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Engineered Biomaterial Surfaces for Neonatal Murine Cardiomyocyte Culture Toward Understanding Cardiac Hypertrophy.

Introduction

Cardiomyocytes, the principal cell type found in the heart orchestrates the cardiac contractions and ensures efficient blood flow throughout the body. Myocardial injury causes significant loss of cardiomyocytes and often results in heart failure owing to its limited regenerative potential, making it one of the leading causes of morbidity and mortality worldwide. In order to study the characteristics of the heart, cardiomyocytes are the principal cells upon which many experiments have been validated. It is a challenge to obtain superior quality cardiomyocytes in terms of viability, spontaneous contractions and increased yield of cells in vitro. One of the challenges in cardiomyocyte culture is to select a suitable substrate or matrix on which the cells can adhere. For this purpose, a variety of extracellular matrix (ECM) proteins such as collagen, fibronectin, laminin and gelatin have been shown to be useful, most of which are sourced from slaughtered animals whilst fibronectin is purified from human plasma. Besides the sacrifice involved, products prepared from animal sources carry a significant risk of contamination and heterogeneity. Recent research has focused on the alternatives for these conventionally used substrate coatings. Over the past decade, a family of intermediate filament proteins, namely keratins has been gaining substantial interest as a substrate for surface coating. Even though they are structural proteins, they possess great potential as biomaterials due to its abundance, biodegradability, bioactivity and the fact that they can be easily harvested from discarded human hair. Keratins are currently being explored for tissue engineering applications and used for making 3D scaffolds, hydrogels and nanofibers that can more realistically mimic the in vivo cell surrounding and behaviors. Although it has previously been shown to be a useful coating material for various cell lines including human mesenchymal stem cells (hMSCs), keratins have never been studied for the cell attachment and growth pattern of primary neonatal cardiomyocytes obtained from murine model systems. In this study, we investigated the efficacy of keratin derived from human hair as an unconventional nanoscale substrate coating for culturing primary cardiomyocytes from neonatal murine hearts.

Outcomes of the study

Physical characterization by SEM and AFM revealed the presence of keratin protein adsorbed as nano structures on the tissue culture polystyrene plate (TCPS) whose surface roughness was also in order of few nanometers (Figure 1). The keratin and fibronectin coated surfaces were found to be of comparable roughness with mean roughness value (Ra) of 1.20 [+ or -] 0.07 nm and 2.16 [+ or -] 0.08 nm, respectively. Previously, Taraballi et al. showed that nanoscale topography of human hair keratin influences cellular adhesion and proliferation in murine fibroblasts. These uniform nanoscale features observed on keratin coated substrates further conferred the use of these substrates to enhance surface compliance for culturing of primary cardiomyocytes in addition to the presence of cell adhesive sites on keratin and its bioactivity.

While other conventionally used substrates such as fibronectin and gelatin are obtained from animal sources and involve cumbersome and expensive extraction procedures, keratin can be easily extracted from human hair using a simple in house procedure, making it a cost effective substitute. Our approximate calculation based on the in house preparation of keratin and commercially procured fibronectin reveals that the cost is reduced by approximately 9095%.

Our optimized protocol for culturing of cardiomyocytes yielded atleast ~[10.sup.6] cells per heart which involved digestion of neonatal murine hearts with 0.2% trypsin, 0.4mg/ml collagenase type II and 0.01M of D-Glucose prepared in phosphate buffer saline (PBS). Characterization of cardiomyocytes with specific markers revealed that they can attach, grow and show spontaneous contractions on keratin coated substrates similar to fibronectin/gelatin coated surfaces. We also observed that the seeding efficiency on keratin coated surfaces was comparable to that on fibronectin coated surface.

Cardiomyocyte differentiation was assessed by immunofluorescence. Cardiac hypertrophy was induced using Phenylephrine (PE) and Isoprotenol (ISO). Various signaling pathways are activated in response to hypertrophic stimuli of which activation of the PI3K/Akt and the ERK1/2 axis plays a crucial role. Activation of both these pathways stimulates mTOR phosphorylation which activates key regulators of translation and hence enhances protein synthesis. Akt is also a critical mediator of cell size and the increased activation of Akt in hypertrophy leads to larger sized cells. Consistent with this we find increased phosphorylation and activation of Akt, ERK1 /2 and mTOR in PE treated cardiomyocytes growing on keratin. We also observed enhanced protein synthesis and increased cell size as well. At the transcriptional level the hypertrophy is accompanied by activation of fetal gene program. We observed increased expression of fetal genes in PE treated cardiomyocytes growing on keratin coated surfaces further confirming the development of hypertrophy. Another hallmark of development of cardiac hypertrophy is the increased levels of ANP and BNP. As circulating hormones originating from heart, they are considered to compensate for failing heart due to their diurectic, natriuretic and vasodilating properties. We were able to show that upon treatment with PE or ISO the cells on keratin coated TCPS showed increased ANP release.

A critical regulator of cardiomyocyte contractibility is calcium ([Ca.sup.2+]) mobilization. We visualized the [Ca.sup.2+] flux in neonatal cardiomyocytes plated on keratin using the fluorescent calcium binding dye, Fluo4. We observed drastic increase in the frequency as well the intensity of the signal spikes in the PE treated cardiomyocytes grown on keratin coated surfaces which is characteristic of cardiomyocyte hypertrophy.

We were further created patterned micro-environments using silicon to achieve anisotropy in cells as is observed in the in vivo histology sections of the heart (Figure 2). Patterned substrates were characterized using SEM. Substrates of varying dimensions (35gm wide grooves and 3-5gm wide ridges) were investigated, with smooth surfaces used as controls. Cell-matrix interaction was studied using SEM. The cells cultured on micro-ridges were found to be elongated and aligned along the grooves forming a well-developed contractile apparatus, as demonstrated by sarcomeric aactinin staining and calcium staining. This not only resembled in vivo tissue in terms of the parallel arrangement of cells but also showed presence of antigrade calcium current as seen in the functional heart tissue. As opposed to cells on the micro-ridges, cells adhered in a random manner on the smooth controls. The calcium current was more diffused and less oriented on the smooth control as compared to the ridges.

Summary

Taken together, these results highlight the biocompatibility, suitability and ease of using keratin coated surfaces for neonatal murine cardiomyocyte culture to study cardiac hypertrophy in vitro (6) and also the emphasize the importance of topography in assessing cardiac function. We describe an economical and efficient protocol, for murine cardiomyocyte culture on surfaces coated with keratin. We further find that our protocol works equally well for cardiomyocytes isolated from both mouse and rat hearts. Importantly, we also validated that our culture protocol can serve as a model for studying cardiac hypertrophy in vitro with important implications for improving human health.

References

[1.] Laflamme MA, Murry CE: Heart regeneration. Nature 2011; 473:326-335

[2.] Reichl S: Films based on human hair keratin as substrates for cell culture and tissue engineering. Biomaterials 2009; 30:6854-6866

[3.] Rouse JG, Van Dyke ME: A Review of Keratin-Based Biomaterials for Biomedical Applications. Materials 2010; 3:999-1014

[4.] Ghosh LD, Ravi V, Sanpui P, Sundaresan NR, Chatterjee K: Keratin mediated attachment of stem cells to augment cardiomyogenic lineage commitment. Colloids and surfaces B, Biointerfaces 2016; 151:178-188

[5.] Sundaresan NR, Gupta M, Kim G, Rajamohan SB, Isbatan A, Gupta MP: Sirt3 blocks the cardiac hypertrophic response by augmenting Foxo3a-dependent antioxidant defense mechanisms in mice. The Journal of clinical investigation 2009; 119:2758-2771

[6.] Jain A, Ravi V, Muhamed J, Chattrjee K, Sundaresan NR: A simplified protocol for culture of murine neonatal cardiomyocytes on nanoscale keratin coated surfaces. International Journal of Cardiology 2017; 232: 160-170

Aditi Jain (a), Jafar Hasan (b), Venkatraman Ravi (c), Jaseer Muhamed (c), Kaushik Chatterjee (#,a,b) and Nagalingam R. Sundaresan (#c)

(a) Centre for Biosystems Science and Engineering, (b) Department of Materials Engineering, (c) Cardiovascular and Muscle Research Laboratory, Department of Microbiology and Cell Biology, Indian Institute of Science, Bengaluru 560012, India

Received 1 October 2017

Accepted 25 October 2017

Published online 30 April 2018

* Coresponding authors.

E-mail address: k.chatterjee@materials.iisc.ernet.in (Dr. Kaushik Chatteejee); E-mail address: rsundaresan@mchl.iisc.ernet.in . Nagalingam R. Sundaresan)

Caption: Figure 1: Cardiomyocytes on keratin coated surfaces

Caption: Figure 2: Cardiomyocytes on surface with microscale topography
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Title Annotation:Original Article
Author:Jain, Aditi; Hasan, Jafar; Ravi, Venkatraman; Muhamed, Jaseer; Chatterjee, Kaushik; Sundaresan, Naga
Publication:Trends in Biomaterials and Artificial Organs
Date:Jan 1, 2018
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