Publication: Characterization of the effect of the substrate over functional and electrophysiological properties during culture of cardiomyocytes
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2015-07
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2015-07-08
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Abstract
Heart failure is a common, costly and potentially fatal condition commonly caused by cardiac cells depletion. Current therapies are aimed at protecting surviving cardiomyocytes, but they are unable to produce cardiac regeneration. Tissue engineered cardiac patches seem promising as cardiac repair tools, but they still pose many limitations as they do not mimic in vivo behavior. Recently, the use of flexible membranes for cardiac cell culture has been suggested as determinant in cardiomyocytic properties. The goal of this study is to compare for the first time key cardiomyocyte functional properties for cardiac repair such as proliferation, migration and displacement, and electrophysiological properties of HL-1 cardiac cell line in two different materials: (1) rigid Petri dishes and (2) flexible PDMS (polydimethylsiloxane) silicon wells. The study was carried out in the Laboratorio de Órganos y Matrices Bioartificiales belonging to the Instituto de Investigación Sanitaria Gregorio Marañón.
In order to assess these properties, HL-1 cells were cultured on both substrates and different tests were performed. Proliferation assay was carried out using alamarBlue® colorimetric test to calculate the percentage of reduction which is directly related to cell proliferation. Migration and displacement were determined by performing wound tests followed up using time-lapse imaging, and quantified using custom software made in Matlab. To study characteristic properties of cardiomyocytes, impulse propagation was recorded using optical mapping techniques, and results were analysed using custom software developed in Matlab to obtain conduction velocity information. At molecular level, expression of genes coding for proteins involved in impulse generation and propagation was analysed using qPCR (quantitative Polymerase Chain Reaction) technique.
Results show that HL-1 cells were able to grow and retain an adult cardiomyocyte phenotype on both substrates and spontaneous contractile activity was kept. In general, proliferation of HL-1 cells was confirmed to be very high, in opposite to migration and displacement. Proliferation was higher at early stages in Petri, but as the culture grew, proliferation rate got higher in silicon. Migration and displacement were quite low as wounds did not close within 60 hours, which is characteristic of adult cardiomyocytes. The results and observations suggest that these cells colonize new areas only by proliferation. Regarding the electrophysiological properties, conduction velocity showed to be higher in silicon wells (150 %). This result was supported by a higher expression of genes involved in action potential.
Results from this study support our hypothesis that the use of flexible membranes induces a more similar cardiac phenotype to that shown in vivo, especially characterized by higher conduction velocities. This opens new insights into cardiac patches, confirming that mechanical characteristics of the substrate play a role in cardiac cell phenotype. Silicon wells and other flexible membranes will allow further mimicking the in vivo environment by growing cardiac cells under mechanical and electrical stimulation.
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Cardiac cell culture, Cardiomyocytes, Electrophysiological properties