Recombinant spider silk protein eADF4(C16)-RGD coatings are suitable for cardiac tissue engineering

Cardiac tissue engineering is a promising approach to treat cardiovascular diseases, which are a major socio-economic burden worldwide. An optimal material for cardiac tissue engineering, allowing cardiomyocyte attachment and exhibiting proper immunocompatibility, biocompatibility and mechanical characteristics, has not yet emerged. An additional challenge is to develop a fabrication method that enables the generation of proper hierarchical structures and constructs with a high density of cardiomyocytes for optimal contractility. Thus, there is a focus on identifying suitable materials for cardiac tissue engineering. Here, we investigated the interaction of neonatal rat heart cells with engineered spider silk protein (eADF4(C16)) tagged with the tripeptide arginyl-glycyl-aspartic acid cell adhesion motif RGD, which can be used as coating, but can also be 3D printed. Cardiomyocytes, fibroblasts, and endothelial cells attached well to eADF4(C16)-RGD coatings, which did not induce hypertrophy in cardiomyocytes, but allowed response to hypertrophic as well as proliferative stimuli. Furthermore, Kymograph and MUSCLEMOTION analyses showed proper cardiomyocyte beating characteristics on spider silk coatings, and cardiomyocytes formed compact cell aggregates, exhibiting markedly higher speed of contraction than cardiomyocyte mono-layers on fibronectin. The results suggest that eADF4(C16)-RGD is a promising material for cardiac tissue engineering.

In 2016, cardiovascular disease (CVD) was responsible for approximately 17.6 million deaths worldwide, an increase of 14.5% compared to 2006. CVD may cause myocardial infarction, which results in the loss of functional myocardium and scar formation and can lead to chronic heart failure. As the human heart does not macroscopically regenerate after damage, cardiac function decreases 1 . Various approaches have been utilized to compensate for lost cardiomyocytes due to cell death, including stem cell therapy, activation of endogenous stem cells, induction of cardiomyocyte proliferation, and cardiac tissue engineering [2][3][4] . Zimmerman et al. were able to significantly increase the function of infarcted hearts in immune-suppressed rats upon implantation of grafts consisting of cardiomyocytes embedded in a mixture of type I collagen and Matrigel 5 , a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells consisting mainly of laminin, nidogen, collagen and heparan sulfate proteoglycans. The potential of cardiac tissue engineering to improve heart function after myocardial infarction has since been validated by several independent groups in small as well as large animal models [6][7][8] . At the same time, clinical trials using engineered cardiac patches in humans have been performed, and their safety could be confirmed. While Menasché et al. proved the technical feasibility to graft fibrin patches containing human embryonic stem cell-derived cardiac progenitor cells onto hearts of heart failure patients 9 , it remains unclear if this procedure will improve heart function in heart failure patients.

Results
Properties of eADF4(C16)-RGD coatings. Due to the inherent negative surface charge of cells and hence preferential attachment to polycationic surfaces 13 , the polyanionic nature of eADF4(C16) and its lack of cell binding sequences prohibits cell attachment. Since an integrin-binding RGD motif has been shown to promote cardiomyocyte attachment 12 , we investigated cardiac cell behavior on coatings made of eADF4(C16)-RGD, a variant of eADF4(C16) in which an RGD-tag is fused C-terminally 29 . Glass coverslips were dip-coated into a solution of eADF4(C16)-RGD in formic acid (Fig. 1a), applying 0.15 mg/cm 2 silk protein as previously published 13 . As the surfaces of both eADF4(C16)-RGD and silicate glass 32 are negatively charged, electrostatic repulsion and hence delamination of the coatings occurred. Therefore, glass coverslips were silanized using (3-aminopropyl)triethoxysilane (APTES). Hoechst 33342 staining was used to confirm successful coating as previously described 13 , showing a blue background illumination ( Supplementary Fig. 1). No obvious signs of degradation were observed during the experiments. This is in agreement with previous studies investigating the stability of silk films and coatings [33][34][35] .
Spider silk coatings prepared from formic acid typically possess thermodynamically stable β-sheet-rich structures making them water-insoluble 36,37 , and this secondary structure could be confirmed using spectroscopy and successive analysis of the amide I band according to Hu et al. 38 using Fourier self-deconvolution (FSD) (Fig. 1b) with 22.53 ± 0.25% beta-sheets, 14.71 ± 0.32% alpha helices, 31.83 ± 1.34% random coils, and 24.83 ± 0.99% turns. As expected, silanized glass showed no signal in the amide I region of FTIR spectra. Next, surface hydrophilicity of eADF4(C16)-RGD coatings was determined in comparison to that of eADF4(C16) coatings and silanized glass using water contact angle measurements (Fig. 1c). Both eADF4(C16)-RGD and eADF4(C16) coatings were more hydrophilic than silanized glass (contact angle: 58.4° ± 3.2°) with contact angles of 37.2° ± 3.9° and 29.5° ± 2.4°, respectively (p < 0.001). eADF4(C16)-RGD coatings are suitable for cardiac cell attachment. To evaluate eADF4(C16)-RGD coatings for cardiac cell adhesion, cells from postnatal day 3 (P3) rat hearts were isolated and cultured on eAD-F4(C16)-RGD coatings. Fibronectin coatings were used as positive control, as fibronectin is a natural component of the cardiac extracellular matrix 21,22 to which neonatal rat cardiomyocytes have been shown to attach 23 . Cells were cultured in the presence of fetal bovine serum (FBS), as serum withdrawal has been associated with reduced cardiomyocyte cell viability in culture 39 . Yet, it is important to examine the interactions between coatings and cardiomyocytes without or at low concentrations of serum, as it has been demonstrated that FBS induces hypertrophy in neonatal rat cardiomyocytes 13 and human pluripotent stem cell-derived cardiomyocytes 40 .
In order to confirm that eADF4(C16)-RGD coatings are nontoxic to cardiac cells, cardiac cells were allowed to attach overnight to eADF4(C16)-RGD and fibronectin coatings, were cultured for 48 h in the presence of 0.2% and 10% FBS (v/v), and a calcein-acetoxymethylester (calcein-AM) and Ethidium-homodimer-1 (EthD-1) live/ dead assay was conducted (Fig. 2a,b). Analysis of the data revealed that both coatings were non-toxic, showing a low level of EthD-1 positive cells. In addition, there was no statistically significant difference between the percentage of live calcein-AM-or dead EthD-1-positive cells cultured on eADF4(C16)-RGD or fibronectin coatings at 0.2% or 10% FBS (Fig. 2c,d) suggesting that eADF4(C16)-RGD coatings are suitable for the adhesion of cardiac cells.
To determine if cardiomyocytes attach to eADF4(C16)-RGD coatings, isolated and enriched cardiomyocytes were allowed to attach overnight to eADF4(C16)-RGD and fibronectin coatings and were cultured for 48 h in the presence of 0.2% and 10% FBS (v/v). Subsequently, cells were fixed, stained for the cardiomyocyte-specific marker sarcomeric α-actinin, and the number of cardiomyocytes per microscopic field was counted (Fig. 3a, for representative images see Fig. 4). Analysis of this data showed that cardiomyocytes attached to eADF4(C16)-RGD coatings, and there was no significant difference between the number of actinin-positive cells between eAD-F4(C16)-RGD and fibronectin coatings or between different serum conditions for either surface. Our data demonstrate that eADF4(C16)-RGD coatings support the attachment of cardiac cells as well as coatings made of fibronectin, a natural component of the cardiac extracellular matrix 21,22 .
Cardiac tissue contains a variety of cells, including cardiomyocytes, fibroblasts and endothelial cells 41 . It has been shown that the presence of non-myocytes in cardiac patches improves the structure and function of tissue 42 and, furthermore, vascularization is required for the survival of a functional patch 43 . Thus, cardiac cells were isolated without cardiomyocyte enrichment, allowed to attach for 3 h or 24 h in the presence of 0.2% or 10% FBS and stained for cell-type-specific markers (actinin, cardiomyocytes; collagen-1, fibroblasts; VE-cadherin, endothelial cells 13 ) and DNA. As shown in Fig. 3, all investigated cardiac cell types attached to fibronectin and eADF4(C16)-RGD coatings. Note, fibroblasts and especially endothelial cells were less frequent than cardiomyocytes. Thus images in Fig. 3 are not representative in regard to cardiomyocyte attachment. In addition, induction of hypertrophy (increase in cell size) of cardiomyocytes by fibronectin gives at a first glance the impression of a difference even at the same cell density.
Taken together, these data demonstrate that eADF4(C16)-RGD coatings are comparable to coatings made of fibronectin, a natural component of the cardiac extracellular matrix 21,22 , with regards to cardiac cell adhesion and cell viability.
Cardiomyocytes cultured on eADF4(C16)-RGD coatings respond to proliferative stimuli. For a cardiac patch to contract properly and thus improve heart function, the presence of a high density of cardiomyocytes is essential. One way to increase cardiomyocyte density after engineering a cardiac tissue is the induction of cardiomyocyte proliferation. Therefore, the response to proliferative stimuli of cardiomyocytes cultured on eADF4(C16)-RGD was assessed using an EdU-incorporation assay (Fig. 4). In this assay, the nucleotide analog EdU is added to the medium and thus can be utilized by the cells during DNA synthesis. This allows visualization of cells undergoing DNA synthesis via fluorescent labelling and the subsequent detection of EdU incorporated in the DNA via fluorescence microscopy.  www.nature.com/scientificreports www.nature.com/scientificreports/ Cardiomyocytes were allowed to attach overnight to the coatings and were then cultured for 48 h in the presence of 0.2% FBS, 10% FBS, or a mixture composed of fibroblast growth factor 1 (FGF1), mitogen-activated protein kinase p38i inhibitor (p38i) and 0.2% FBS. Previously, it has been shown that FGF-1/p38i induces proliferation 44 and 10% FBS promotes cell cycle re-entry in neonatal cardiomyocytes 45 . Quantitative analysis demonstrated that 10% FBS, as well as FGF1/p38i/0.2% FBS, induced DNA synthesis in cardiomyocytes attached to eADF4(C16)-RGD or fibronectin coatings, with no statistically significant difference between the two coatings ( Fig. 4d). In addition, as observed previously for neonatal rat cardiomyocytes cultured on gelatin 44,46,47 and eAD-F4(κ16) 13 , cardiomyocytes stimulated with FGF1/p38i/0.2% FBS presented an elongated morphology on eAD-F4(C16)-RGD coatings (Fig. 4C). These data indicate that cardiomyocytes behave similarly on eADF(C16)-RGD coatings as on the inert material gelatin, and that eADF4(C16)-RGD coatings allow attached cardiomyocytes to properly respond to proliferative stimuli.
Cardiomyocytes cultured on eADF4(C16)-RGD coatings respond to hypertrophic stimuli. In vivo, cardiomyocytes display a physiological hypertrophic response, i.e. an increase in cellular size, upon stimuli such as hormones or growth factors released during times of increased cardiovascular stress, e.g. in athletes or during pregnancy 48 . Previously, it has been shown that fibronectin exhibits a hypertrophic effect on neonatal rat cardiomyocytes in culture 49 . Notably, cardiomyocytes stimulated with 0.2% FBS or FGF1/p38i/0.2% FBS appeared smaller on eADF4(C16)-RGD coatings than on fibronectin coatings (Fig. 4a,c), suggesting that www.nature.com/scientificreports www.nature.com/scientificreports/ eADF4(C16)-RGD coatings may have no hypertrophic effect on cardiomyocytes. In order to evaluate whether eADF4(C16)-RGD coatings indeed exhibit no hypertrophic effect and whether cardiomyocytes cultured on eAD-F4(C16)-RGD coatings are able to respond properly to strong (10% FBS) and weak (phenylephrine, PE) hypertrophic stimuli 13 , cells were cultured for 48 h in the presence of these factors, and 0.2% FBS was used as a control. Gelatin, an established material for tissue engineering 11 , was used as a control substrate 12,13,50 , since fibronectin could not be used due to hypertrophic effects on neonatal rat cardiomyocytes in culture 49 and associated pathophysiological hypertrophy in vivo 21 . Cells were stained for atrial natriuretic factor (ANF), which is expressed around the nucleus in hypertrophic cardiomyocytes 51 and has been successfully used as a marker for cardiomyocyte hypertrophy 13 (Fig. 5).
Our data show that the number of ANF-positive cardiomyocytes stimulated with 0.2% FBS is not significantly different on eADF4(C16)-RGD coatings compared to that on gelatin (Fig. 5d). In addition, stimulation of cardiomyocytes cultured on eADF4(C16)-RGD coatings with 10% FBS or 50 µM PE resulted in a significant increase in ANF-positive cardiomyocytes comparable to that on the other matrices (Fig. 5d). These data indicate www.nature.com/scientificreports www.nature.com/scientificreports/ that eADF4(C16)-RGD coatings exhibit no significant hypertrophic effect and allow cardiomyocytes to respond adequately to hypertrophic stimuli.
Cardiomyocytes cultured on eADF4(C16)-RGD coatings exhibit normal beating behavior. Naturally, for a bioengineered cardiac patch to perform well in vivo, cardiomyocytes must beat regularly on/within the scaffold. Kymograph software (Fiji) was used to determine the number of beats per minute of cultured cardiomyocytes by measuring beating cardiomyocytes' membrane deflection in 10-second videos (Fig. 6a). Example videos are included as part of the supplementary material ( Supplementary Movie 1-4). Analysis of the videos indicated that cardiomyocytes cultured on eADF4(C16)-RGD coatings beat regularly (Fig. 6a), with no statistically significant difference between eADF4(C16)-RGD and fibronectin coatings. Yet, there was a trend towards a higher beating frequency on eADF4(C16)-RGD coatings (Fig. 6b). These data suggest that cardiomyocytes cultured on eADF4(C16)-RGD coatings exhibit normal beating behavior. www.nature.com/scientificreports www.nature.com/scientificreports/ A closer analysis of the videos revealed that cardiomyocytes grown on eADF4(C16)-RGD coatings tend to cluster or aggregate. Kymograph analysis showed that these clusters do not all beat synchronously (Fig. 6c). Finally, MUSCLEMOTION analyses of the videos demonstrated a higher speed of contraction for cardiomyocytes cultured on eADF4(C16)-RGD coatings at low serum conditions compared to those on fibronectin coatings (Fig. 6d), which was statistically significant (Fig. 6f). This suggests that cardiomyocytes cultured on eADF4(C16)-RGD coatings exhibit improved contractility (see also Discussion). Notably, speed of contraction for cardiomyocytes cultured on eADF4(C16)-RGD coatings was not improved at high serum concentrations ( Fig. 6d-g). In contrast, speed of contraction for cardiomyocytes cultured on fibronectin coatings was markedly improved reaching the levels observed for eADF4(C16)-RGD coatings. This indicates that cardiomyocytes cultured on eADF4(C16)-RGD coatings are less dependent on external stimuli in order to maintain basic contractile properties. This may allow a more constant contractile performance in future cardiac tissue engineering applications. All other measured parameters showed no significant differences between eADF4(C16)-RGD and fibronectin coatings (Supplementary Fig. 2) and no obvious differences were observed in cell eccentricity, cell shape, sarcomere alignment, and sarcomere density. These data indicate that eADF4(C16)-RGD allows cardiomyocytes to exhibit proper beating behavior.

Discussion
We conclude that the recombinant spider silk protein eADF4(C16)-RGD is a promising material for the application in cardiac tissue engineering and offers the many benefits of silk materials, such as low immunogenicity and biodegradability 33,34 . Cell types vital for cardiac tissue engineering, cardiomyocytes, endothelial cells, and fibroblasts, attach in a similar manner to eADF4(C16)-RGD and fibronectin coatings. Although fibronectin is a component of the native cardiac extracellular matrix 20,23 , fibronectin is not optimal for cardiac tissue engineering as it contributes to pathological cardiac hypertrophy but not physiological growth 21 . Coatings made of eADF4(C16)-RGD are noncytotoxic without obvious pharmacological properties, and do not hinder cardiomyocytes to respond adequately to extracellular stimuli. Finally, cardiomyocytes form compact cellular aggregates on eADF4(C16)-RGD coatings. One highly interesting feature of eADF4(C16)-RGD, which provides an outlook for future studies, is its 3D printability by robotic dispensing without requiring crosslinking additives or thickeners for mechanical stabilization 30,31 . Investigation on fabrication of scaffolds for clinical application using bioinks based on eADF4(C16)-RGD and stem cell-derived cardiomyocytes will be the next steps to evaluate putative applications.
In recent years, major developments have been made in the field of cardiac tissue engineering based on mouse model results indicating that engineered cardiac patches can improve cardiac function after myocardial infarction 5 leading to first clinical safety trials 4,9 . Yet, several hurdles have to be overcome to establish a therapy. One major issue is that current artificial constructs do not exhibit the force measured in human adult heart muscle 52 , despite identifying several parameters improving force development such as mechanical strain/load, chronic electric pacing, and supplementation of medium with L-thyroxin 53 . This might be due to the fact that the utilized cells still do not reach an adult-like phenotype and the inability of generating thick, vascularized cardiac tissue in which cells are hierarchically organized. Several approaches are currently used to address this problem such as the use of prefabricated scaffolds (decellularized matrices or matrices generated in a non-cell-compatible manner) and 3D printing 53 . Yet, these approaches are currently limited by a low number of available materials. Our data, in conjunction with previously published data, suggest that eADF4(C16)-RGD is a promising material for coating prefabricated scaffolds as well as 3D printing in cardiac tissue engineering.
In order to generate a functional cardiac patch, materials are required that allow attachment not only of cardiomyocytes, but also of non-myocytes, which are known to improve the structure and function of engineered cardiac tissues 42 . Furthermore, vascularization is necessary for patch survival 43 . Here, we show that fibroblasts and endothelial cells adhere to eADF4(C16)-RGD coatings. In addition, eADF4(C16)-RGD coatings appeared to exert no obvious effect on cardiomyocyte function. Importantly, cardiomyocytes grown on eADF4(C16)-RGD coatings responded to hypertrophic stimuli. This suggests that cardiac patches based on eADF4(C16)-RGD will respond to extracellular stimuli in vivo to adapt heart function to altered physiological conditions during periods of increased strain, such as in athletes or during pregnancy 48 .
The generation of thick cardiac constructs, which are densely packed with cardiomyocytes, remains a major challenge due to for example challenges in seeding prefabricated scaffolds. A solution could be the promotion of cardiomyocyte proliferation 54 , which could compensate for initially low cardiomyocyte densities. Here, we show that cardiomyocytes grown on eADF4(C16)-RGD coatings responded properly to an inducer of cardiomyocyte proliferation 44 . In addition, we observed that cardiomyocytes on eADF4(C16)-RGD coatings agglomerate to compact cellular structures. This might be a very useful property in generating a more compact cardiac tissue construct. Yet, in this study, local compaction was associated with light arrhythmia. Further studies will show if this hampers the functionality and whether this can for example be compensated by the inclusion of electroconductive materials 10,55 .
Kymograph and MUSCLEMOTION video analyses show that cardiomyocytes grown on eADF4(C16)-RGD or fibronectin coatings exhibit a similar contractile behavior. However, cardiomyocytes exhibited a significantly higher speed of contraction on eADF4(C16)-RGD than on fibronectin coatings when cultured at low serum concentrations. Cardiac contractility is discernible by the maximum contraction speed and its ejection fraction 56 . A higher speed of contraction therefore translates directly to improved cardiac function. In addition, a trend, albeit not statistically significant, was observed towards a faster relaxation time of cardiomyocytes on eAD-F4(C16)-RGD coatings. The speed at which ventricular cardiomyocytes are able to undergo relaxation is of vital importance for the modulation of the heart rate in vivo. If, e.g. due to stimulation by the sympathetic nervous system, the heart beat is sped up in healthy humans, the length of the diastole is shortened more radically compared to that of the systole relative to the length of the cardiac cycle 57 . www.nature.com/scientificreports www.nature.com/scientificreports/ Taken together, our data suggest that the recombinant spider silk protein eADF4(C16)-RGD is a promising material for cardiac tissue engineering. Future studies will show whether the 3D printability of eADF4(C16)-RGD together with the use of cardiomyocytes and non-myocytes derived from human induced pluripotent stem cells allow the generation of cardiac patches for clinical application.

Methods preparation of recombinant silk protein. Engineered Araneus diadematus fibroin 4
(C16) with an RGD tag (eADF4(C16)-RGD) comprises 16 repeats of a C-module (sequence: GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP) and an RGD tag (GGSGGRGDSPG) for integrin binding 29 . The recombinant silk protein was produced and purified as described previously 58 . Silanization procedure. Glass coverslips (Ø 12 mm, Thermo Fisher Scientific) were silanized according to Verné et al. 59 using (3-aminopropyl)triethoxysilane (APTES, sigma aldrich). Glass coverslips were cleaned for 5 min in acetone in an ultrasound bath, quickly washed with H 2 O MQ , followed by another acetone, water and acetone treatment. After this washing procedure, the silanization reaction was carried out using APTES in ethanol (0.2 ml APTES in 50 ml ethanol p.a.) for 5 h under gentle movement. The coverslips were then washed twice with 100% (v/v) ethanol before a thermal treatment at 100 °C for 1 h was performed to consolidate bonding of the glass surface with silane. Directly before coating, the coverslips were washed again with H 2 O MQ and ethanol.
Spider silk coating procedure. The silanized glass coverslips were coated as described previously 13 . The lyophilized spider silk protein eADF4(C16)-RGD was dissolved in formic acid and diluted with H 2 O MQ (5:1, formic acid:H 2 O MQ ; silk concentration 0.34% w/v) before the coverslips were dip-coated in the eADF4(C16)-RGD solution and allowed to dry on Parafilm M.

Water contact angle measurements. A Surftens-universal (OEG GmbH, Germany) in combination
with SCA-20 software was used to determine the water contact angle of silanized-and silk-covered glass coverslips. Uniform drops were deposited and the contact angles were determined after equilibration at room temperature using the sessile drop method. 20 measurements were done (n = 20) per sample.

Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. A Bruker
Tensor 27 spectrometer (Bruker, Germany) equipped with a Ge-crystal was used to record ATR-FTIR spectra of eADF4(C16)-RGD coatings on glass, and silanized glass was used as a control. The spectra are accumulations of 100 scans from 4000-800 cm −1 with a resolution of 4 cm −1 . Fourier self-deconvolution (FSD) was performed to determine the secondary structure content by analyzing the amide I region (1595-1705 cm −1 ) according to Hu et al. 38 Five samples were analyzed using the Opus software (Bruker, Germany) (n = 5). neonatal rat cardiomyocyte isolation and cell culture. All experiments were executed in accordance to the Guide for the Care and Use of Laboratory Animals Directive 2010/63/EU, European Parliament. Organ extraction and primary cell culture preparation was approved by the local Animal Ethics Committee of Erlangen, conforming to governmental and international regulations on animal experimentation (protocol TS-9/16 Nephropatho). Ventricular cardiomyocyte isolation was performed as previously published 13 . In brief, ventricles from 3 days-old (P3) Sprague Dawley rats were digested using the gentleMACS Dissociation kit (Miltenyl Biotec, 130-098-373) according to the manufacturer's instructions. Afterwards, cardiomyocytes were enriched by pre-plating for 90 min at 37 °C. The suspension of non-attached cells enriched in cardiomyocytes was collected, centrifuged for 5 min at 330 × g, resuspended in DMEM F12 + GLUTAMAX containing 100 U/mg/ml penicillin/ streptomycin and cultured in medium containing supplements: 3.0 mM Na-pyruvate (Sigma, S8636), 2.0 mM L-glutamine (Invitrogen, 25030081), 0.1 mM ascorbic acid (Sigma A4034), 1:200 insulin/transferrin/Na-selenite supplement (Sigma A4034), and 0.2% bovine serum albumin (BSA, Sigma A7409), if not stated otherwise. For the assessment of non-myocyte attachment, cardiac cells were isolated as described by Sadoshima et al. with minor modifications 60 . Ventricular cardiac cells from P3 Sprague Dawley rats were isolated utilizing 0.14 mg/ ml collagenase II (Gibco 17101-015) and 0.5 mg/ml pancreatin (Sigma P3292). Isolated cells were cultured without preplating in the above-described supplemented medium. Cells were seeded at a density of 150,000/500 µl/ well on glass coverslips (Ø 12 mm, Thermo Fisher Scientific) in 24-well tissue culture plates. For video analysis experiments, a cell density of 100,000/250 µl/well was seeded in Thermo Scientific Nunc Lab-Tek II CC2 8-well chambers. For proliferation assays, cells were seeded in DMEM F12 + GLUTAMAX containing 100 U/mg/ml penicillin/streptomycin and 1% fetal bovine serum (FBS).
Live/dead cell viability assay. Cells were seeded in DMEM F12 + GLUTAMAX + 100 U/mg/ml penicillin/streptomycin + supplements and were allowed to attach overnight. They were then washed once with DPBS, and the medium was changed, supplemented with either 0.2% FBS or 10% FBS. After incubation for 48 h, cells were washed three times in DPBS for 5 min and incubated with calcein-AM (0.25 µl/ml) in PBS and ethidium-homodimer 1 (1 µl/ml) in PBS (Life Technologies, L2334) for 10 min at 37 °C and 5% CO 2 . Subsequently, cells were fixed in 3.7% PFA for 15 min, washed in DPBS, mounted and fixed on the slides using nail polish to allow immediate microscopy. The percentage of calcein-and ethidium-homodimer-positive cells per microscopic field was assessed for 10 randomly chosen microscopic fields (0.1 mm²) per experiment. cell adhesion. Cells were allowed to attach for 3 h and 24 h (cardiac cells without preplating) or 48 h (enriched cardiomyocytes). Subsequently, cells were stained for DNA, sarcomeric α-actinin, collagen 1, or for DNA, sarcomeric α-actinin, and VE cadherin. To determine the average number of attached cardiomyocytes per microscopic field, experimental data from the performed hypertrophy assay (see below) were used to assess the average number of actinin-positive cardiomyocytes per microscopic field for 10 randomly chosen microscopic fields (0.1 mm²) per experiment.
Hypertrophy assay. Cells were seeded in DMEM F12 + GLUTAMAX + 100 U/mg/ml penicillin/streptomycin + supplements and were allowed to attach overnight. They were then washed with DPBS, and the medium was changed, supplemented with either 0.2% FBS, 10% FBS, or 50 µM PE (phenylephrine, Sigma Aldrich, P6126). After another 48 h, the cells were fixed and stained to determine the average percentage of atrial natriuretic factor (ANF)-positive cardiomyocytes per microscopic field for 10 randomly chosen microscopic fields (0.1 mm²) per experiment. proliferation assay. Cells were seeded in DMEM F12 + GLUTAMAX + 100 U/mg/ml penicillin/streptomycin + 1% FBS without supplements and allowed to attach overnight. After washing with DPBS, the medium was changed to DMEM F12 + GLUTAMAX + 100 U/mg/ml penicillin/streptomycin + 0.2% FBS, + 10% FBS, or + 0.2% FBS, 50 ng/ml FGF1 (R&D Systems, 132-FA) and 10 µM p38i (Tocris, SB203580-HCl). To the corresponding wells, additional p38i (10 µM) was added every 24 h for the next 48 h. 5-ethynyl-2'-deoxyuridine (EdU, Life Technologies, C10337) was added after the medium change after 24 h and 48 h to yield a concentration of 30 µM/ well. 72 h after the medium was changed, cells were fixed and stained using the kit according to the manufacturer's protocol. Subsequently, the average percentage of EdU-positive cardiomyocytes per microscopic field was determined for 10 randomly chosen microscopic fields (0.1 mm²) per experiment. Video analysis. Cells were seeded in DMEM F12 + GLUTAMAX + 100 U/mg/ml penicillin/streptomycin + supplements and incubated overnight. They were then washed with DPBS, and the medium was changed to medium containing 0.2% FBS or 10% FBS. After 48 h, the cells were washed, the medium was changed once again with the respective FBS concentrations and incubated overnight. Then, for a period of 10 seconds, the number of beats during this time was either counted or videos recorded and the number of beats was determined using the Fiji Kymograph-plugin four times per experiment. Briefly, when a line segment is drawn on a video file, the Kymograph-plugin can generate a time-series image from the video's constituent images that the line's coordinates refer to. In the resulting images, the x-axis represents time with the width of the image covering the entire 10-second period or a portion of the video, while the y-axis represents the axis of movement. MUSCLEMOTION analyses of beating movies of cardiomyocytes was done based on a previously published method. 61 Statistical analysis. n is the number of independent experiments. Each independent experiment was performed with 2 technical replicates. 10 randomly chosen microscopic fields (0.1 mm 2 ) from each 2 technical replicates were chosen, meaning 30 fields when n was 3, and used for quantitative analyses. Data are expressed as the mean ± SD of at least three independent experiments. The statistical significance of the differences between means was evaluated by a two-tailed Student's t-test (Microsoft Excel) or where appropriate by one way ANOVA followed by Bonferroni's post-hoc test (IBM SPSS Statistics Version 21). p < 0.05 was considered statistically significant.