Background— Cardiac hypertrophy is classically regarded as a compensatory response, yet the active tissue remodeling processes triggered by various types of mechanical stress can enhance or diminish the function of the heart. Despite the disparity in outcomes, there are similarities in the hypertrophic responses. We hypothesized that a generic genetic response that is not dependent on the particular nature of the hypertrophic stimulus exists. To test our hypothesis, we compared the temporal evolution of transcriptomes measured in hearts subjected to either adaptive (exercise-induced) or maladaptive (aortic banding–induced) hypertrophy.

The heart is a complex organ system composed of a highly diverse set of muscle and nonmuscle cells. Understanding the pathways that drive the formation, migration, and assembly of these cells into the heart muscle tissue, the pacemaker and conduction system, and the coronary vasculature is a central problem in developmental biology. Efforts to unravel the biological complexity of in vivo cardiogenesis have identified a family of closely related multipotent cardiac progenitor cells. These progenitors must respond to non-cell-autonomous signaling cues to expand, differentiate, and ultimately integrate into the three-dimensional heart structures. Coupling tissue-engineering technologies with patient-specific cardiac progenitor biology holds great promise for the development of human cell models of human disease and may lay the foundation for novel approaches in regenerative cardiovascular medicine.

The capability to selectively and reversibly control protein-protein interactions in antibody-doped polypyrrole (PPy) was accomplished by changing the voltage applied to the polymer. Polypyrrole was doped with sulfate polyanions and monoclonal anti-human fibronectin antibodies (alphaFN). The ability to toggle the binding and dissociation of fibronectin (FN) to alphaFN-doped polypyrrole was demonstrated. Staircase potential electrochemical impedance spectroscopy (SPEIS) was performed to characterize the impedance and charge transfer characteristics of the alphaFN-doped PPy as a function of applied voltage, frequency, and FN concentration. Impedance measurements indicated oxidation of alphaFN-doped PPy promoted selective binding of FN to alphaFN antibodies and reduction of the polymer films facilitated FN dissociation. Moreover, SPEIS measurements suggested that the apparent reversibility of antigen binding to antibody-doped PPy is not due to the suppression of hydrophobic binding forces between antibody and antigen. Instead, our data indicate that reversible antigen binding to antibody-doped PPy can be attributed to the minimization of charge in the polymer films during oxidation and reduction. Furthermore, alphaFN-doped PPy was utilized to collect real-time, dynamic measurements of varying FN concentrations in solution by repeatedly binding and releasing FN. Our data demonstrate that antibody-doped PPy represents an electrically controllable sensing platform which can be exploited to collect rapid, repeated measurements of protein concentrations with molecular specificity.

Morphogenesis is often considered a function of transcriptional synchrony and the spatial limits of diffusing mitogens; however, physical constrainment by the cell microenvironment represents an additional mechanism for regulating self-assembly of subcellular structures. We asked whether myocyte shape is a distinct signal that potentiates the organization of myofibrillar arrays in cardiac muscle myocytes. We engineered the shape of neonatal rat ventricular myocytes by culturing them on microfabricated fibronectin islands, where they spread and assumed the shape of the island. Myofibrillogenesis followed, both spatially and temporally, the assembly of unique actin networks whose architecture was predictable given the shape of the island. Subsequently, the z lines of the sarcomeres aligned and registered in distinct patterns in different regions of the myocytes in such a way that orthogonal axes of contraction could be distinctly engineered. These data suggest that physical constrainment of muscle cells by extracellular matrix may be an important regulator of myofibrillar organization.

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 α-actinin images revealed invariant sarcomeric registration irrespective of myocyte aspect ratio. Since the sarcomere subunits 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.

We describe a design algorithm to build a cardiac myocyte with specific spatial dimensions and physiological function. Using a computational model of a cardiac muscle cell, we modeled calcium (Ca2+) wave dynamics in a cardiac myocyte with controlled spatial dimensions. The modeled myocyte was replicated in vitro when primary neonate rat ventricular myocytes were cultured on micropatterned substrates. The myocytes remodel to conform to the two dimensional boundary conditions and assume the shape of the printed extracellular matrix island. Mechanical perturbation of the myocyte with an atomic force microscope results in calcium-induced calcium release from intracellular stores and the propagation of a Ca2+ wave, as indicated by high speed video microscopy using fluorescent indicators of intracellular Ca2+. Analysis and comparison of the measured wavefront dynamics with those simulated in the computer model reveal that the engineered myocyte behaves as predicted by the model. These results are important because they represent the use of computer modeling, computer-aided design, and physiological experiments to design and validate the performance of engineered cells. The ability to successfully engineer biological cells and tissues for assays or therapeutic implants will require design algorithms and tools for quality and regulatory assurance.

The spatial and temporal scales of cardiac organogenesis and pathogenesis make engineering of artificial heart tissue a daunting challenge. The temporal scales range from nanosecond conformational changes responsible for ion channel opening to fibrillation which occurs over seconds and can lead to death. Spatial scales range from nanometre pore sizes in membrane channels and gap junctions to the metre length scale of the whole cardiovascular system in a living patient. Synchrony over these scales requires a hierarchy of control mechanisms that are governed by a single common principle: integration of structure and function. To ensure that the function of ion channels and contraction of muscle cells lead to changes in heart chamber volume, an elegant choreography of metabolic, electrical and mechanical events are executed by protein networks composed of extracellular matrix, transmembrane integrin receptors and cytoskeleton which are functionally connected across all size scales. These structural control networks are mechanoresponsive, and they process mechanical and chemical signals in a massively parallel fashion, while also serving as a bidirectional circuit for information flow. This review explores how these hierarchical structural networks regulate the form and function of living cells and tissues, as well as how microfabrication techniques can be used to probe this structural control mechanism that maintains metabolic supply, electrical activation and mechanical pumping of heart muscle. Through this process, we delineate various design principles that may be useful for engineering artificial heart tissue in the future.

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.

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.

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.

Coordinated, cohort cell migration plays an important role in the morphogenesis of tissue patterns in metazoa. However, individual cells intrinsically move in a random walk-like fashion when studied in vitro. Hence, in the absence of an external orchestrating influence or template, the emergence of cohort cell migration must involve a symmetry-breaking event. To study this process, we used a novel experimental system in which multiple capillary endothelial cells exhibit spontaneous and robust cohort migration in the absence of chemical gradients when cultured on micrometer-scale extracellular matrix islands fabricated using microcontact printing. A computational model suggested that directional persistence of random-walk and dynamic mechanical coupling of adjacent cells are the critical control parameters for this symmetry-breaking behavior that is induced in spatially-constrained cell ensembles. The model predicted our finding that fibroblasts, which exhibit a much shorter motility persistence time than endothelial cells, failed to undergo symmetry breaking or produce cohort migration on the matrix islands. These findings suggest that cells have intrinsic motility characteristics that are tuned to match their role in tissue patterning. Our results underscore the importance of studying cell motility in the context of cell populations, and the need to address emergent features in multicellular organisms that arise not only from cell-cell and cell-matrix interactions, but also from properties that are intrinsic to individual cells.

Mechanochemical and mechanoelectrical signaling is imperative for cardiac organogenesis and underlies pathophysiological events. New techniques for engineering cardiac tissue allow unprecedented means of modeling these phenomena in vitro. However, experimental design is often hampered by a lack of models that can be adapted to the ideal conditions these methods allow. To address these deficiencies, we developed a mathematical model to calculate the distribution of stress and strain in fibrous cardiac tissue. The fluid-fiber-collagen model characterizes the mechanical behavior of cardiac tissue and is solved analytically for the distributions of stress and strain along the myocardial fibers. An example application of the model is presented: modeling the distribution of strains in the vicinity of an ischemic region. The ischemic region is stretched during systole, as has been shown in previous one-dimensional models. Our model predicts a complex distribution of stretch in the border zone surrounding the ischemic region and in nonischemic regions surrounding the border zone. These strain patterns may predict patterns of mechanochemical coupling that results in localized fibrosis, altered gene expression, or the mechanoelectrical signaling events that potentiate cardiac arrhythmias.

Structural and functional cardiac anisotropy varies with the development, location, and pathophysiology in the heart. The goal of this study was to design a cell culture model system in which the degree, change in fiber direction, and discontinuity of anisotropy can be controlled over centimeter-size length scales. Neonatal rat ventricular myocytes were cultured on fibronectin on 20-mm diameter circular cover slips. Structure-function relationships were assessed using immunostaining and optical mapping. Cell culture on microabraded cover slips yielded cell elongation and coalignment in the direction of abrasion, and uniform, macroscopically continuous, elliptical propagation with point stimulation. Coarser microabrasion (wider and deeper abrasion grooves) increased longitudinal (23.5 to 37.2 cm/s; r=0.66) and decreased transverse conduction velocity (18.1 to 9.2 cm/s; r=−0.84), which resulted in increased longitudinal-to-transverse velocity anisotropy ratios (1.3 to 3.7, n=61). A thin transition zone between adjacent uniformly anisotropic areas with 45° or 90° difference in fiber orientation acted as a secondary source during 2× threshold field stimulus. Cell culture on cover slips micropatterned with 12- or 25-μm wide fibronectin lines and previously coated with decreasing concentrations of background fibronectin yielded transition from continuous to discontinuous anisotropic architecture with longitudinally oriented intercellular clefts, decreased transverse velocity (16.9 to 2.6 cm/s; r=−0.95), increased velocity anisotropy ratios (1.6 to 5.6, n=70), and decreased longitudinal velocity (36.4 to 14.6 cm/s; r=−0.85) for anisotropy ratios >3.5. Cultures of cardiac myocytes with controlled degree, uniformity and continuity of structural, and functional anisotropy may enable systematic 2-dimensional in vitro studies of macroscopic structure-related mechanisms of reentrant arrhythmias. The full text of this article is available at http://www.circresaha.org.

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.

Stretch-activated ion channels have been identified as transducers of mechanoelectric coupling in the heart, where they may play a role in arrhythmogenesis. The role of the cytoskeleton in ion channel control has been a topic of recent study and the transmission of mechanical stresses to stretch-activated channels by cytoskeletal attachment has been hypothesized. We studied the arrhythmogenic effects of stretch in 16 Langendorff-perfused rabbit hearts in which we pharmacologically manipulated the microtubular network of the cardiac myocytes. Group 1 (n=5) was treated with colchicine, which depolymerizes microtubules, and Group 2 (n=6) was treated with taxol, which polymerizes microtubules. Stretch-induced arrhythmias were produced by transiently increasing the volume of a fluid-filled left ventricular balloon with a volume pump driven by a computer-controlled stepper motor. Electrical events were recorded by a contact electrode which provided high-fidelity recordings of monophasic action potentials and stretch-induced depolarizations. The probability of eliciting a stretch-induced arrhythmia increased (0.22+/-0.11 to 0.62+/-0.19, p=0.001) in hearts treated with taxol (5 microM), whereas hearts treated with colchicine (100 microM) showed no statistically significant change. We conclude that proliferation of microtubules increased the arrhythmogenic effect of transient left ventricle diastolic stretch. This result indicates a possible mode of arrhythmogenesis in chemotherapeutic patients and patients exhibiting uncompensated ventricular hypertrophy. The data would indicate that the cytoskeleton represents a possible target for antiarrhythmic therapies.

Mechanical stretch has been demonstrated to have electrophysiological effects on cardiac muscle, including alteration of the probability of excitation, alteration of the action potential waveform, and stretch-induced arrhythmia (SIA). We demonstrate that regional ventricular ischemia due to coronary artery occlusion increases arrhythmogenic effects of transient diastolic stretch, whereas globally ischemic hearts showed no such increase. We tested our hypothesis that, during phase Ia ischemia, regionally ischemic hearts may be more susceptible to triggered arrhythmogenesis due to transient diastolic stretch. During the first 20 min of regional ischemia, the probability of eliciting a ventricular SIA (PSIA) by transient diastolic stretch increased significantly. However, after 30 min, PSIA decreased to a value comparable with baseline measurements, as expected during phase Ib, where most ventricular arrhythmias are of reentrant mechanisms. We also suggest that mechanoelectrical coupling may contribute to the nonreentrant mechanisms underlying reperfusion-induced arrhythmia. When coronary artery occlusion was relieved after 30 min of ischemia, we observed an increase in PSIA and the maintenance of this elevated level throughout 20 min of reperfusion. We conclude that mechanoelectrical coupling may underlie triggered arrhythmogenesis during phase 1a ischemia and reperfusion.

We have developed a computationally simple model for calculating the magnetic-field strength at a point due to a single motor unit compound action potential (SMUCAP). The motor unit is defined only in terms of its anatomical features, and the SMUCAP is approximated using the tripole model. The distributed current density J is calculated within the volume defined by the motor unit. The law of Biot and Savart can then be cast in a form necessitating that J be integrated only over the region containing current sources or conductivity boundaries. The magnetic-field strength is defined as the summation of the contributions to the field made by every muscle fiber in the motor unit. Applying this model to SMUCAP measurements obtained using a high-resolution SUper Conducting Quantum Interference Device (SQUID) magnetometer may yield information regarding the distribution of action currents (AC's) and the anatomical properties of single motor units within a muscle bundle.