Emilia Entcheva

Contact Fluorescence Imaging of Reentry in Monolayers of Cultured Neonatal Rat Ventricular Myocytes

Reentry in Cell Monolayers Imaged by CFI. Introduction: We present a novel contact fluorescence imaging (CFI) approach to monitor transmembrane potentials in monolayers of cultured neonatal rat ventricular cells. We apply CFI to demonstrate, for the first time, long‐term recordings as well as electrical induction and termination of reentrant activity in this in vitro model.

Loss of Cardiac Phosphoinositide 3-Kinase p110α Results in Contractile Dysfunction

Background— Phosphoinositide 3-kinase (PI3K) p110&agr; plays a key role in insulin action and tumorigenesis. Myocyte contraction is initiated by an inward Ca2+ current (ICa,L) through the voltage-dependent L-type Ca2+ channel (LTCC). The aim of this study was to evaluate whether p110&agr; also controls cardiac contractility by regulating the LTCC. Methods and Results— Genetic ablation of p110&agr; (also known as Pik3ca), but not p110&bgr; (also known as Pik3cb), in cardiac myocytes of adult mice reduced ICa,L and blocked insulin signaling in the heart. p110&agr;-null myocytes had a reduced number of LTCCs on the cell surface and a contractile defect that decreased cardiac function in vivo. Similarly, pharmacological inhibition of p110&agr; decreased ICa,L and contractility in canine myocytes. Inhibition of p110&bgr; did not reduce ICa,L. Conclusions— PI3K p110&agr; but not p110&bgr; regulates the LTCC in cardiac myocytes. Decreased signaling to p110&agr; reduces the number of LTCCs on the cell surface and thus attenuates ICa,L and contractility.

Learning and detecting emergent behavior in networks of cardiac myocytes

We address the problem of specifying and detecting emergent behavior in networks of cardiac myocytes, spiral electric waves in particular, a precursor to atrial and ventricular fibrillation. To solve this problem we: (1) apply discrete mode abstraction to the cycle-linear hybrid automata (CLHA) we have recently developed for modeling the behavior of myocyte networks; (2) introduce the new concept of spatial superposition of CLHA modes; (3) develop a new spatial logic, based on spatial superposition, for specifying emergent behavior; (4) devise a new method for learning the formulae of this logic from the spatial patterns under investigation; and (5) apply bounded model checking to detect the onset of spiral waves. We have implemented our methodology as the EMERALD tool suite, a component of our EHA framework for specification, simulation, analysis, and control of excitable hybrid automata. We illustrate the effectiveness of our approach by applying EMERALD to the scalar electrical fields produced by our CELLEXCITE simulation environment for excitable-cell networks.

The Role of Cardiac Tissue Alignment in Modulating Electrical Function

Introduction: Most cardiac arrhythmias are associated with pathology‐triggered ion channel remodeling. However, multicellular effects, for example, exaggerated anisotropy and altered cell‐to‐cell coupling, can also indirectly affect action potential morphology and electrical stability via changed electrotonus. These changes are particularly relevant in structural heart disease, including hypertrophy and infarction. Recent computational studies showed that electrotonus factors into stability by altering dynamic properties (restitution). We experimentally address the question of how cell alignment and connectivity alter tissue function and whether these effects depend on the direction of wave propagation.

Cardiac cell networks on elastic microgrooved scaffolds

We sought to construct a model-engineered cardiac construct having anisotropic properties and consisting of inter-connected cardiac cells with syncytial tissuelike behavior. We report basic structural, electrophysiological and mechanical characterization of multicellular tissuelike engineered constructs developed using elastic matrices with 3-D surface microtopography. To properly assess functionality of the constructs in the tissue setting, we employed spatial optical fluorescence techniques enabling measurements at the micro- and macro-scale.

Hypertrophic phenotype in cardiac cell assemblies solely by structural cues and ensuing self-organization

In vitro models of cardiac hypertrophy focus exclusively on applying “external” dynamic signals (electrical, mechanical, and chemical) to achieve a hypertrophic state. In contrast, here we set out to demonstrate the role of “self‐organized” cellular architecture and activity in reprogramming cardiac cell/tissue function toward a hypertrophic phenotype. We report that in neonatal rat cardiomyocyte culture, subtle out‐of‐plane microtopographic cues alter cell attachment, increase biomechanical stresses, and induce not only structural remodeling, but also yield essential molecular and electrophysiological signatures of hypertrophy. Increased cell size and cell binucleation, molecular up‐regulation of released atrial natriuretic peptide, altered expression of classic hypertrophy markers, ion channel remodeling, and corresponding changes in electrophysiological function indicate a state of hypertrophy on par with other in vitro and in vivo models. Clinically used antihypertrophic pharmacological treatments partially reversed hypertrophic behavior in this in vitro model. Partial least‐squares regression analysis, combining gene expression and functional data, yielded clear separation of phenotypes (control: cells grown on flat surfaces;hypertrophic: cells grown on quasi‐3‐dimensional surfaces and treated). In summary, structural surface features can guide cardiac cell attachment, and the subsequent syncytial behavior can facilitate trophic signals, unexpectedly on par with externally applied mechanical, electrical, and chemical stimulation.—Chung, C., Bien, H., Sobie, E. A., Dasari, V., McKinnon, D., Rosati, B., Entcheva, E. Hypertrophic phenotype in cardiac cell assemblies solely by structural cues and ensuing self‐organization. FASEB J. 25, 851–862 (2011). www.fasebj.org

Modeling and simulation of cardiac tissue using hybrid I/O automata

We propose a new biological framework based on the Lynch et al. theory of Hybrid I/O Automata (HIOAs) for modeling and simulating excitable tissue. Within this framework, we view an excitable tissue as a composition of two main kinds of component: a diffusion medium and a collection of cells, both modeled as an HIOA. This approach yields a notion of decomposition that allows us to describe a tissue as the parallel composition of several interacting tissues, a property that could be exploited to parallelize, and hence improve, the efficiency of the simulation process. We also demonstrate the feasibility of our HIOA-based framework to capture and mimic different kinds of wave-propagation behavior in 2D isotropic cardiac tissue, including normal wave propagation along the tissue; the creation of spiral waves; the break-up of spiral waves into more complex patterns such as fibrillation; and the recovery of the tissue to the rest via electrical defibrillation.

Modelling excitable cells using cycle-linear hybrid automata

Cycle-linear hybrid automata (CLHAs), a new model of excitable cells that efficiently and accurately captures action-potential morphology and other typical excitable-cell characteristics such as refractoriness and restitution, is introduced. Hybrid automata combine discrete transition graphs with continuous dynamics and emerge in a natural way during the (piecewise) approximation process of any nonlinear system. CLHAs are a new form of hybrid automata that exhibit linear behaviour on a per-cycle basis but whose overall behaviour is appropriately nonlinear. To motivate the need for this modelling formalism, first it is shown how to recast two recently proposed models of excitable cells as hybrid automata: the piecewise-linear model of Biktashev and the nonlinear model of Fenton-Karma. Both of these models were designed to efficiently approximate excitable-cell behaviour. We then show that the CLHA closely mimics the behaviour of several classical highly nonlinear models of excitable cells, thereby retaining the simplicity of Biktashevs model without sacrificing the expressiveness of Fenton-Karma. CLHAs are not restricted to excitable cells; they can be used to capture the behaviour of a wide class of dynamic systems that exhibit some level of periodicity plus adaptation.

Expression of skeletal but not cardiac Na+ channel isoform preserves normal conduction in a depolarized cardiac syncytium

AIMS Reentrant arrhythmias often develop in the setting of myocardial infarction and ensuing slow propagation. Increased Na(+) channel expression could prevent or disrupt reentrant circuits by speeding conduction if channel availability is not limited by membrane depolarization within the diseased myocardium. We therefore asked if, in the setting of membrane depolarization, action potential (AP) upstroke and normal conduction can be better preserved by the expression of a Na(+) channel isoform with altered biophysical properties compared to the native cardiac Na(+) channel isoform, namely having a positively shifted, voltage-dependent inactivation. METHODS AND RESULTS The skeletal Na(+) channel isoform (SkM1) and the cardiac Na(+) channel isoform (Nav1.5) were expressed in newborn rat ventricular myocyte cultures with a point mutation introduced in Nav1.5 to increase tetrodotoxin (TTX) sensitivity so native and expressed currents could be distinguished. External K(+) was increased from 5.4 to 10 mmol/L to induce membrane depolarization. APs, Na(+) currents, and conduction velocity (CV) were measured. In control cultures, elevated K(+) significantly reduced AP upstroke ( approximately 75%) and CV ( approximately 25%). Expression of Nav1.5 did not protect AP upstroke from K(+) depolarization. In contrast, in SkM1 expressing cultures, high K(+) reduced AP upstroke <50% and conduction was not significantly reduced. In a simulated anatomical reentry setting (using a void), the angular velocity (AV) of induced reentry was faster and the excitable gap shorter in SkM1 cultures compared to control for both normal and high K(+). CONCLUSION Expression of SkM1 but not Nav1.5 preserves AP upstroke and CV in a K(+)-depolarized syncytium. The higher AV and shorter excitable gap observed during reentry excitation around a void in SkM1 cultures would be expected to facilitate reentry self-termination. SkM1 Na(+) channel expression represents a novel gene therapy for the treatment of reentrant arrhythmias.

Acoustic micromachining of three-dimensional surfaces for biological applications.

We present the use of an accessible micromachining technique (acoustic micromachining) for manufacturing micron-feature surfaces with non-discretely varying depth. Acoustic micromachining allows for non-photolithographic production of metal templates with programmable spatial patterns and involves the use of standard acoustic, cutting and electroplating equipment for mass production of vinyl records. Simple 3D patterns were transferred from an acoustic signal into working nickel templates, from which elastic polymer molds were obtained, featuring deep surface grooves and non-discrete (smooth) variations in the z-dimension. Versatility and applicability of the method is demonstrated in obtaining microfluidics structures, manufacturing high-surface area wavy polymer fibers, assembly of cell networks on scaffolds with 3D topography, and microcontact printing of proteins and cells.