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

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.

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.

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.

Cancer cells become less deformable and more invasive with activation of β-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. β-adrenergic receptor (βAR) signaling drives invasion and metastasis, but the effects on cell deformability are not known.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).