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.

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. METHODS AND RESULTS: We show that cardiac cell arrangement can alter electrical stability in an in vitro cardiac tissue model by mechanisms both dependent and independent of the direction of wave propagation, and local structural remodeling can be felt beyond a space constant. Notably, restitution of action potential duration (APD) and conduction velocity was significantly steepened in the direction of cell alignment. Furthermore, prolongation of APD and calcium transient duration was found in highly anisotropic cell networks, both for longitudinal and transverse propagation. This is in contrast to expected correlation between wave propagation direction and APD based on electrotonic effects only, but is consistent with our findings of increased cell size and secretion of atrial natriuretic factor, a hypertrophy marker, in the aligned structures. CONCLUSION: Our results show that anisotropic structure is a potent modulator of electrical stability via electrotonus and molecular signaling. Tissue alignment must be taken into account in experimental and computational models of arrhythmia generation and in designing effective treatment therapies.

Electrospun fine-textured scaffolds for heart tissue constructs.

The structural and functional effects of fine-textured matrices with sub-micron features on the growth of cardiac myocytes were examined. Electrospinning was used to fabricate biodegradable non-woven poly(lactide)- and poly(glycolide)-based (PLGA) scaffolds for cardiac tissue engineering applications. Post-processing was applied to achieve macro-scale fiber orientation (anisotropy). In vitro studies confirmed a dose-response effect of the poly(glycolide) concentration on the degradation rate and the pH value changes. Different formulations were examined to assess scaffold effects on cell attachment, structure and function. Primary cardiomyocytes (CMs) were cultured on the electrospun scaffolds to form tissue-like constructs. Scanning electron microscopy (SEM) revealed that the fine fiber architecture of the non-woven matrix allowed the cardiomyocytes to make extensive use of provided external cues for isotropic or anisotropic growth, and to some extent to crawl inside and pull on fibers. Structural analysis by confocal microscopy indicated that cardiomyocytes had a preference for relatively hydrophobic surfaces. CMs on electrospun poly(L-lactide) (PLLA) scaffolds developed mature contractile machinery (sarcomeres). Functionality (excitability) of the engineered constructs was confirmed by optical imaging of electrical activity using voltage-sensitive dyes. We conclude that engineered cardiac tissue structure and function can be modulated by the chemistry and geometry of the provided nano- and micro-textured surfaces. Electrospinning is a versatile manufacturing technique for design of biomaterials with potentially reorganizable architecture for cell and tissue growth.

Functional cardiac cell constructs on cellulose-based scaffolding.

Cellulose and its derivatives have been successfully employed as biomaterials in various applications, including dialysis membranes, diffusion-limiting membranes in biosensors, in vitro hollow fibers perfusion systems, surfaces for cell expansion, etc. In this study, we tested the potential of cellulose acetate (CA) and regenerated cellulose (RC) scaffolds for growing functional cardiac cell constructs in culture. Specifically, we demonstrate that CA and RC surfaces are promoting cardiac cell growth, enhancing cell connectivity (gap junctions) and electrical functionality. Being optically clear and essentially non-autofluorescent, CA scaffolds did not interfere with functional optical measurements in the cell constructs. Molding to follow fine details or complex three-dimensional shapes are additional important characteristics for scaffold design in tissue engineering. Biodegradability can be controlled by hydrolysis, de-acetylization of CA and cytocompatible enzyme (cellulase) action, with glucose as a final product. Culturing of cardiac cells and growth of tissue-like cardiac constructs in vitro could benefit from the versatility and accessibility of cellulose scaffolds, combining good adhesion (comparable to the standard tissue-culture treated polystyrene), molding capabilities down to the nanoscale (comparable to the current favorite in soft lithography-polydimethylsiloxane) with controlled biodegradability.