Stiffness of the substrate influences the phenotype of embryonic chicken cardiac myocytes

Abstract

Abstract:We examined the effect of substrate stiffness on the beating rate, force of contraction, and cytoskeletal structure of embryonic chicken cardiac myocytes by culturing them on laminin-coated polyacrylamide (PA) substrates. Cells cultured on PA substrates with elasticity comparable to that of the native myocardium (18 kPa) exhibited the highest beating rate during the first few days of culture. The initial beating rate of individual cells on all the substrates varied significantly but began to converge within 5 days. We also examined the focal adhesions (FAs) and cytoskeletal structure on different substrates via confocal microscopy and found a higher percentage of FAs on tissue culture (TC) plastic dishes compared with the softer PA gels. Furthermore, highly aligned sarcomeric striations were clearly visible on 18 kPa, 50 kPa, and TC dish, whereas cells on 1 kPa only showed nonaligned diffused striations. The force of contraction on these substrates was measured using a micro-electromechanical system force sensor, which showed that the force of contraction for the cells on TC dishes (F ¼ 71.30 6 6.38 nN) was significantly larger than those cultured on the 18-kPa PA gel (F ¼ 30.16 6 3.83 nN). This is most likely due to the formation of higher percentage of FAs on the TC dishes compared with fewer FAs on the softer gels. Our cumulative findings can have a significant impact on the design of 3D cardiac tissue engineered scaffolds.

Effect of Substrate Mechanics on Cardiomyocyte Maturation and Growth

“…5,6,16,21,22 A main reason that substrate mechanics affect the CMs function is the fact that CMs are characterized by their rhythmic beating, 11 as previously mentioned. During contraction, the calcium transient size and duration regulates the contractile force.…”

“…14,15 There are various forms of mechanical loadings that develop in cardiac patches and affect cell maturation and development, and these are due to the microenvironmental cues, including cell shape, extracellular matrix (ECM), biomolecules, neighboring cells, substrate stiffness, applied strain, and topology. The CM development is best on substrates of comparable stiffness to the native tissue 16 ( Table 1 summarizes the elasticity of different types of heart tissue). However, the influence of all mechanical factors on the CMs maturation should be understood in order to aid in the development of cardiac cell therapies.…”

“…This confirmed the hypertrophy that the differentiation of CM phenotypes is influenced by the substrate stiffness showing that softer substrates induce a more mature phenotype. 33 Furthermore, the effect of substrate stiffness on CM beating has also been investigated, 16 by seeding CMs, derived from embryonic (day 8) chicks, on PA gel substrates of different stiffness for 1-5 days. The PA gel substrates that were considered had a stiffness of 1, 18, and 50 kPa.…”

“…16 CMs beat synchronously when there is direct contact between each other, exerting more force compared with individual cells 34 ; hence, after the fifth day of culture, the beating frequencies across the different substrates became more uniform, as the CMs had proliferated and came into contact with each other, with the fastest beating on the 50 kPa gel. This explains why some studies show that CMs function better on relatively stiffer substrates compared with other studies.…”

“…Thus, soft matrices over time showed less organized sarcomeres as compared with the stiff matrices that had wellorganized and aligned myofibrils. 16 Although myofibril formation in CMs has been extensively studied in vitro, the difficulty of obtaining a rhythmically contractile phenotype on hard substrates is clearly illustrated. 5 Rhythmic beating of cultured CMs depends on the efficient spreading of the cells on the substrate and on myofibril reassembly.…”