Scholarly article on topic 'The functional properties of nephronectin: An adhesion molecule for cardiac tissue engineering'

The functional properties of nephronectin: An adhesion molecule for cardiac tissue engineering Academic research paper on "Basic medicine"

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{Nephronectin / "Tissue engineering" / RGD / "Cell adhesion" / Cardiomyocytes / "Connexin 43"}

Abstract of research paper on Basic medicine, author of scientific article — Chinmoy Patra, Filomena Ricciardi, Felix B. Engel

Abstract Despite significant advances in preventive cardiovascular medicine and therapy for acute and chronic heart failure, cardiovascular diseases remain among the leading causes of death worldwide. In recent years cardiac tissue engineering has been established as a possible future treatment option for cardiac disease. However, the quality of engineered myocardial tissues remains poor. In tissue engineering it is important that the scaffold allows cells to attach, spread, maintain their differentiation status or differentiate into functional cells in order to exhibit their physiological function. Here, we have investigated the suitability of the natural cardiac extracellular matrix component nephronectin as an adhesive material for cardiac tissue engineering. Primary neonatal rat cardiomyocytes were seeded on collagen-, fibronectin- or nephronectin-coated glass coverslips and analyzed for cell adhesion, cellular metabolic activity, response to extracellular stimuli, cell-to-cell communication, differentiation and contractility. Our data demonstrate that most neonatal cardiomyocytes attached in an RGD domain-dependent manner within 18 h to nephronectin. The cells exhibited high metabolic activity, responded to growth factor stimuli and maintained their differentiation status. Moreover, nephronectin promoted sarcomere maturation and alignment, cell-to-cell communication and synchronous contractions. In conclusion, our findings demonstrate that nephronectin has excellent properties for cardiomyocyte adhesion and function and thus has the potential to improve current cardiac tissue engineering approaches.

Academic research paper on topic "The functional properties of nephronectin: An adhesion molecule for cardiac tissue engineering"

Biomaterials

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Biomaterials

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The functional properties of nephronectin: An adhesion molecule for cardiac tissue engineering

Chinmoy Patra, Filomena Ricciardi, Felix B. Engel*

Department of Cardiac Development and Remodelling, Max-Planck-Institute for Heart and Lung Research, Parkstrasse 1, 61231 Bad Nauheim, Germany

ARTICLE INFO ABSTRACT

Despite significant advances in preventive cardiovascular medicine and therapy for acute and chronic heart failure, cardiovascular diseases remain among the leading causes of death worldwide. In recent years cardiac tissue engineering has been established as a possible future treatment option for cardiac disease. However, the quality of engineered myocardial tissues remains poor. In tissue engineering it is important that the scaffold allows cells to attach, spread, maintain their differentiation status or differentiate into functional cells in order to exhibit their physiological function. Here, we have investigated the suitability of the natural cardiac extracellular matrix component nephronectin as an adhesive material for cardiac tissue engineering. Primary neonatal rat cardiomyocytes were seeded on collagen-, fibronectin- or nephronectin-coated glass coverslips and analyzed for cell adhesion, cellular metabolic activity, response to extracellular stimuli, cell-to-cell communication, differentiation and contractility. Our data demonstrate that most neonatal cardiomyocytes attached in an RGD domain-dependent manner within 18 h to nephronectin. The cells exhibited high metabolic activity, responded to growth factor stimuli and maintained their differentiation status. Moreover, nephronectin promoted sarcomere maturation and alignment, cell-to-cell communication and synchronous contractions. In conclusion, our findings demonstrate that nephronectin has excellent properties for cardiomyocyte adhesion and function and thus has the potential to improve current cardiac tissue engineering approaches.

© 2012 Elsevier Ltd. All rights reserved.

Article history: Received 16 February 2012 Accepted 5 March 2012 Available online 20 March 2012

Keywords: Nephronectin Tissue engineering RGD

Cell adhesion Cardiomyocytes Connexin 43

1. Introduction

Cardiovascular diseases (CVDs) represent a major socioeconomic burden. They are responsible for around 30% of death worldwide claiming as many lives as cancer, chronic lower respiratory diseases, accidents and diabetes combined. The estimated cost of CVD in 2008 was US$ 297.7 billion in the US alone accounting for around 16% of the total health expenditures and the future burden of heart disease is expected to further increase [1].

The primary cause of most CVDs is reduced heart function due to the irreversible loss of heart muscle cells, the cardiomyocytes [2—4]. The mammalian heart cannot regenerate and a possible endogenous regenerative capacity can so far not be enhanced to generate enough cardiomyocytes to improve heart function after injury [5]. The only available therapy to effectively replace damaged hearts is at the moment heart transplantation. Unfortunately, there are not enough donor hearts to match the demand [6,7]. Therefore,

* Corresponding author. Tel.: +49 6032 705248; fax: +49 6032 705211. E-mail address: felix.engel@mpi-bn.mpg.de (F.B. Engel).

0142-9612/$ - see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2012.03.021

it is critically important to develop technologies aimed at providing new cardiomyocytes to diseased hearts.

One approach to reverse heart disease is the application of stem cells. In the past it has been proven that transplanted fetal and neonatal cardiomyocytes could functionally integrate and enhance recipient cardiac function. Thus, multiple stem cell types have been tested for re-population of the injured myocardium. However, there appears little or no differentiation of the engrafted cells into cardiomyocytes in vivo [8—10]. In addition, injection of stem cells in the heart muscle causes severe cell loss due to shear stress. Injection in the blood circulation requires a homing signal to the damaged tissue, which is still elusive. One solution to this problem might be the integration of tissue engineering and stem cell biology towards creating functional heart muscle in vitro [11,12]. The engineered tissue can be adjusted according to the needs of a patient before implantation and materials used as scaffolds might enhance cardiomyocyte differentiation. Importantly, it has been shown that engineered cardiac tissue based on type I collagen gel, Matrigel and primary rat postnatal cardiomyocytes improves LV function after myocardial infarction [13]. However, the used material appears in its present form not suitable for clinical

application. Thus, there is a need for other scaffold materials or modifications of existing scaffolds [14,15].

It has been shown that the natural cardiac extracellular matrix (ECM) after decellularization is suitable to engineer cardiac tissue [16]. It provides adhesion substrates, imparts structural support, stores and sequesters soluble factors, and transduces mechanical signals [17]. In addition, it plays an important role in directing tissue formation during development. For this reason, it is important to define natural components of the cardiac ECM during development and to analyze their properties. In the future this might help to engineer scaffolds by incorporating ECM-derived peptides into biomaterials to mimic biochemical signals required to form complex cardiac tissue patches [15].

Recently, we have discovered nephronectin (Npnt) as a transiently expressed ECM protein regulating valve formation during cardiac development [18]. It contains an N-terminal signal peptide followed by EGF-like repeats, an RGD sequence and a C-terminal MAM domain [19]. Nephronectin is expressed in cardiomyocytes throughout the heart and is secreted into the cardiac jelly, which is in direct contact to cardiomyocytes and endocardial cells [18]. The presence of an RGD sequence suggest that it will affect car-diomyocyte behavior as it has been demonstrated that RGD immobilization in an alginate scaffold increased neonatal rat car-diomyocyte adhesion, prevented cell apoptosis and accelerated cardiac tissue regeneration [20]. Besides its importance for valve formation Npnt has been shown to play a role in kidney development [21], arrector pili muscle formation [22] and osteoblast differentiation [23]. However, the function of Npnt remains poorly understood.

In this study we have investigated the potential of Npnt as an adhesion molecule for 3-days-old cardiac cells by analyzing cellular metabolic activity, cell-to-cell communication, cell cycle activity, differentiation status and contractility. For comparison we utilized gelatin and fibronectin.

2. Materials and methods

2.1. Rat neonatal cardiac cell isolation and cell culture

Animal experiments were approved by the local Committee for Care and Use of Laboratory Animals (Regierungspräsidium Darmstadt, Gen. Nr. B 2/202) and conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Cardiac cells were isolated from 3-days-old Sprague Dawley rats as previously reported [24]. Cardiomyocytes were enriched by preplating for 2 h (2 mM L-glutamine, 10% FBS and 100 U/mg/ml Pen/Strep in DMEM/F12 medium). Non-attached cells were collected, centrifuged, resuspended in neonatal cardiomyocyte medium (3 mM Na pyruvate, 2 mM L-glutamine, 0.1 mM ascorbic acid, 1 mg/ml insulin/transferrin/selenium, 0.2% BSA and 100 U/mg/ml Pen/Strep in DMEM/F12 medium) supplemented with 5% horse serum and seeded in a 24-well plate at 500 ml per well for 48 h if not stated otherwise. Cells were cultured at 37 °C in a 5% CO2/95% air humidified atmosphere.

2.2. Coating procedure for cell culture

Cells were cultured on coated glass coverslips (0 12 mm, Karl Hecht GmbH, Germany), which were placed in 24-well tissue culture plates. Before coating coverslips were treated once with 70% ethanol for 5 min and twice with 100% ethanol for 3 min. For gelatin coating air-dried coverslips were incubated with 1% (w/v) gelatin (Sigma)/water for 2 h at 37 °C. Gelatin was afterwards aspirated and coverslips were treated for 30 min with UV light. For Npnt (R&D Systems GmbH, Germany) and fibronectin (PromoCell, Germany) coating, air-dried coverslips were incubated 2 h with 100 ml of 10 mg/ml fibronectin or Npnt in PBS at 37 °C. Cells were seeded immediately after the aspiration of the protein solutions.

2.3. Immunocytochemistry

Cells were fixed with 3.7% formaldehyde (Sigma) for 15 min. Different antibodies were diluted in blocking buffer (5% goat serum, 0.2% Tween-20 in PBS), and all steps were carried out at room temperature. For permeabilization, samples were treated with 0.5% Triton X-100/PBS for 10 min. BrdU incorporation assays were performed by incubating permeabilized cells in 2 N HCl/1%Triton X-100 for 30 min. Samples

were blocked with blocking buffer for 20 min and incubated for 1 h with primary antibodies (mouse monoclonal anti-a-smooth muscle actin, 1:100, Sigma; mouse monoclonal anti-sarcomeric a-actinin; rat monoclonal anti-BrdU, 1:100, Abcam; rabbit polyclonal anti-connexin 43, anti-troponin 1,1:50, Santa Cruz Biotechnology, and rat monoclonal anti CD31,1:50, BD Pharmingen). Either ALEXA 488- or ALEXA 594-conjugated antibodies (1:200, Molecular Probes) were used to detect immune complexes. 4',6'-diamidino-2-phenylindole (DAPI, Sigma) (0.5 mg/mL PBS) was used to visualize DNA. 1solectin staining was performed on live cells as previously described [25]. Briefly, cells were washed with 10% FCS supplemented PBS (FCS-PBS), incubated for 30 min on ice with Alexa 488 conjugated Isolectin (20 mg/ml, Molecular probes, Germany)/FCS-PBS, again washed in FCS-PBS and analyzed. All images were captured using a Leica fluorescence microscope.

2.4. Cell attachment assay

Protein coated glass coverslips were seeded with 1.2 x 105 cells in 500 ml neonatal medium supplemented with 5% HS. After 6,18 and 48 h the number of attached cells was quantified by calculating the average number of attached cells from 10 randomly chosen microscopic fields for each independent experiment.

2.5. Cellular metabolic activity

1n order to determine the cell viability and cytocompatibility of different matrices 0.4 x 105 cardiomyocyte-enriched cardiac cells were seeded and cultured for up to 8 days with media changes on every second day. Metabolic activity was determined by MTT assays (Sigma) after 2,4, 6 and 8 days of culture.

2.6. Integrin receptors blocking assays

Cardiomyocyte-enriched cells were incubated for 30 min with 300 mM of an Arg-Gly-Glu-Ser (RGES) peptide (American peptide Company, Sunnyvale, CA) as control or an Arg-Gly-Asp-Ser (RGDS) peptide (Calbiochem, La Jolla, CA) at 37 °C [26]. Afterward cells were allowed to attach either for 24 h or 48 h.

2.7. Cell cycle activity

1n order to analyze cell cycle activity coated coverslips were seeded with 0.8 x 105 cells for 48 h. Subsequently, cells were cultured for another 2 days in neonatal medium and stimulated either with 5% horse serum or with a single treatment of 50 ng/mL FGF1 (R&D Systems) and everyday treatment of 10 mM SB 203580 HCl (p38i) (Tocris). BrdU (30 mM, Sigma) was added for the last 24 h. Cell cycle activity was quantified after 48 h of stimulation by counting 500 to 700 car-diomyocytes from 6 to 9 randomly chosen microscopic fields for each independent experiment.

2.8. Video image analysis

Cardiomyocytes were cultured with 5% HS on different matrices. Movies of beating cardiomyocytes at day 2 were recorded with a Sony HDR-SR12 camcorder and formatted (Wondershare or iSkysoft video converter).

2.9. RNA isolation and gene expression analysis

Total RNA was extracted from cardiomyocyte-enriched cells after 2 days of culture on different matrices by RNeasy mini kit (Qiagen, Germany). cDNA was synthesized by using random hexamer (Fermentas) following standard protocol. Quantitative RT-PCR amplification was performed in a single color real time poly-merase chain reaction system using iQSYBR Green Supermix (Bio-Rad) and primer pairs mentioned in Supplemental Table 1. Semi-quantitative polymerase chain reaction was performed with the same primers following standard protocols.

2.10. Statistical analysis

Data are presented as the mean ± SEM of at least three independent experiments. Statistical significance of differences was evaluated by one way ANOVA followed by Bonferroni's post-hoc test (GraphPad Prism). p < 0.05 was considered statistically significant.

3. Results

3.1. Attachment of cardiac cells

One important criteria for scaffolds used in tissue engineering is their attachment properties. Thus, we cultured 3-days-old postnatal rat cardiomyocytes on nephronectin (Npnt) and determined its adhesive properties. As positive control served fibronectin, a natural cardiac ECM component and established surface coating

material to culture cardiomyocytes [27,28]. Gelatin was used as a negative control as it has poor adhesive properties.

Quantitative analysis of attached cardiac cells after cardiomyocyte-specific staining revealed that cardiomyocyte attachment depended on the surface coating material and the length of the seeding period (Fig. 1A,B). At 6 and 18 h a significantly higher number of cardiomyocytes was attached on Npnt compared to gelatin. However, there was no difference to the positive control fibronectin. In addition, spreading of cardiomyocytes was clearly visible on Npnt and fibronectin but not on gelatin (Fig. 1A). At 48 h the number of attached cardiomyocytes was similar among the different matrices. These data suggest that Npnt is a good matrix for cardiomyocyte attachment.

In the adult heart cardiomyocytes cover around 75% of the heart volume. However, 50%-70% of the cells in the heart are non-myocytes. The main cell types are fibroblasts, endothelial cells, and vascular smooth muscle cells [29,30]. One goal in tissue engineering is to generate a tissue patch that is as similar as possible to cardiac tissue. Thus, it is important to determine if cardiac cells other than cardiomyocytes can attach to Npnt. Our data demonstrate that non-myocyte attachment was neither dependent on time nor on the type of matrix. The majority of non-myocytes (troponin 1-negative) was attached at 6 h (Fig. 1A,C). In car-diomyocyte cultures we found at 44 h beside cardiomyocytes (orange and red arrow) also vascular smooth muscle cells (SMA-positive and troponin 1-negative, yellow arrow) (Fig. 2A). To test how well non-myocytes attach to Npnt we seeded freshly isolated cardiac cells for 2 h. 1mmunostaining analyses demonstrated that there are only very few cardiomyocytes attached but not spread yet (Fig. 2B, orange arrow). 1n contrast, vascular smooth muscle cells (SMA-positive and troponin 1-negative, yellow arrow) and

endothelial cells (CD31- or Isolectin-positive, green arrow) were attached on Npnt-coated coverslips and spread (Fig. 2B,C).

1n conclusion, our data demonstrate that Nephronectin is not only a good substrate for cardiomyocyte attachment but also for other cellular components of the heart.

3.2. Metabolic activity

MTT assays were performed to better evaluate the cyto-compatibility of Npnt. This assay is an indirect measurement of the cellular metabolic activity and was performed on neonatal car-diomyocytes cultured for up to 8 days on the indicated 2D substrates. Cardiomyocyte cultures grown on Npnt and fibronectin exhibited a similar metabolic activity at all examined time points, which was significantly higher at days 2 and 4 compared to the activity of cardiomyocytes grown on gelatin (Fig. 3A). After 6 days of culture we did not observe any significant difference among the cells grown on gelatin, Npnt and fibronectin in regards to cellular activity and the ration of cardiomyocytes to non-myocytes (Fig. 3A,B). 1n summary, Npnt has in comparison to fibronectin no negative effect on the metabolic activity of cardiomyocytes.

3.3. RGD-dependent cell attachment

Similar to fibronectin, Npnt contains an RGD sequence and it has been shown that this RGD sequence can bind with a number of integrin receptors [31,32]. In contrast, gelatin does not have any RGD sequence. Several studies have demonstrated that the RGD sequence in natural as well as synthetic molecules stimulate cell attachment and spreading via activation of cell surface integrin receptors [33]. To investigate whether the superior attachment

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Fig. 1. Cardiomyocytes attachment on Npnt. (A) Cardiomyocyte-enriched cells seeded 6,18 and 48 h on different matrices were stained with anti-troponin 1 antibodies (cardiomyocytes, green) and DAP1 (nuclei, blue). Scale bars: 50 mm (B, C) Quantification of attached cardiomyocytes (B) and non-myocytes (C) (n = 3, mean ± SEM, *: p < 0.05, ns: not significant). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2. Non-myocyte attachment on Npnt. (A) Neonatal rat cardiomyocyte-enriched cells seeded on Npnt for 44 h were stained with anti-troponin I (cardiomyocytes, green) and anti-smooth muscle a-actin (SMA) antibodies (marks smooth muscle cells and a subpopulation of cardiomyocytes, red) and with DAPI (nuclei, blue). Yellow arrow: smooth muscle cell (SMA-positive, troponin I-negative). Orange arrow: mature cardiomyocyte (troponin I-positive). Red arrow: less matured cardiomyocyte (SMA-positive, troponin I-positive). (B) Freshly isolated neonatal rat cardiac cells seeded for 2 h were stained as in A. Orange arrow: attached but not spread cardiomyocyte (troponin I-positive). Yellow arrow: smooth muscle cell (SMA-positive, troponin I-negative). (C) Endothelial cells were identified by CD31 (red) or isolectin staining (green, combined with Differential Interference Contrast, DIC). Nuclei were visualized with DAPI. Scale bars: 50 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

properties of Npnt and fibronectin compared to gelatin are due to their RGD sequences, we performed integrin receptor blocking assays by using RGDS peptides. In order to block all integrin receptors, we incubated cardiomyocyte-enriched cells with RGDS peptides for 30 min at 37 °C prior to cell seeding. RGES peptides were used as a control. MTT assays were performed to determine cellular activity after 24 and 48 h of seeding. We set the relative metabolic activity as 100% for the cells incubated with RGES (negative control) for each substrate independently. The cellular metabolic activity of cells cultured on gelatin was not affected by incubation with RGDS or RGES peptides (Fig. 4A,B). In contrast, there was a significant reduction in the activity of cardiomyocytes seeded on Npnt (100 ± 5.8% to 72.6 ± 7.1%, p < 0.05) and fibronectin (100 ± 6.1% to 70.3 ± 3.9%, p < 0.05) at 24 h (Fig. 4A). At 48 h we observed still a weak effect of the RGDS peptide but the effect was not significant (Fig. 4B). In summary, our data suggest that the cellular activity of cardiomyocytes on Npnt depends on its RGD sequences.

The RGD sequence can modulate cell adhesion properties [34,35]. Therefore, the reduced metabolic activity after integrin receptor blocking might be due to reduced numbers of attached cardiomyocytes. To investigate the effects of integrin blocking on cardiomyocytes attachment, RGDS-treated cells were allowed to

attach for 24 h and were subsequently stained for cardiomyocyte-specific actinin to determine the number of attached cardiomyocytes. Our quantitative analyses revealed that significantly reduced numbers of cardiomyocytes attached on Npnt after RGDS treatment (Fig. 4C,D). However the number of attached cardiomyocytes on gelatin and fibronectin was not affect by RGDS treatment. In addition, spreading of cardiomyocytes on Npnt as well as fibronectin was affected by integrin blocking.

3.4. Induction of cell cycle activity by growth factors

To further demonstrate that Npnt has no negative effect on cardiomyocytes we tested their responsiveness to extracellular stimuli. To explore the effect of growth factors on the car-diomyocytes seeded on different matrices, we have stimulated cardiomyocytes either with horse serum (HS) or fibroblast growth factor 1 (FGF1) along with an inhibitor of the mitogen-activated protein kinase p38 (p38i) that are known to induce cell cycle activity [36]. Stimulation with HS results usually in a low activation of DNA synthesis in cardiomyocytes seeded on gelatin. Quantitative analyses indicated, that there is a significant increase of BrdU-positive cardiomyocytes seeded on Npnt (21.6 ± 5.3%) compared to gelatin (11.4 ± 3.1%). The number of cardiomyocytes

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Fig. 3. Relative metabolic activity of cardiomyocytes cultured on Npnt. (A) Neonatal rat cardiomyocyte-enriched cells were cultured on different matrices for up to 8 days. Histogram represents an MTT assay-based quantitative analysis of the relative cellular metabolic activity (n = 3, mean ± SEM, *: p < 0.05, ns: not significant). (B) Representative immunofluorescence images of cardiomyocytes at day 8 of culture that were used for quantitative analysis in A. Cardiomyocytes were stained for the cardiac specific protein sarcomeric a-actinin (green) and nuclei were visualized by DAPI (blue). Most of the attached cells are cardiomyocytes. Scale bars: 50 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

incorporating BrdU on Npnt was comparable to cardiomyocytes seeded on fibronectin (25.5 ± 3.8%) (Fig. 5A,B). Stimulation with FGF1/p38i is a strong inducer of cardiomyocyte cell cycle activity and induced BrdU incorporation in 28.2 ± 5.3% of cardiomyocytes seeded on gelatin (Fig. 5A,C). Stimulation of cardiomyocytes seeded on Npnt or fibronectin was significantly more effective (45.5 ± 7.4% and 45.3 ± 7.6%, respectively) (Fig. 5A,C). Taken together, cardiomyocytes attached on Npnt responded well to extracellular stimuli.

3.5. Connexin 43 expression and contractility

The contractile force of an engineered cardiac tissue is a good indicator for the cardiac tissue quality. It depends amongst others on well-differentiated sarcomeres that are aligned in parallel and electrical cell-to-cell coupling enabling synchronous contractions. Cardiomyocyte grown on Npnt and fibronectin displayed at 2 days clearly visible aligned sarcomeres based on actinin and troponin I expression (Fig. 6A). In contrast, the sarcomeric structure in car-diomyocytes grown on gelatin were mainly not aligned and appeared immature.

The gap junctional protein connexin 43 is essential for electrical signal propagation between cardiomyocytes [37]. We observed that cardiomyocytes grown on Npnt and fibronectin expressed high amounts of connexin 43 along their intercellular junctions (Fig. 6B). In contrast, connexin 43 expression at the intercellular junctions of cardiomyocytes grown on gelatin remained low or non-detectable. Quantitative analysis of the beating frequency indicated that the

number of contraction—relaxation cycles was significantly higher in cardiomyocytes grown on Npnt than on fibronectin or gelatin (Fig. 6C). In accordance to the connexin 43 expression, cardiomyocytes seeded on gelatin showed independent focal beating whereas cardiomyocytes seeded on Npnt or fibronectin contracted synchronously (Supplementary Movies 1—3). These data demonstrate that Npnt promotes intercellular communication, sarcomere maturation and alignment as well as synchronous contractions.

Supplementary video related to this article can be found at doi: 10.1016/j.biomaterials.2012.03.021.

3.6. nppa and nppb expression

The quality of an engineered cardiac tissue depends on the maturity of the seeded cardiomyocytes. Thus it is important that the scaffold material used for tissue engineering maintains or even promotes maturation of cardiomyocytes. The molecular phenotype of the cells in our cultures was investigated by measuring the relative levels of mRNA for natriuretic peptide precursor nppa and nppb. 18S-RNA levels were used for normalizing gene expression data. Fetal heart ventricles express higher levels of nppa and nppb than adult ventricles. Ventricular reexpression of nppa in adults is associated with ventricular hypertrophy. Therefore, nppa and nppb, which are preferentially expressed in atrial, immature ventricular, or hypertrophic ventricular tissue but not in mature, healthy ventricular tissue were used as an indicator for cardiomyocyte maturation [38]. A semi-quantitative gene expression analyses indicated that the expression of nppa and nppb were significantly

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Fig. 4. Cardiomyocyte attachment on Npnt is integrin-dependent. (A,B) Rat neonatal cardiomyocyte-enriched cells were seeded either for 24 h (A) or 48 h (B) on gelatin, Npnt or fibronectin in the presence of RGDS or RGES (control) peptides. (A,B) Histogram represents an MTT assay-based quantitative analysis of the relative cellular metabolic activity. For each matrix, control was set as 100%. (C) Quantitative analysis of attached cardiomyocytes at 24 h. For each matrix control was set as 100% (n = 3, mean ± SEM, *: p < 0.05, ns: not significant). (D) Representative immunofluorescence images used for C. Cardiomyocyte-enriched cells seeded for 24 h on the indicated matrices incubated either with RGDS or RGES (control) peptides were stained for sarcomeric a-actinin (cardiomyocytes, green) and nuclei were visualized by DAP1 (blue). Scale bars: 50 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. Npnt stimulates growth factor-induced cell cycle activity. DNA synthesis (BrdU incorporation) of neonatal rat cardiomyocytes were analyzed after 2 days of stimulation either with 5% horse serum (HS) or a combination of FGF1 + p38 inhibitor. (A) Representative immunofluorescence images of cultured cardiomyocyte-enriched cells after 48 h of stimulation stained with anti-troponin I (cardiomyocytes, green) and anti-BrdU antibodies (replicating cells, red) and with DAPI (nuclei, blue). Orange arrow indicates a BrdU-positive cardiomyocyte. Yellow arrow indicates a BrdU-positive non-myocyte. Scale bars: 50 mm. (B,C) Quantitative analyses (n = 3, mean ± SEM, *: p < 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 6. Npnt promotes intercellular communication and contractility. (A) Representative immunofluorescence images of 2 days-cultured cardiomyocytes stained either for sarcomere a-actinin (cardiomyocytes, green) or troponin I (cardiomyocytes, green). Nuclei were visualized with DAPI (blue). Cardiomyocytes seeded on Npnt contain well-established and aligned sarcomeres (yellow arrows). (B) Representative immunofluorescence images of cardiomyocytes seeded on different matrices and subsequently stained with anti-sarcomeric a-actinin (cardiomyocytes, green) and anti-connexin 43 antibodies (intercellular junctions, red) and with DAPI (nuclei, blue). Importantly cardiomyocytes on Npnt express connexin 43 (yellow arrow heads) indicating well-established intercellular communication. (C) Quantitative analysis of the cardiomyocyte beating frequency 48 h after seeding (n = 3, mean ± SEM, **: p < 0.01). Scale bars: 20 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

reduced in cardiomyocytes cultured on gelatin compared to expression levels in freshly isolated non-cultured neonatal cardiomyocytes at 2 days of culture (Fig. 7A). Real time PCR confirmed this result and demonstrated that cardiomyocytes cultured on fibronectin express higher levels of nppa and nppb. In contrast, nppa and nppb expression levels of cardiomyocytes cultured on Npnt were not significantly different from the expression levels in freshly isolated non-cultured cardiomyocytes (Fig. 7B). Our data suggest that Npnt maintains the maturation state of cardiomyocytes. This is further supported by the observation that most cardiomyocytes cultured on Npnt did not express alpha smooth muscle actin (2A). During in vivo cardiogenesis, cardiomyocytes express different actin isoforms: skeletal (SKA), cardiac (CAA) and smooth muscle (SMA) alpha actin. SMA marks the onset of cardiomyocyte differentiation. Later in development it is sequentially replaced by SKA and CAA isoforms [39,40]. When cardiomyocytes from neonatal or

adult rats are cultured in vitro, they usually re-express SMA, which is a sign of dedifferentiation and most importantly affects the contractile properties of the cells [41-43]. Our data demonstrate that Npnt prevents the reexpression of SMA.

4. Discussion

Cardiac tissue engineering is a promising approach to partially reverse cardiovascular diseases including myocardial infarction and congenital heart disease. The goal is to generate ex vivo a tissue-like structure that should be as similar to native myocardium as possible. It should contain cardiomyocytes but also fibroblasts, endothelial cells, and vascular smooth muscle cells [29,30]. In order to maximize the contractile force of cardiac patches they have to contain a high number of cardiomyocytes, which are electrical coupled, are mature containing aligned sarcomeres. In recent years

Fig. 7. Cardiomyocytes seeded on Npnt express similar level of nppa and nppb as freshly isolated neonatal cardiomyocytes. (A,B) Semi-quantitative (A) and quantitative (B) PCR analysis of nppa and nppb mRNA expression in freshly isolated neonatal cardiomyocytes (not cultured) or 2 days-cultured cardiomyocytes on different matrices. 18S-RNA was used as loading control. Ladder indicates a size marker (n = 3, mean ± SEM, *: p < 0.05, ns: not significant).

a number of scaffold-forming materials have been used to generate in vitro cardiac patches, but none of them reached the quality for therapeutic application. One major limitation is the generated contractile force. Forces of isolated native myocardium are 50 mN/mm2 [44]. Reported values for engineered cardiac patches range between 0.05 and 2 mN/mm2 [45]. Thus, it is important to identify materials that can enhance cardiomyocyte attachment, maturation and electrical coupling.

Recently, it has been shown that the natural ECM of the heart provides a suitable scaffold for cardiac tissue engineering [16]. Moreover, it has been shown that the ECM composition changes during heart development, is required to maintain or acquire new cellular functions and is crucial for directing tissue specification. Thus, it is important to characterize the natural components of the cardiac ECM during development [17]. Especially the poor adhesive properties of cardiomyocytes hampers the generation of cardiac tissues. Thus, recent strategies in tissue engineering focus on incorporating ECM-derived peptides into biomaterials in order to mimic the natural matrix [15]. In this study we studied Npnt, which we have recently shown to be developmentally regulated. The data presented here suggest that Npnt has the potential to become useful for cardiac tissue engineering. We have shown that all major cellular components of the heart like cardiomyocytes, cardiac fibroblasts, vascular smooth muscle cells and endothelial cells can attach and spread on Npnt. Cardiomyocytes attached and spread markedly faster on Npnt than on gelatin. Our data suggest that this is in part due to the presence of the integrin binding RGD sequence in Npnt [46]. In addition, cardiomyocytes grown on Npnt exhibited a matured contractile apparatus with well-aligned sarcomeres. Connexin 43 expression data suggest moreover that the cells were electrical coupled. Consequently, cardiomyocytes on Npnt contracted synchronously and exhibited a higher beating frequency than cardiomyocytes on gelatin or fibronectin. Finally, we analyzed the expression of nppa, nppb and SMA in order to assess the differential status of cardiomyocytes [38,41—43]. Cardiomyocytes grown Npnt exhibited the same expression pattern as freshly isolated cardiomyocytes (nppa and nppb) or did not re-express fetal genes (SMA). Taken together, our data indicate that Npnt maintains differentiation, promotes sarcomere maturation as well as electrical signal propagation and consequently cardiomyocyte contractility.

In the future it will be important to investigate if Npnt or Npnt peptides can be used for surface modifications or bulk

modifications of 3D scaffolds to provide a more natural microenvironment to generate advanced cardiac tissue patches.

5. Conclusions

Nephronectin (Npnt) enables at least in part due to its RGD domain the efficient attachment of the main cell types of the heart: cardiomyocytes, endothelial cells, and vascular smooth muscle cells. Cardiomyocytes growing on Npnt contain mature well-aligned sarcomeres, express connexin 43 and couple electrically with each other resulting in synchronous beating at least for 8 days. Moreover, most cardiomyocytes on Npnt were SMA-negative supporting the notion that Npnt maintains the differentiation state of the cells. Npnt was superior to fibronectin, which is considered as one of the best cardiac adhesive. Cardiomyocytes on Npnt displayed a higher beating frequency and maintained on a molecular level a more differentiated phenotype. Our findings suggest that Npnt is a cardiac adhesive molecule with the potential to improve cardiac tissue engineering. This study underlines the importance to elucidate the composition of the cardiac ECM during development to reveal ECM components to further optimize the fabrication of scaffolds for the generation of cardiac patches.

Disclosure statement

No competing financial interests exist.

Acknowledgments

We are grateful to Petra Freund, Sandra Ruhl and Ingrid Hauck-Schmalenberger for technical support. This work was supported by a grant from the Alexander von Humboldt Foundation (Sofja Kovalevskaja Award to F. B. E.), the International Research Training Group 1566 (PROMISE, DFG), and the Excellence Cluster Cardio-Pulmonary System (DFG).

Appendix A. Supplementary material

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.biomaterials.2012. 03.021.

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