Human Skeletal Muscle Cells on Engineered 3D Platform Express Key

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Tissue Engineering and Regenerative Medicine

Human skeletal muscle cells on engineered 3D platform express key growth and developmental proteins Akshata R. Naik, Sebastian Pernal, Kenneth T. Lewis, Yaobin Wu, Hongkai Wu, Nicholas J Carruthers, Paul M. Stemmer, and Bhanu P Jena ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01338 • Publication Date (Web): 18 Jan 2019 Downloaded from http://pubs.acs.org on January 19, 2019

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ACS Biomaterials Science & Engineering

Human skeletal muscle cells on engineered 3D platform express key growth and developmental proteins Akshata R. Naik1, Sebastian Pernal1, Kenneth T. Lewis1, Yaobin Wu5, Hongkai Wu5, Nicholas J. Carruthers3, Paul M. Stemmer3, Bhanu P. Jena1,2,3,*

1Department of Physiology, School of Medicine, 2NanoBioScience Institute, 3Center for

Molecular Medicine & Genetics, 4Institute of Environment Health Sciences, Wayne State University, MI 48201, USA 5Department of Chemistry, Hong Kong University of Science & Technology, Hong Kong, China

* Corresponding author at: Wayne State University School of Medicine, 540 E. Canfield, 5245, 5215 Scott Hall, Detroit, MI 48201, USA. Tel: 313-577-1532; Fax: 313-993-4177 E-mail address: [email protected] (B.P. Jena)

1 ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Current approaches in regenerative medicine to develop human skeletal muscle replicating native tissue for engrafts and high throughput drug screening and gene therapy is still in its infancy and has not proven to recapitulate the behavior and regulatory processes present in endogenous skeletal muscle tissue. This stems at least in part from the lack of a comprehensive understanding of the emergent properties of in vitro skeletal muscle growth and development. To address this gap in our current knowledge, we have developed a stretchable micropatterned 3D human skeletal muscle platform that recapitulates organized and parallel growth of muscle cells and fibers as opposed to the randomly oriented cells growth on a 2D glass surface. Mass spectrometry of the muscle cells growing on the 3D platform express key myogenic proteins such as myoferlin for myoblast fusion required in the formation of muscle tissue, and proteins involved in mitochondrial health and biogenesis, in contrast to cells growing on 2D glass surface. These results demonstrate that the engineered human muscle cells grown on the 3D platform holds great promise to further establish the emergent properties of in vitro skeletal muscle growth and development for a wide range of biomedical applications.

KEYWORDS: 3D Microphysiological Platform, Human skeletal muscle proteome, Muscleon-a-Chip, Tandem mass spectrometry


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INTRODUCTION Muscle contraction is essential for proper physiological functioning from the beating of the heart to locomotion, involving the molecular motor myosin and ATP. Muscle contraction occurs by the sliding action of the thick (myosin) and thin (actin) filaments, driven by the hydrolysis of ATP produced in the mitochondria. No treatments are currently available for skeletal muscle atrophy as a consequence of disuse in the intensive care unit (ICU). Similarly, a large body of work1,2 demonstrate that when humans are exposed to microgravity as in the International Space Station, a major detriment is the accelerated wasting of skeletal muscles and loss of function. While skeletal muscle tissue is capable of repair following minor injury, current approaches are severely limiting for volumetric muscle loss as a consequence of disuse in the ICU, microgravity and induced by trauma, requiring engineered muscle tissue engrafts. Similarly, while animal models of disease and drug testing for use in treatment and therapy in humans have provided a wealth of information, over 70% of drugs with stellar results in animals demonstrate alarmingly low efficacy in human trials3. To overcome these limitations, we have developed a stretchable microphysiological micropatterned 3D platform4 to engineer human muscle tissue, both for tissue engrafts and to test how human skeletal muscle tissue responds to various exercise regimens, gene therapy, and drug treatments. The current study was undertaken to assess changes in morphology and proteome of the mitochondrial and motor proteins in human skeletal muscle cells grown on the 3D platform compared to on glass surface. As



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opposed to the random organization of muscle cells growing on a 2D surface, our microphysiological 3D platform provides controlled and organized growth of human muscle tissue. Our results further demonstrate an upregulation of expression of a widespectra of both mitochondria and motor proteins in HSkMC cells growing on the 3D platform as opposed to on the glass surface. These results demonstrate that the 3D platform holds great promise in muscle graft development, in addition to evaluating the efficacy of various gene therapies and drugs, to overcome a wide range of skeletal muscle disorders.

MATERIALS AND METHODS Human primary skeletal muscle cells were obtained from Lonza Bioscience Inc. (Walkersville, Maryland, USA). Cells were handled, stored and cultured, according to the published procedures and protocols approved by the institutional review board of Wayne State University. Cells stored frozen in liquid nitrogen were thawed, resuspended in muscle cell growth medium (Lonza Bioscience Inc., Walkersville, Maryland, USA) and seeded on both glass-bottom petri dishes and the 3D micropatterned platform. Cells were cultured at 37 C, 5% CO2 and 100% humidity condition.

3D Platform Development The stretchable microphysiological micropatterned 3D platform was constructed according to minor modification of our published procedure4 (Figure 1). Microgrooved Poly-L-lysine-polydopamine (PDA)-coated parafilm membranes with precision micropatterned 10 µm groove spacings were prepared using master


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fabricated stamps developed using photolithography using SU-8 photoresist. Parafilm (Pechiney Plastic Packaging Company, USA) was covered by a flat poly (methyl methacrylate) (PMMA) chip and the PDMS stamp, that was heated to 60 °C using a hot compressor (Taiming, Inc., USA) for a period of 3s. The sandwich assembly was then cooled to room temperature, prior to slowly peeling off the PMMA chip from the stamp. The micropatterned parafilm was next treated with oxygen plasma for 5 min, dipped into a 2 mg/mL dopamine (Sigma-Aldrich, USA) Tris buffer (10 mM) solution at pH 8.5 for 8h. The polydopamine coating was generated by the autopolymerization of dopamine. The patterned PDA-coated membrane was then incubated in 0.1% Poly-L-lysine in phosphate-buffered saline (PBS) pH 7.4 for 1h, prior to 10 min exposure to UV, washed with culture medium, and cell seeding. This 3D platform developed by our published procedure4 exhibits a significantly lower water contact angle, indicating that abundant hydrophilic groups were created on the surface of parafilm. Additionally, the surface energy of the PDA-coated parafilm is approximately 16-fold higher than that of pristine parafilm, which significantly promotes cell adhesion, growth and survival.

Light and Fluorescent Microscopy Light microscopy was performed using a FSX100 Olympus microscope, and images



(Figure 2) were acquired through a 10x, 40x and 63x objective lens. To determine the position of the cell nucleus, cells were exposed to 4',6- diamidino-2-phenylindole (DAPI) for nuclear stain (Molecular Probes, Life Technologies, Carlsbad, CA). To determine the distribution of myosin IIB in human skeletal muscles growing on glass and the 3D platform, immunofluorescence studies were performed according to published procedures5,6. Myosin IIB fibers were stained with a 1:200 dilution of a mouse monoclonal heavy chain primary antibody, followed by a 1:500 dilution of AF 488 donkey anti-mouse secondary antibody. An immunofluorescence FSX100 Olympus microscope was used to acquire immuno-fluorescent images through a 63x objective lens (numerical aperture, 1.40) with illumination at 488nm. Myosin IIB distribution was examined from the acquired images using the ImageJ software. Understanding the arrangement and distribution of intracellular structures had remained qualitative until recently when cell and nuclei alignment measurements could be made4,7-9, and following the development of FibrilTool10. FibrilTool is an ImageJ plug-in tool based on the concept of nematic tensor, which provides a quantitative description of the anisotropy of organelle arrays and their average orientation in cells, directly from the raw images obtained from micrographs. In images of the cell, the assessment of fibrillar structures such as cytoskeletal elements had remained primarily descriptive. However, following the development of


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FibrilTool, an ImageJ plugin, alignment of structures in micrographs can be better accessed, as demonstrated in this study.

A multitude of data in microscopy images

take the form of fibrillar structures such as cytoskeletal elements. In addition to evaluating fluorescence intensity of these structures, other parameters, such as the length, orientation, and alignment of multiple fibril structures, are important for understanding cellular structure-function. FibrilTool makes this possible through computing the nematic tensor, a metric of alignment in 2D or 3D space, for each structure, making it possible to obtain statistics on the alignment of all structures within a cell at nanometer resolution. In the current study, the FibrilTool provided the average orientation and anisotropy of nucleus arrays in primary human skeletal muscle cells growing on a glass surface and the micropatterned 3D platform (Figure 3). Tryptic Digest of Human Skeletal Muscle Cells In studies using in-solution digestion, human skeletal muscle cells on the glass platform and on the 3D platform were solubilized separately in triplicates in lysis buffer, followed by precipitation in cold methanol as previously described11. The resulting protein pellets (25 µg) were solubilized in 0.05% ProteaseMax and 40 mM TRIS, pH 8.0.

After reduction with 5 mM dithiothreitol and alkylation with 15 mM

iodoacetamide, trypsin (Promega Gold) was added to the diluted sample solution



(1:20 w/w) for overnight digestion at 37 C.

LC-MS/MS Analysis and Database Search LC-MS/MS was performed according to minor modification of our published procedure12. Analyses were made using an Acclaim PepMap RSLC, 75 m x 25 cm column with LC-MS/MS performed on an Orbitrap Fusion mass spectrometer. Tandem mass spectra were extracted by Proteome Discoverer version 1.4. Charge state deconvolution and deisotoping were not performed. All MS/MS samples were analyzed using Sequest-HT (Thermo Fisher Scientific; version 2.1.1.21) and X! Tandem (The GPM, the gpm.org; version CYCLONE (2010.12.01.1)). The Uniprot human complete database (downloaded 2017.17.14, 42,139 entries) was searched assuming the digestion enzyme trypsin.

The fragment ion mass tolerance was 0.60 Da and the

parent ion tolerance was 10.0 parts per million (ppm).

Carbamidomethyl of cysteine

was specified as a fixed modification. Deamidation of asparagine and glutamine and oxidation of methionine were specified in Sequest-HT as variable modifications. Glu>pyro-Glu of the n-terminus, ammonia-loss of the n-terminus, gln->pyro-Glu of the n-terminus, deamidation of asparagine and glutamine and oxidation of methionine were specified in X! Tandem as variable modifications. Scaffold (version 4.8.6, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide


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and protein identifications.

Peptide identifications were accepted if they could be

established at greater than 99.0% probability. Peptide Probabilities from Sequest-HT were assigned by the Scaffold Local FDR algorithm. Peptide Probabilities from X! Tandem were assigned by the Peptide Prophet algorithm with Scaffold delta-mass correction13. Protein identifications were accepted if they could be established at greater than 99.0% probability to achieve an FDR less than 1.0% and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm14. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Principal component analysis (PCA) was conducted using Origin 2018 (OriginLab Corp., Northampton MA). Inclusion criteria for PCA were proteins that were present in all experiments, and any null proteins were excluded.

RESULTS AND DISCUSSION

In our earlier study4, a PDA-coated stretchable micropatterned membrane was developed for osteogenic differentiation of stem cells, demonstrating the capability for organized cell growth. The current study was conducted using a modification of the previously developed platform4, to test the hypothesis that organized parallel growth of human muscle cells can be achieved with complete efficiency using the



stretchable 3D platform mimicking native physiological conditions as opposed to randomly oriented cells growing on a 2D glass surface. Furthermore, the current study was carried out to determine whether the 3D platform is able to express optimally motor and mitochondrial proteins required for muscle activity, and therefore amenable to the development of engineered skeletal muscle tissue for future use in grafts, drug screening and gene therapy.

Substrate microarchitecture on cell morphology and behavior have been demonstrated to be critical in tissue engineering15-22. Earlier studies report that cells prefer and therefore migrate from a 2D growing substrate to a 3D platform23, and that the substrate stiffness closely mimicking native tissue plays a critical role24. Different cell types are known to have different surface adhesion properties influencing their division, growth and survival. Since in our earlier studies on osteogenic differentiation of stem cells using the stretchable and micropatterned polydopamine (PDA)-coated parafilm membrane, we observed several fold increased adhesion, growth and cellular gene expression over parafilm, the primary human skeletal muscle cells in the present study was first cultured using the PDA-coated micropatterned parafilm membrane. Since a typical human skeletal muscle cell is 1050 m in thickness, the 10 m patterned groove was chosen to culture them. Interestingly, primary human skeletal muscle cell failed to adhere and hence survive


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on the PDA-coated micropatterned parafilm membrane (data not shown). Poly-Llysine, being a synthetic positively charged and well-established polymer, known to promote cell adhesion yet undegradable by cellular proteases, was therefore chosen to surface-coat the platform post PDA-functionalization. The 2D glass surfaces to grow cells in comparison, were therefore coated with poly-L-lysine.

Results from the current study demonstrates that the morphology of human skeletal muscle cells grown on the 3D platform express organized parallel arrangement of cells as reflected by the arrangement of the nuclei and muscle fiber growth, including myosin IIB, as opposed to the randomly oriented cells growing on the glass surface (Figure 2, 3). Although, within individual skeletal muscle cells growing on glass surface the myosin IIB appear to be somewhat parallelly arranged in an organized fashion (Figure 3A), the random arrangement of cells on the glass (Figure 2D, 2F) resulting in an unorganized orientation is reflected in the arrangement of the cell nuclei (Figure 3D). This is opposed to the observed organized arrangement of skeletal muscle cells within the parallel groves of the 3D micropatterned membrane platform (Figure 3E, 3F).

These results provide morphological evidence that the 3D

micropatterned platform helps in the organized parallel orientation and arranged growth of human skeletal muscle cells required for cell fusion and the consequent development of the multinucleated skeletal muscle tissue25-28.



To further test the hypothesis that micropatterned substrates influence cellular protein expression more aligned with the native tissue, mass spectrometry of both mitochondrial and motor protein expression in human skeletal muscle cells growing on the 3D platform and on the glass surface, was performed (Figures 4 and Table I). Results from the study demonstrate upregulation of a large spectra of both mitochondrial and motor proteins in the 3D platform over glass, suggesting great promise in muscle graft development in addition to being able to evaluate the potency and efficacy of various gene therapies and drug targets to overcome a wide range of myopathies. Normal myoblast fusion in the formation of skeletal muscle requires myoferlin29, which is upregulated in the 24h cells growing on the 3D platform (Table I). As previously iterated, the growth of skeletal muscle occurs during embryonic development and continues in adult life for regeneration, where single nucleated myoblasts fuse with each other to form myotubes. Furthermore, during muscle growth, mononucleated myoblasts can also fuse to form large syncytial myofibers as a mechanism for increasing muscle mass without increasing myofiber number. Myoblast fusion however requires the alignment and fusion of two apposed lipid bilayers which is potentiated by myoferlin. MYO1C expression in muscles cells growing on the 3D platform reflects the increased capability of glucose transport into these cells. Similarly, ACL6A expression in cells growing on the 3D platform is


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evidence of increased transcriptional activation by its capacity to remodel chromatin. Another example of the expression in 3D muscle culture, is the expression of SYIM, the isoleucine tRNA ligase or synthase involved in mitochondrial respiratory chain enzyme function and higher expression levels of skeletal muscles. Similarly, there is upregulation of many critically important proteins for muscle growth, development and health, among them the ATP-dependent RNA helicase DDX3 upregulated in human muscle cells growing on the 3D platform (Table I). ATP-dependent RNA helicase DDX3 is known for its protein quality control in the mitochondria30. Inactivation of DDX3 leads to the accumulation of mitochondrial reactive oxygen species associated with defect in hydrogen peroxide detoxification, resulting in mitochondrial membrane potential loss, fragmentation, and cell death. Furthermore, it is reported30 that in the absence of DDX3, there is increase in components of the unfolded protein response and polyubiquitinated proteins resulting in mitochondrial damage. Principal component analysis (PCA) on the MS data (Figures 4) demonstrated statistical significance (p

Human Skeletal Muscle Cells on Engineered 3D Platform Express Key

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