A simplified protocol for culture of murine neonatal cardiomyocytes on nanoscale keratin coated surfaces

Jain, Aditi and Ravi, Venkatraman and Muhamed, Jaseer and Chatterjee, Kaushik and Sundaresan, Nagalingam R. (2017) A simplified protocol for culture of murine neonatal cardiomyocytes on nanoscale keratin coated surfaces. In: International Journal of Cardiology, 232 . pp. 160-170. ISSN 01675273

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Abstract

Objective We aim to develop a simple, efficient and cost-effective protocol for culturing the neonatal cardiomyocytes using keratin derived from human hair, which can be used for studying cardiac hypertrophy in vitro. Methods Keratin was extracted from human hair and applied as nanoscale coating onto the culture dishes. Physical parameters such as surface morphology and roughness of the coating were studied by SEM and AFM. Cardiomyocyte specific markers were assessed by immunofluorescence. Signaling pathways activated in hypertrophy were analyzed by western blotting and changes in the expression of fetal genes were analyzed by qPCR. The changes in the calcium fluxes were observed microscopically using Fluo-4. Results Keratin coated surfaces displayed a uniform coating and comparable roughness across dishes. Our optimized protocol for isolating cardiomyocytes yielded up to ~ 106 cells per heart. Characterization of cardiomyocytes with specific markers revealed that they can attach, grow and show spontaneous contractions on keratin-coated substrates similar to fibronectin-coated surfaces. Phenylephrine (PE) treated cardiomyocytes grown on keratin-coated substrates exhibited increased cell size, sarcomere organization and perinuclear ANP expression indicating the development of cardiac hypertrophy. In addition, we observed increased activation of Akt and ERK pathways, induction of the fetal genes and increased protein synthesis upon PE treatment, which are characteristics of cardiomyocyte hypertrophy. The protocol was extended to mouse cardiomyocytes and found to show similar results upon examination. Conclusion We demonstrate that keratin can act as an efficient yet cost effective alternative substrate for the attachment, growth and differentiation of neonatal murine cardiomyocytes.

Item Type:	Journal Article
Publication:	International Journal of Cardiology
Publisher:	Elsevier Ireland Ltd
Additional Information:	The copyright of this article belongs to the Elsevier Ireland Ltd.
Keywords:	Cardiac hypertrophy; Cardiomyocytes; Human hair; Keratin; Nanoscale coating
Department/Centre:	Division of Interdisciplinary Sciences > Centre for Biosystems Science and Engineering Division of Mechanical Sciences > Materials Engineering (formerly Metallurgy)
Date Deposited:	30 May 2022 04:55
Last Modified:	30 May 2022 04:55
URI:	https://eprints.iisc.ac.in/id/eprint/72840

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