Harold Bien

Stimulating Cardiac Muscle by Light: Cardiac Optogenetics by Cell Delivery

Background— After the recent cloning of light-sensitive ion channels and their expression in mammalian cells, a new field, optogenetics, emerged in neuroscience, allowing for precise perturbations of neural circuits by light. However, functionality of optogenetic tools has not been fully explored outside neuroscience, and a nonviral, nonembryogenesis-based strategy for optogenetics has not been shown before. Methods and Results— We demonstrate the utility of optogenetics to cardiac muscle by a tandem cell unit (TCU) strategy, in which nonexcitable cells carry exogenous light-sensitive ion channels, and, when electrically coupled to cardiomyocytes, produce optically excitable heart tissue. A stable channelrhodopsin2 (ChR2)-expressing cell line was developed, characterized, and used as a cell delivery system. The TCU strategy was validated in vitro in cell pairs with adult canine myocytes (for a wide range of coupling strengths) and in cardiac syncytium with neonatal rat cardiomyocytes. For the first time, we combined optical excitation and optical imaging to capture light-triggered muscle contractions and high-resolution propagation maps of light-triggered electric waves, found to be quantitatively indistinguishable from electrically triggered waves. Conclusions— Our results demonstrate feasibility to control excitation and contraction in cardiac muscle by light, using the TCU approach. Optical pacing in this case uses less energy, offers superior spatiotemporal control and remote access and can serve not only as an elegant tool in arrhythmia research but may form the basis for a new generation of light-driven cardiac pacemakers and muscle actuators. The TCU strategy is extendable to (nonviral) stem cell therapy and is directly relevant to in vivo applications.Background— After the recent cloning of light-sensitive ion channels and their expression in mammalian cells, a new field, optogenetics, emerged in neuroscience, allowing for precise perturbations of neural circuits by light. However, functionality of optogenetic tools has not been fully explored outside neuroscience, and a nonviral, nonembryogenesis-based strategy for optogenetics has not been shown before. Methods and Results— We demonstrate the utility of optogenetics to cardiac muscle by a tandem cell unit (TCU) strategy, in which nonexcitable cells carry exogenous light-sensitive ion channels, and, when electrically coupled to cardiomyocytes, produce optically excitable heart tissue. A stable channelrhodopsin2 (ChR2)-expressing cell line was developed, characterized, and used as a cell delivery system. The TCU strategy was validated in vitro in cell pairs with adult canine myocytes (for a wide range of coupling strengths) and in cardiac syncytium with neonatal rat cardiomyocytes. For the first time, we combined optical excitation and optical imaging to capture light-triggered muscle contractions and high-resolution propagation maps of light-triggered electric waves, found to be quantitatively indistinguishable from electrically triggered waves. Conclusions— Our results demonstrate feasibility to control excitation and contraction in cardiac muscle by light, using the TCU approach. Optical pacing in this case uses less energy, offers superior spatiotemporal control and remote access and can serve not only as an elegant tool in arrhythmia research but may form the basis for a new generation of light-driven cardiac pacemakers and muscle actuators. The TCU strategy is extendable to (nonviral) stem cell therapy and is directly relevant to in vivo applications.

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.

PI3K Regulation of RAC1 Is Required for KRAS-Induced Pancreatic Tumorigenesis in Mice

BACKGROUND & AIMS New drug targets are urgently needed for the treatment of patients with pancreatic ductal adenocarcinoma (PDA). Nearly all PDAs contain oncogenic mutations in the KRAS gene. Pharmacological inhibition of KRAS has been unsuccessful, leading to a focus on downstream effectors that are more easily targeted with small molecule inhibitors. We investigated the contributions of phosphoinositide 3-kinase (PI3K) to KRAS-initiated tumorigenesis. METHODS Tumorigenesis was measured in the Kras(G12D/+);Ptf1a(Cre/+) mouse model of PDA; these mice were crossed with mice with pancreas-specific disruption of genes encoding PI3K p110α (Pik3ca), p110β (Pik3cb), or RAC1 (Rac1). Pancreatitis was induced with 5 daily intraperitoneal injections of cerulein. Pancreata and primary acinar cells were isolated; acinar cells were incubated with an inhibitor of p110α (PIK75) followed by a broad-spectrum PI3K inhibitor (GDC0941). PDA cell lines (NB490 and MiaPaCa2) were incubated with PIK75 followed by GDC0941. Tissues and cells were analyzed by histology, immunohistochemistry, quantitative reverse-transcription polymerase chain reaction, and immunofluorescence analyses for factors involved in the PI3K signaling pathway. We also examined human pancreas tissue microarrays for levels of p110α and other PI3K pathway components. RESULTS Pancreas-specific disruption of Pik3ca or Rac1, but not Pik3cb, prevented the development of pancreatic tumors in Kras(G12D/+);Ptf1a(Cre/+) mice. Loss of transformation was independent of AKT regulation. Preneoplastic ductal metaplasia developed in mice lacking pancreatic p110α but regressed. Levels of activated and total RAC1 were higher in pancreatic tissues from Kras(G12D/+);Ptf1a(Cre/+) mice compared with controls. Loss of p110α reduced RAC1 activity and expression in these tissues. p110α was required for the up-regulation and activity of RAC guanine exchange factors during tumorigenesis. Levels of p110α and RAC1 were increased in human pancreatic intraepithelial neoplasias and PDAs compared with healthy pancreata. CONCLUSIONS KRAS signaling, via p110α to activate RAC1, is required for transformation in Kras(G12D/+);Ptf1a(Cre/+) mice.

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.

Tension Development and Nuclear Eccentricity in Topographically Controlled Cardiac Syncytium

The goal of this study was to use topographic control by microfabricated scaffolds with 3-dimensional surfaces to induce active tension development and enhanced contractility in engineered cardiac syncytium (a high density cardiac cell structure with reconstituted cell-to-cell connections and synchronized tissue-like behavior). Deeply microgrooved (feature height 50μm) elastic scaffolds were designed using polydimethylsiloxane molding, and neonatal rat cardiomyocytes were grown on them to confluency. Engineered cardiac cell constructs on the topographically modified (T) scaffolds showed higher order of intra and intercellular organization (fiber-like structures) compared to those grown on various flat surfaces (F), and developed self-organized persisting electrical and mechanical activity. These structural and functional changes were accompanied by a statistically significant (p < 0.001) increase in nuclear eccentricity (mean ± S.E.: 0.79 ∓ 0.01, n=137 in T vs. 0.64 ± 0.01, n=863 in F), and a preferential nuclear orientation, deviating from the axis of the grooves at a shallow angle. The orientation of the nuclei correlated well with the actin fiber arrangement in the T-samples, as well as with the direction of maximum displacement. Topography-induced nuclear deformation, a sign of tension development, implies further functional changes in transcription and cell signaling. In conclusion, we demonstrate topographic control of electromechanics in engineered cardiac syncytium, without external mechanical or electrical stimulation. These findings suggest a possibility to use controled microenvironments in the design of biological autonomous force generators with reconstituted excitable tissue.

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

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.

Microtopographical effects of natural scaffolding on cardiomyocyte function and arrhythmogenesis

A natural myocardial patch for heart regeneration derived from porcine urinary bladder matrix (UBM) was previously reported to outperform synthetic materials (Dacron and expanded polytetrafluoroethylene (ePTFE)) used in current surgical treatments. UBM, an extracellular matrix prepared from urinary bladder, has intricate three-dimensional architecture with two distinct sides: the luminal side with a smoother surface relief; and the abluminal side with a fine mesh of nano- and microfibers. This study tested the ability of this natural scaffold to support functional cardiomyocyte networks, and probed how the local microtopography and composition of the two sides affects cell function. Cardiomyocytes isolated from neonatal rats were seeded in vitro to form cardiac tissue onto luminal (L) or abluminal (Ab) UBM. Immunocytochemistry of contractile cardiac proteins demonstrated growth of cardiomyocyte networks with mature morphology on either side of UBM, but greater cell compactness was seen in L. Fluorescence-based imaging techniques were used to measure dynamic changes in intracellular calcium concentration upon electrical stimulation of L and Ab-grown cells. Functional differences in cardiac tissue grown on the two sides manifested themselves in faster calcium recovery (p<0.04) and greater hysteresis (difference in response to increasing and decreasing pacing rates) for L vs Ab side (p<0.03). These results suggest that surface differences may be leveraged to engineer the desired cardiomyocyte responses and highlight the potential of natural scaffolds for fostering heart repair.

Mechanical and spatial determinants of cytoskeletal geodesic dome formation in cardiac fibroblasts

This study tests the hypothesis that the cell cytoskeletal (CSK) network can rearrange from geodesic dome type structures to stress fibers in response to microenvironmental cues. The CSK geodesic domes are highly organized actin microarchitectures within the cell, consisting of ordered polygonal elements. We studied primary neonatal rat cardiac fibroblasts. The cues used to trigger the interconversion between the two CSK architectures (geodesic domes and stress fibers) included factors affecting spatial order and the degree of CSK tension in the cells. Microfabricated three-dimensional substrates with micrometre sized grooves and peaks were used to alter the spatial order of cell growth in culture. CSK tension was modified by 2,3-butanedione 2-monoxime (BDM), cytochalasin D and the hyphae of Candida albicans. CSK geodesic domes occurred spontaneously in about 20% of the neonatal rat cardiac fibroblasts used in this study. Microfabricated structured surfaces produced anisotropy in the cell CSK and effectively converted geodesic domes into stress fibers in a dose-dependent manner (dependence on the period of the features). Affectors of actin structure, inhibitors of CSK tension and cell motility, e.g. BDM, cytochalasin D and the hyphae of C. albicans, suppressed or eliminated the geodesic domes. Our data suggest that the geodesic domes, similar to actin stress fibers, require maintenance of CSK integrity and tension. However, microenvironments that promote structural anisotropy in tensed cells cause the transformation of the geodesic domes into stress fibers, consistent with topographic cell guidance and some previous CSK model predictions.

Lenses and effective spatial resolution in macroscopic optical mapping

Optical mapping of excitation dynamically tracks electrical waves travelling through cardiac or brain tissue by the use of fluorescent dyes. There are several characteristics that set optical mapping apart from other imaging modalities: dynamically changing signals requiring short exposure times, dim fluorescence demanding sensitive sensors and wide fields of view (low magnification) resulting in poor optical performance. These conditions necessitate the use of optics with good light gathering ability, i.e. lenses having high numerical aperture. Previous optical mapping studies often used sensor resolution to estimate the minimum spatial feature resolvable, assuming perfect optics and infinite contrast. We examine here the influence of finite contrast and real optics on the effective spatial resolution in optical mapping under broad-field illumination for both lateral (in-plane) resolution and axial (depth) resolution of collected fluorescence signals.