Chronic stretch of engineered heart tissue induces hypertrophy and functional improvement

Abstract

To examine the influence of chronic mechanical stretch on functional behavior of cardiac myocytes, we reconstituted embryonic chick or neonatal rat cardiac myocytes to a 3-dimensional engineered heart tissue (EHT) by mixing freshly isolated cells with neutralized collagen I and culturing them between two Velcro-coated silicone tubes, held at a fixed distance with a metal spacer. After 4 days, EHTs were subjected to a phasic unidirectional stretch for 6 days in serum-containing medium. Compared to unstretched controls, RNA/DNA and protein/cell ratios increased by 100% and 50%, respectively. ANF mRNA and alpha-sarcomeric actin increased by 98% and 40%, respectively. Morphologically, stretched EHTs exhibited improved organization of cardiac myocytes into parallel arrays of rod-shaped cells, increased cell length and width, longer myofilaments, and increased mitochondrial density. Thus, stretch induced phenotypic changes, generally referred to as hypertrophy. Concomitantly, force of contraction was two- to fourfold higher both under basal conditions and after stimulation with calcium or the beta-adrenergic agonist isoprenaline. Contraction kinetics were accelerated with a 14-44% decrease in twitch duration under all those conditions. In summary, we have developed a new in vitro model that allows morphological, molecular, and functional consequences of stretch to be studied under defined conditions. The main finding was that stretch of EHTs induced cardiac myocyte hypertrophy, which was accompanied by marked improvement of contractile function.

A Novel Miniaturized Multimodal Bioreactor for Continuous In Situ Assessment of Bioartificial Cardiac Tissue During Stimulation and Maturation

“…A reduction in a-MHC expression levels, reflecting changes in gene expression more toward a fetal phenotype, 20 was also described for three-dimensional cultures of murine embryonic stem cell-derived cardiomyocytes after application of 10% stretch at 1 Hz; however, higher frequencies led to the opposite effect. 31 At the same time, we were able to detect upregulation of ANF expression both after mechanical and b-adrenergic stimulation, as was described before for stretched monolayer 19 and threedimensional cultures of rat cardiomyocytes, 14 as well as ventricles of the hypertrophic rat heart. 33 From the quantitative real-time PCR data and the force measurements, we conclude that the mechanical stimulation applied in our bioreactor led to moderate hypertrophy in cardiomyocytes within the BCTs, which had a positive effect on systolic forces.…”

“…To investigate the influence of mechanical load on engineered cardiac tissue, motorized stretching devices for the application of defined strain have been described. 14,15,31 Traditional mechanical testing devices to measure mechanical properties of tissueengineered constructs typically require discontinuation of the cultivation process and therefore provide only end-point measurements of the respective samples. On the other hand, sophisticated bioreactor systems have been developed allowing for long-term monitoring of contractile activity via software-based analysis of video-optically captured contractions.…”

“…Therefore, the aim of this study was to generate a bioreactor for the development and testing of miniaturized bioartificial tissue, enabling smallscale stem cell-based cardiac tissue engineering. It has been proposed that mechanical stretch can improve tissue formation and contractility of bioartificial cardiac grafts generated from primary chicken cardiomyocytes 14 or cardiomyocytes of neonatal and fetal rodent origin. 15,16 This has also been demonstrated for human heart cells seeded on gelatin scaffolds.…”

“…However, it has been difficult to directly monitor the effects of mechanical stretch and other stimulants continuously during the course of tissue formation. Usually, measurements require transfer of the generated tissue from a cultivation vessel to a measurement device or organ bath chamber 14,15,22 ; therefore, force measurement has been predominantly used as an end-point analysis. With this approach, long-term data acquisition can only be managed with numerous replicate samples requiring a multiple of the cell numbers that would be needed in continuous analyses.…”

Endothelial cells guided by immobilized gradients of vascular endothelial growth factor on porous collagen scaffolds

“…Neonatal rat heart cells are cultivated in a mixture of collagen gel, Matrigel® and later on growth factors and subjected to unidirectional and cyclic mechanical stetch at 10% strain and 2 Hz. Under these conditions, the cells formed highly differentiated cardiac muscle synctium that exhibited contractile and electrophysiological properties of working myocardium [24]. Multiple circular strips of constructs were combined to form a large-scale implantable tissue patch.…”