Beating behavior of primary neonatal cardiomyocytes and cardiac-differentiated P19.CL6 cells on different extracellular matrix components

Abstract

Stem cell-based therapy in cardiac tissue engineering is an emerging field that shows great potential for treating heart diseases. However, even preliminary issues, such as the ideal niche for cardiomyocytes, have not been clarified yet. In the present study, the effects of extracellular matrix (ECM) components on the beating duration of neonatal rat cardiomyocytes (RCMs) and on the cardiac differentiation of P19.CL6 carcinoma stem cells were studied. RCMs were cultured on gelatin-, fibronectin-, and collagen type I-coated dishes and on noncoated polystyrene dishes, and their beating rate, beating duration, and cardiac gene expression were evaluated. The beating period and the expression of troponin T type-2 (TNNT2) and troponin C type-1 (TNNC1) of cardiomyocytes cultured on gelatin-coated dishes were longer and higher than for those on dishes with other coatings. For the cardiac differentiation of P19.CL6 cells, troponin T type-2 expression on gelatin- and fibronectin-coated dishes was five times that on collagen type I-coated dishes or polystyrene dishes 11 days after induction. These results indicate that a gelatin-coated surface has a high ability not only to maintain the cardiac phenotype but also to enhance cardiac differentiation.

Effects of Various Extracellular Matrix Proteins on the Growth of HL-1 Cardiomyocytes

“…However, after the initial growth period, within 24 h, cells cultured on fibronectin-treated and laminintreated surfaces showed significantly high levels of adhe- sion. These results support previous work showing that fibronectin and laminin play crucial roles in increasing cell-surface adhesion for various cell types [von der Mark and Ocalan, 1989;Adams and Watt, 1993;Hidalgo-Bastida et al, 2007;Miskon et al, 2009]. In contrast, the collagen I-treated and gelatin-treated surfaces showed low levels of cell adhesion, and cell numbers were sparse in comparison.…”

“…Previous studies have shown that the action potential and beating duration of cardiomyocytes were higher on gelatin-treated surfaces than on fibronectin-treated or laminin-treated surfaces [Miskon et al, 2009]. In addition, considerably higher levels of troponin were found on fibronectin-treated and gelatin-treated surfaces than on collagen-treated surfaces.…”

“…Although the cellular environment is complex, the addition of even one physical or biochemical cue in vivo can affect the behavior of cultured cells. For example, treating the surfaces of cell culture systems with various ECM proteins significantly affects cell growth, adhesion, segmentation, proliferation, characteristics, metabolism and function [Kim et al, 2006;Akhyari et al, 2008;Miskon et al, 2009]. Therefore, pretreatment of culture substrates with appropriate ECM proteins is essential for cell culture and analysis [Miskon et al, 2009].…”

Increasing Thermal Stability of Gelatin by UV-Induced Cross-Linking with Glucose

“…Gelatin has many properties which make it an ideal starting material for cell scaffolds. Native gelatin is easily degraded by proteases, possesses minimal antigenicity [52, 53], and is very soluble in aqueous solution [54]. Based on our results, cross-linking gelatin renders it insoluble at physiological temperatures; prior work also correlates greater cross-linking density with reduced rate of enzymatic degradation [32].…”

Differential responses of induced pluripotent stem cell-derived cardiomyocytes to anisotropic strain depends on disease status

“…The molds were removed and immediately coated with a pre-mixed solution of an extracellular matrix protein (5μg/cm 2 ) in serum-free medium. Four protein coatings were tested; gelatin as it increases beating behavior and functions of cardiomyocytes (Koch-Schneidemann et al, 1994; Miskon et al, 2009), laminin as it improves cell attachment of adult cardiomyocytes (Koch-Schneidemann et al, 1994), fibronectin as it enhances the functions of cardiomyocytes derived from stem cells (Burridge et al, 2014; Gandaglia et al, 2012), and vitronectin because it increases the adhesion of cardiomyocytes (Braam et al, 2008; Michel, 2003). The resulting samples were covered with aluminum foil and left overnight on a shaker plate at 4°C prior to seeding cells.…”