Sean P. Sheehy

Muscular Thin Films for Building Actuators and Powering Devices

We demonstrate the assembly of biohybrid materials from engineered tissues and synthetic polymer thin films. The constructs were built by culturing neonatal rat ventricular cardiomyocytes on polydimethylsiloxane thin films micropatterned with extracellular matrix proteins to promote spatially ordered, two-dimensional myogenesis. The constructs, termed muscular thin films, adopted functional, three-dimensional conformations when released from a thermally sensitive polymer substrate and were designed to perform biomimetic tasks by varying tissue architecture, thin-film shape, and electrical-pacing protocol. These centimeter-scale constructs perform functions as diverse as gripping, pumping, walking, and swimming with fine spatial and temporal control and generating specific forces as high as 4 millinewtons per square millimeter.

Sarcomere Alignment is Regulated by Myocyte Shape

Cardiac organogenesis and pathogenesis are both characterized by changes in myocyte shape, cytoskeletal architecture, and the extracellular matrix (ECM). However, the mechanisms by which the ECM influences myocyte shape and myofibrillar patterning are unknown. We hypothesized that geometric cues in the ECM align sarcomeres by directing the actin network orientation. To test our hypothesis, we cultured neonatal rat ventricular myocytes on islands of micro-patterned ECM to measure how they remodeled their cytoskeleton in response to extracellular cues. Myocytes spread and assumed the shape of circular and rectangular islands and reorganized their cytoskeletons and myofibrillar arrays with respect to the ECM boundary conditions. Circular myocytes did not assemble predictable actin networks nor organized sarcomere arrays. In contrast, myocytes cultured on rectangular ECM patterns with aspect ratios ranging from 1:1 to 7:1 aligned their sarcomeres in predictable and repeatable patterns based on highly localized focal adhesion complexes. Examination of averaged alpha-actinin images revealed invariant sarcomeric registration irrespective of myocyte aspect ratio. Since the sarcomere sub-units possess a fixed length, this observation indicates that cytoskeleton configuration is length-limited by the extracellular boundary conditions. These results indicate that modification of the extracellular microenvironment induces dynamic reconfiguring of the myocyte shape and intracellular architecture. Furthermore, geometric boundaries such as corners induce localized myofibrillar anisotropy that becomes global as the myocyte aspect ratio increases.

Biohybrid thin films for measuring contractility in engineered cardiovascular muscle

In vitro cardiovascular disease models need to recapitulate tissue-scale function in order to provide in vivo relevance. We have developed a new method for measuring the contractility of engineered cardiovascular smooth and striated muscle in vitro during electrical and pharmacological stimulation. We present a growth theory-based finite elasticity analysis for calculating the contractile stresses of a 2D anisotropic muscle tissue cultured on a flexible synthetic polymer thin film. Cardiac muscle engineered with neonatal rat ventricular myocytes and paced at 0.5 Hz generated stresses of 9.2 +/- 3.5 kPa at peak systole, similar to measurements of the contractility of papillary muscle from adult rats. Vascular tissue engineered with human umbilical arterial smooth muscle cells maintained a basal contractile tone of 13.1 +/- 2.1 kPa and generated another 5.1 +/- 0.8 kPa when stimulated with endothelin-1. These data suggest that this method may be useful in assessing the efficacy and safety of pharmacological agents on cardiovascular tissue.

Recapitulating maladaptive, multiscale remodeling of failing myocardium on a chip

The lack of a robust pipeline of medical therapeutic agents for the treatment of heart disease may be partially attributed to the lack of in vitro models that recapitulate the essential structure–function relationships of healthy and diseased myocardium. We designed and built a system to mimic mechanical overload in vitro by applying cyclic stretch to engineered laminar ventricular tissue on a stretchable chip. To test our model, we quantified changes in gene expression, myocyte architecture, calcium handling, and contractile function and compared our results vs. several decades of animal studies and clinical observations. Cyclic stretch activated gene expression profiles characteristic of pathological remodeling, including decreased α- to β-myosin heavy chain ratios, and induced maladaptive changes to myocyte shape and sarcomere alignment. In stretched tissues, calcium transients resembled those reported in failing myocytes and peak systolic stress was significantly reduced. Our results suggest that failing myocardium, as defined genetically, structurally, and functionally, can be replicated in an in vitro microsystem by faithfully recapitulating the structural and mechanical microenvironment of the diseased heart.

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.

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.

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.

The contribution of cellular mechanotransduction to cardiomyocyte form and function.

Myocardial development is regulated by an elegantly choreographed ensemble of signaling events mediated by a multitude of intermediates that take a variety of forms. Cellular differentiation and maturation are a subset of vertically integrated processes that extend over several spatial and temporal scales to create a well-defined collective of cells that are able to function cooperatively and reliably at the organ level. Early efforts to understand the molecular mechanisms of cardiomyocyte fate determination focused primarily on genetic and chemical mediators of this process. However, increasing evidence suggests that mechanical interactions between the extracellular matrix (ECM) and cell surface receptors as well as physical interactions between neighboring cells play important roles in regulating the signaling pathways controlling the developmental processes of the heart. Interdisciplinary efforts have made it apparent that the influence of the ECM on cellular behavior occurs through a multitude of physical mechanisms, such as ECM boundary conditions, elasticity, and the propagation of mechanical signals to intracellular compartments, such as the nucleus. In addition to experimental studies, a number of mathematical models have been developed that attempt to capture the interplay between cells and their local microenvironment and the influence these interactions have on cellular self-assembly and functional behavior. Nevertheless, many questions remain unanswered concerning the mechanism through which physical interactions between cardiomyocytes and their environment are translated into biochemical cellular responses and how these signaling modalities can be utilized in vitro to fabricate myocardial tissue constructs from stem cell-derived cardiomyocytes that more faithfully represent their in vivo counterpart. These studies represent a broad effort to characterize biological form as a conduit for information transfer that spans the nanometer length scale of proteins to the meter length scale of the patient and may yield new insights into the contribution of mechanotransduction into heart development and disease.

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.

Quality Metrics for Stem Cell-Derived Cardiac Myocytes

Summary Advances in stem cell manufacturing methods have made it possible to produce stem cell-derived cardiac myocytes at industrial scales for in vitro muscle physiology research purposes. Although FDA-mandated quality assurance metrics address safety issues in the manufacture of stem cell-based products, no standardized guidelines currently exist for the evaluation of stem cell-derived myocyte functionality. As a result, it is unclear whether the various stem cell-derived myocyte cell lines on the market perform similarly, or whether any of them accurately recapitulate the characteristics of native cardiac myocytes. We propose a multiparametric quality assessment rubric in which genetic, structural, electrophysiological, and contractile measurements are coupled with comparison against values for these measurements that are representative of the ventricular myocyte phenotype. We demonstrated this procedure using commercially available, mass-produced murine embryonic stem cell- and induced pluripotent stem cell-derived myocytes compared with a neonatal mouse ventricular myocyte target phenotype in coupled in vitro assays.