricular cardiomyocyte pairs. To assess the correlation between Cx43 immunosignal and g(j), we developed a method to determine both parameters from the same cell pair. Neonatal rat ventricular cardiomyocytes were seeded on micropatterned islands of fibronectin. This allowed formation of cell pairs with reproducible shapes and facilitated tracking of cell pair locations. Moreover, cell spreading was limited by the fibronectin pattern, which allowed us to increase cell height by reducing the surface area of the pattern. Whole cell dual voltage clamp was used to record g(j) of cell pairs after 3-5 days in culture. Fixation of cell pairs before removal of patch electrodes enabled preservation of cell morphology and offline identification of patched pairs. Subsequently, pairs were immunostained, and the volume of junctional Cx43 was quantified using confocal microscopy, image deconvolution, and three-dimensional reconstruction. Our results show a linear correlation between g(j) and Cx43 immunosignal within a range of 8-50 nS.

Self-organization of muscle cell structure and function.

The organization of muscle is the product of functional adaptation over several length scales spanning from the sarcomere to the muscle bundle. One possible strategy for solving this multiscale coupling problem is to physically constrain the muscle cells in microenvironments that potentiate the organization of their intracellular space. We hypothesized that boundary conditions in the extracellular space potentiate the organization of cytoskeletal scaffolds for directed sarcomeregenesis. We developed a quantitative model of how the cytoskeleton of neonatal rat ventricular myocytes organizes with respect to geometric cues in the extracellular matrix. Numerical results and in vitro assays to control myocyte shape indicated that distinct cytoskeletal architectures arise from two temporally-ordered, organizational processes: the interaction between actin fibers, premyofibrils and focal adhesions, as well as cooperative alignment and parallel bundling of nascent myofibrils. Our results suggest that a hierarchy of mechanisms regulate the self-organization of the contractile cytoskeleton and that a positive feedback loop is responsible for initiating the break in symmetry, potentiated by extracellular boundary conditions, is required to polarize the contractile cytoskeleton.

Comparison of scanning ion conductance microscopy with atomic force microscopy for cell imaging.

We present the first direct comparison of scanning ion conductance microscopy (SICM) with atomic force microscopy (AFM) for cell imaging. By imaging the same fibroblast or myoblast cell with both technologies in series, we highlight their advantages and disadvantages with respect to cell imaging. The finite imaging force applied to the sample in AFM imaging results in a coupling of mechanical sample properties into the measured sample topography. For soft samples such as cells this leads to artifacts in the measured topography and to elastic deformation, which we demonstrate by imaging whole fixed cells and cell extensions at high resolution. SICM imaging, on the other hand, has a noncontact character and can provide the true topography of soft samples at a comparable resolution.

Nuclear morphology and deformation in engineered cardiac myocytes and tissues.

Cardiac tissue engineering requires finely-tuned manipulation of the extracellular matrix (ECM) microenvironment to optimize internal myocardial organization. The myocyte nucleus is mechanically connected to the cell membrane via cytoskeletal elements, making it a target for the cellular response to perturbation of the ECM. However, the role of ECM spatial configuration and myocyte shape on nuclear location and morphology is unknown. In this study, printed ECM proteins were used to configure the geometry of cultured neonatal rat ventricular myocytes. Engineered one- and two-dimensional tissue constructs and single myocyte islands were assayed using live fluorescence imaging to examine nuclear position, morphology and motion as a function of the imposed ECM geometry during diastolic relaxation and systolic contraction. Image analysis showed that anisotropic tissue constructs cultured on microfabricated ECM lines possessed a high degree of nuclear alignment similar to that found in vivo; nuclei in isotropic tissues were polymorphic in shape with an apparently random orientation. Nuclear eccentricity was also increased for the anisotropic tissues, suggesting that intracellular forces deform the nucleus as the cell is spatially confined. During systole, nuclei experienced increasing spatial confinement in magnitude and direction of displacement as tissue anisotropy increased, yielding anisotropic deformation. Thus, the nature of nuclear displacement and deformation during systole appears to rely on a combination of the passive myofibril spatial organization and the active stress fields induced by contraction. Such findings have implications in understanding the genomic consequences and functional response of cardiac myocytes to their ECM surroundings under conditions of disease.

Controlling the orientation and synaptic differentiation of myotubes with micropatterned substrates.

Micropatterned poly(dimethylsiloxane) substrates fabricated by soft lithography led to large-scale orientation of myoblasts in culture, thereby controlling the orientation of the myotubes they formed. Fusion occurred on many chemically identical surfaces in which varying structures were arranged in square or hexagonal lattices, but only a subset of patterned surfaces yielded aligned myotubes. Remarkably, on some substrates, large populations of myotubes oriented at a reproducible acute angle to the lattice of patterned features. A simple geometrical model predicts the angle and extent of orientation based on maximizing the contact area between the myoblasts and patterned topographic surfaces. Micropatterned substrates also provided short-range cues that influenced higher-order functions such as the localization of focal adhesions and accumulation of postsynaptic acetylcholine receptors. Our results represent what we believe is a new approach for musculoskeletal tissue engineering, and our model sheds light on mechanisms of myotube alignment in vivo.

Control of myocyte remodeling in vitro with engineered substrates.

Tissue microenvironments can regulate cell behavior by imposing physical restrictions on their geometry and size. An example of these phenomena is cardiac morphogenesis, where morphometric changes in the heart are concurrent with changes in the size, shape, and cytoskeleton of ventricular myocytes. In this study, we asked how myocytes adapt their size, shape, and intracellular architecture when spatially confined in vitro. To answer this question, we used microcontact printing to physically constrain neonatal rat ventricular myocytes on fibronectin islands in culture. The myocytes spread and assumed the shape of the islands and reorganized their cytoskeleton in response to the geometric cues in the extracellular matrix. Cytoskeletal architecture is variable, where myocytes cultured on rectangular islands of lower aspect ratios (length to width ratio) were observed to assemble a multiaxial myofibrillar arrangement; myocytes cultured on rectangles of aspect ratios approaching those observed in vivo had a uniaxial orientation of their myofibrils. Using confocal and atomic force microscopy, we made precise measurements of myocyte volume over a range of cell shapes with approximately equal surface areas. When myocytes are cultured on islands of variable shape but the same surface area, their size is conserved despite the changes in cytoskeletal architecture. Our data suggest that the internal cytoskeletal architecture of the cell is dependent on extracellular boundary conditions while overall cell size is not, suggesting a growth control mechanism independent of the cytoskeleton and cell geometry.

Multidimensional detection and analysis of Ca2+ sparks in cardiac myocytes.

Examining calcium spark morphology and its relationship to the structure of the cardiac myocyte offers a direct means of understanding excitation-contraction coupling mechanisms. Traditional confocal line scanning achieves excellent temporal spark resolution but at the cost of spatial information in the perpendicular dimension. To address this, we developed a methodology to identify and analyze sparks obtained via two-dimensional confocal or charge-coupled device microscopy. The technique consists of nonlinearly subtracting the background fluorescence, thresholding the data on the basis of noise level, and then localizing the spark peaks via a generalized extrema test, while taking care to detect and separate adjacent peaks. In this article, we describe the algorithm, compare its performance to a previously validated spark detection algorithm, and demonstrate it by applying it to both a synthetic replica and an experimental preparation of a two-dimensional isotropic myocyte monolayer exhibiting sparks during a calcium transient. We find that our multidimensional algorithm provides better sensitivity than the conventional method under conditions of temporally heterogeneous background fluorescence, and the inclusion of peak segmentation reduces false negative rates when spark density is high. Our algorithm is robust and can be effectively used with different imaging modalities and allows spark identification and quantification in subcellular, cellular, and tissue preparations.

Microtubules can bear enhanced compressive loads in living cells because of lateral reinforcement.

Cytoskeletal microtubules have been proposed to influence cell shape and mechanics based on their ability to resist large-scale compressive forces exerted by the surrounding contractile cytoskeleton. Consistent with this, cytoplasmic microtubules are often highly curved and appear buckled because of compressive loads. However, the results of in vitro studies suggest that microtubules should buckle at much larger length scales, withstanding only exceedingly small compressive forces. This discrepancy calls into question the structural role of microtubules, and highlights our lack of quantitative knowledge of the magnitude of the forces they experience and can withstand in living cells. We show that intracellular microtubules do bear large-scale compressive loads from a variety of physiological forces, but their buckling wavelength is reduced significantly because of mechanical coupling to the surrounding elastic cytoskeleton. We quantitatively explain this behavior, and show that this coupling dramatically increases the compressive forces that microtubules can sustain, suggesting they can make a more significant structural contribution to the mechanical behavior of the cell than previously thought possible.

Targeting of Helicobacter pylori vacuolating toxin to lipid raft membrane domains analysed by atomic force microscopy.

The Helicobacter pylori vacuolating toxin VacA causes several effects on mammalian cells in vitro, including intracellular vacuolation, formation of pores in the plasma membrane and apoptosis. When added to cells, VacA becomes associated with detergent-resistant membranes, indicating that it binds preferentially to lipid rafts. In the present study, we have used atomic force microscopy to examine directly the association of VacA with lipid domains in supported lipid bilayers. VacA did not bind to lipid bilayers at pH 7.6. In contrast, at pH 4.0, VacA associated with the bilayers in the form of 26-nm oligomeric complexes. VacA bound to bilayers produced from either brain lipids or SM (sphingomyelin) plus cholesterol, each of which lacked detectable lipid domains. Bilayers composed of DOPC (dioleoylphosphatidylcholine), SM and cholesterol contained clearly visible raft-like domains, and VacA preferentially associated with these rafts. VacA bound poorly to raft-like domains in DOPC/SM bilayers, indicating that cholesterol is required for efficient association of VacA with lipid domains. When PS (phosphatidylserine), an anionic phospholipid that does not partition significantly into rafts, was added to the mixture of DOPC, SM and cholesterol, VacA was excluded from the rafts, indicating that it binds more avidly to PS than to the raft components. A typical plasma membrane exhibits pronounced lipid asymmetry, with SM enriched in the outer leaflet and PS in the inner leaflet. Therefore it is probable that the association of VacA with rafts in DOPC/SM/cholesterol bilayers represents a useful model for understanding the interactions of VacA with membranes in vivo.

Lipid rafts: feeling is believing.

In the late 1990s, accumulated evidence led to the proposal that biological membranes are composed of microdomains of different lipids, which form functional "rafts." Recent work using atomic force microscopy has given us new insights into the factors influencing the formation and behavior of these physiological microenvironments

Directional control of lamellipodia extension by constraining cell shape and orienting cell tractional forces.

Directed cell migration is critical for tissue morphogenesis and wound healing, but the mechanism of directional control is poorly understood. Here we show that the direction in which cells extend their leading edge can be controlled by constraining cell shape using micrometer-sized extracellular matrix (ECM) islands. When cultured on square ECM islands in the presence of motility factors, cells preferentially extended lamellipodia, filopodia, and microspikes from their corners. Square cells reoriented their stress fibers and focal adhesions so that tractional forces were concentrated in these corner regions. When cell tension was dissipated, lamellipodia extension ceased. Mechanical interactions between cells and ECM that modulate cytoskeletal tension may therefore play a key role in the control of directional cell motility.

Evidence for direct interaction between actin and the cystic fibrosis transmembrane conductance regulator.

Previous studies have demonstrated that actin filament organization controls the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel function. The precise molecular nature of the interaction between actin and CFTR, however, remains largely unknown. In this report, interactions between actin and purified human epithelial CFTR were directly assessed by reconstitution of the channel protein in a lipid bilayer system and by atomic force microscopy (AFM). CFTR-containing liposomes in solution were deposited on freshly cleaved mica and imaging was performed in tapping-mode AFM. CFTR function was also determined in identical preparations. Images of single CFTR molecules were obtained, and addition of monomeric actin below its critical concentration showed the formation of actin filaments associated with CFTR. The data indicate a direct interaction between actin and CFTR exists, which may explain the regulatory role of the cytoskeleton in ion channel function. This was confirmed by functional studies of CFTR single-channel currents, which were regulated by addition of various conformations of actin. The present study indicates that CFTR may directly bind actin and that this interaction helps affect the functional properties of this channel protein.