Variation in stiffness regulates cardiac myocyte hypertrophy via signaling pathways.

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Variation in stiffness regulates cardiac myocyte hypertrophy via signaling pathways

Jieli Li1, Michael Mkrtschjan2, Ying-Hsi Lin1, Brenda Russell1,2* 1

Department of Physiology and Biophysics, Center for Cardiovascular Research, University of

Illinois at Chicago 2

Department of Bioengineering, University of Illinois at Chicago

851 South Morgan Street Chicago, IL 60607 *corresponding author Mailing Address University of Illinois at Chicago, MC 901 835 South Wolcott Avenue Chicago, IL 60612-7342 Tel: 1-312-413-0407 Email: [email protected]

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Abstract Much diseased human myocardial tissue is fibrotic and stiff, which increases the work that the ventricular myocytes must perform to maintain cardiac output. The hypothesis tested is that the increased load due to greater stiffness of the substrata drives sarcomere assembly of cells, thus strengthening them. Neonatal rat ventricular myocytes (NRVM) were cultured on polyacrylamide or polydimethylsiloxane substrates with stiffness of 10 kPa, 100kPa, 400 kPa, or glass with stiffness of 61.9 GPa. Cell size increased with stiffness. Two signaling pathways were explored, phosphorylation of focal adhesion kinase (p-FAK) and lipids by phosphatidylinositol 4,5bisphosphate (PIP2). Subcellular distributions of both were determined in the sarcomeric fraction by antibody localization, and total amounts were measured by Western or dot blotting, respectively. More p-FAK and PIP2 distributed to the sarcomeres of NRVM grown on stiffer substrates. Actin assembly involves the actin capping protein Z, CapZ. Both actin and CapZ dynamic exchange were significantly increased on stiffer substrates when assessed by fluorescence recovery after photobleaching (FRAP) of green fluorescent protein tags. Blunting of actin FRAP by FAK inhibition implicates linkage from mechano-signalling pathways to cell growth. Thus, increased stiffness of cardiac disease can be modeled with polymeric materials to understand how the microenvironment regulates cardiac hypertrophy.

Key words: Mechano-transduction; focal adhesion kinase; lipid signaling; actin assembly; substrate stiffness

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Introduction The mechanical properties of the local microenvironment influence the function of cells (Yang et al. 2014). This is particularly critical following a myocardial infarction, in which stiff, fibrotic scar tissue replaces the normally compliant ventricle with adverse functional consequences. The ventricular myocytes must work harder to maintain cardiac output, which they mainly accomplish by cell hypertrophy. Thus, the processes that link mechanosensing of increased load to the strengthening of myocytes by cell hypertrophy are of major significance in heart diseases. Multiple mechanosensors detect increased mechanical loading (Hoshijima 2006), but the feedback linking sensing to local actin filament assembly is not yet fully understood. Here, we culture cardiac myocytes on substrata of defined stiffness to analyze cell responses. The study of mechanisms of cell growth requires altering the load in a controlled manner, which is difficult to do at the cellular level since cells are usually cultured on hard, plastic surfaces, which poorly mimic the external forces existing in living tissue. Stiffness of the surface on which cells grow significantly affects maturation and differentiation into myocytes (Jacot et al. 2010) and also force generation (Bhana et al. 2010; Broughton and Russell 2015). The stiffness in the heart can vary from embryonic/neonatal of 5-10 kPa (Bhana et al. 2010; de Tombe 2003) to the normal adult rat myocardium of 10-70kPa (Borbély et al. 2005). Infarct stiffness and collagen content increased with time post-infarct up to 400 kPa (Fomovsky et al. 2010a; Fomovsky et al. 2010b; Holmes et al. 2005). Therefore, we fabricated novel substrata out of polyacrylamide (PAA) (Engler et al. 2008) and polydimethylsiloxane (PDMS) in the physiologic and pathologic range (10 to 400 kPa) (Broughton and Russell 2015) in order to model loading of cardiac myocytes and resultant signaling pathways. In muscle, focal adhesions are the primary biomechanical sensors found at the Z discs where integrins are anchored at the costamere to the extracellular matrix. The focal adhesion 3


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kinase, FAK, binds to the cytoskeletal domain of the integrin complex and responds to mechanical stimuli (Senyo et al. 2007). Mechanical stimulation rapidly phosphorylates and activates FAK possibly by unfolding of the protein to expose a phosphorylation site (Chu et al. 2011; Franchini et al. 2000). Interestingly, FAK is activated by cyclic strain at Tyr-397 and distributes along the myofilaments (Torsoni et al. 2003), which might suggest that the distribution of p-FAK regulates actin assembly. Another actin assembly site is at the Z-disc where the actin capping protein, CapZ, is able to slow down filament assembly (Edwards et al. 2014). On mechanical stimulation of myocytes, the CapZβ1 C-terminus may control capping of the actin filament (Lin et al. 2013). Moreover, mechano-transduction arising from stress or strain may also modify the function of CapZ by phosphatidylinositol 4,5-bisphosphate (PIP2), a phospholipid (Li and Russell 2013). In this report, cell signaling and actin, thin filament assembly was assessed by fluorescence recovery after photobleaching (FRAP) to determine the capping dynamics of CapZ and actin. Understanding how fibrotic stiffness in the microenvironment regulates cardiac hypertrophy may be important in cardiac disease states.

Materials and methods Substrata fabrication Polymers with varying stiffness were used to coat glass surfaces with a layer approximately 100 microns thick. The goals were to attain the stiffness range from physiologic (10 kPa) to pathologic (400 kPa) while retaining cell adhesion for culture, good optics for immuno-localization, and protein isolation for Western and dot blotting. PDMS is viscoelastic below 100 kPa, requiring the use of PAA at lower stiffnesses (Wei et al. 2015). Cells can be scraped for Western and dot blotting from stiff PDMS but not from soft PAA below 100 kPa. To 4


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control for potential differences in material properties, 100 kPa surfaces were produced with both PDMS and PAA.

Preparation of glass base for application of polymer layer Cell culture glass bottom dishes and 10 mm circular coverslips were treated using a modified protocol (Poellmann and Wagoner-Johnson 2013; Tse and Engler 2010). The center portions of glass bottom dishes were treated for 30 minutes with 10 N NaOH to expose hydroxyl groups, then washed thoroughly with deionized water. The glass was silanated with 3(trimethoxysilyl) propyl methacrylate (Cat# M6514, Sigma USA) for 90 minutes. Dishes were washed 3 times with 70% EtOH and dried on a 100ºC hotplate. Coverslips necessary for creating flat surfaces were washed with 70% EtOH in petri dishes, air-dried, then silanated by placing them in a desiccator with 20 µl of tridecafluoro-(1,1,2,2-tetrahydrooctyl)-1-trichlorosilane (UCT T2492) for 90 minutes. Following treatment, coverslips were washed with 70% EtOH and dried on a 100ºC hotplate.

Polyacrylamide substrata 40% unpolymerized acrylamide (Cat#161-0140, Bio-Rad, USA) and 2% Bis solution (Cat#161-0142, Bio-Rad, USA) were diluted in water to concentrations necessary to develop 10 kPa (final concentration 5% acrylamide, 0.3% Bis) and 100 kPa (final concentration 30% acrylamide, 0.3% Bis) substrata, respectively. Ammonium persulfate was added at 1% by volume and tetraethylmethylenediamine at 0.1% by volume in order to begin the polymerization reaction. 10 µl of solutions were then added to glass bottom dishes and each covered with a treated coverslip. Substrata were allowed to polymerize for 10 minutes, then coverslips were

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gently pried up, leaving behind a flat, circular substrate. Dishes were washed 3 times in deionized water for 10 minutes at a time to remove unpolymerized acrylamide. To functionalize polyacrylamide substrata for cell adhesion, they were treated twice by drying sulfo-SANPAH (Cat#22589, Thermo Fisher, USA) in HEPES (50 mM, pH 8.5) on each for 90 minutes at 57ºC. A UV cross-linker (UV Crosslinker, Spectronics Corporation, USA) with a 365 nm bulb, placed approximately 10 cm away for 10 minutes, was then used to cross-link sulfo-SANPAH to the substrate surface. Substrates were washed with HEPES between treatments. Following sulfo-SANPAH treatment, substrates were washed 3 times using 50 mM HEPES, then HEPES containing fibronectin (10 µg/ml) was added to dishes and incubated at 37ºC for 2 hours before UV-sterilizing in water for 20 minutes.

Polydimethylsiloxane substrata Polydimethylsiloxane (PDMS) (DowCorning, Midland, MI) was mixed in a 10:1 (400 kPa) or 50:1 (100 kPa) elastomer base to curing agent ratio for approximately 10 min. PDMS mixtures were degassed using a vacuum desiccator for approximately 30 min. PDMS was then spun onto cell culture glass-bottom dishes (In Vitro Scientific, CA), creating a PDMS thickness of approximately 50 µm, or added to 6-well plates. PDMS substrata were then cured for 24 h in a 57ºC oven. After curing, PDMS was cooled and ready for activation for fibronectin coating. PDMS substrata were treated with 5% (3-Aminopropyl) triethoxysilane in 90.25% ethanol solution for 10 min, then washed with 100% ethanol. Substrata were placed in a 57ºC oven for 20 min, washed with 95% ethanol and twice with PBS, then coated with 10 µg/ml fibronectin in DMEM for 2 hours at 37ºC.

Measurement of stiffness 6


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Stiffness of both materials was measured by Young’s Modulus using atomic force microscopy. For PDMS, our lab found 50:1 ratio to yield a Young’s Modulus of 98.4 +/- 7.36 kPa, while a 10:1 ratio yields a 397.4 +/- 30.79, which are rounded off to 100 and 400 kPa, respectively (Broughton 2015); for PAA, mixtures of 5%/0.3% and 30%/0.3% acrylamide/Bis yielded ~9.3+/- 0.26 and 101.4 +/- 1.6 kPa, respectively by atomic force microscopy ([Asylum 1D, Asylum Research; Santa Barbara, CA) and indented by a pyramid-tipped probe (Veeco; Santa Barbara, CA)] (data provided by Dr. Engler at UCSD, Engler et al. 2006). The glass stiffness reported as 61.9 GPa (Wang et al. 2012).

Neonatal rat ventricular myocyte culture Primary heart cultures were obtained from neonatal rats according to Institutional Animal Care and Use Committee and NIH guidelines for the care and use of laboratory animals that are equivalent to the Canadian Council on animal care (CCAC) regulations. Hearts were removed and cells isolated from 1-2 day old Sprague-Dawley rats with collagenase type II (Worthington, Lakewood, NJ USA) as previously described (Boateng et al. 2003). Neonatal rat ventricular myocytes (NRVMs) were re-suspended, filtered through a metal sieve to remove large material and plated at high density (1,000 cells/mm2) in PC-1 medium (Lonza Group Ltd, U.S.A). Unattached cells were removed by aspiration and PC-1 media was replenished. Myocytes were plated on fibronectin coated (10 µg/ml) dishes at 1560 cells/mm2 for Western blotting or 520 cells/mm2 for immunostaining. Stiffnesses used for PAA were 10 and 100 kPa; for PDMS were 100 and 400 kPa; and on glass-bottom dishes (61.9 GPa). Cells were cultured for 3 days in a 5% CO2 incubator.

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Localization of p-FAK (Tyr397) or PIP2 in the cytoskeletal fraction by immunostaining and microscopy For isolation of the cytoskeletal fraction, the Calbiochem® ProteoExtract® Subcellular Proteome Extraction Kit was used (EMD Millipore, Billerica, MA), following a previously described detergent-based protocol (Boateng et al. 2007). The remaining myofibrillar cytsokeleton was immunostained with α-actinin antibody (1: 200, Cell Signaling Technology, Inc., Danvers, MA USA), and either with a p-FAK (Tyr397) antibody (1: 200, Cell Signaling Technology, Inc., Danvers, MA USA) or a PIP2 antibody (1:200, mouse IgG, Abcam, Cambridge, MA USA). Cardiomyocytes were observed by microscopy (Observer Z1, Zeiss), and by confocal microscopy (LSM 710, Zeiss). Cell surface areas were measured by ImageJ software. In each case, three independent experiments were performed, values were calculated and 20 cells from each condition were randomly chosen and used to calculate cell areas. Experiments were repeated at least three times on PDMS (100 kPa, 400 kPa) or glass. The selective extraction method for the subcellular components on the polyacrylamide hydrogels detached the cells from the surface. Efforts to retain cell attachment by shortening the exposure times and altering the detergents were unsuccessful.

PIP2 levels by dot blots Whole cell lysates extracted from NRVMs grown on 100 kPa, 400 kPa, PDMS substrata, or 61.9 GPa (glass) were spotted onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA, USA). These were probed with PIP2 antibody (mouse IgG, Abcam, Cambridge, MA USA) at a 1:500 dilution and detected using a horseradish peroxidase conjugated secondary antibody (anti-mouse, HRP, Cell Signaling Technology, Boston, MA USA) and ECL. 8


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To exclude any non-specific binding of the PIP2 antibodies, we detected the signal with a positive control PIP2 (Echelon Biosciences, Cat#P-4516) (data not shown). Experiments were repeated at least three times. Cell scraping for traditional dot blots could not be performed on PAA (10 kPa or 100 kPa) due to the structurally weak nature of polyacrylamide hydrogels.

Fluorescence recovery after photobleaching (FRAP) for actin dynamics Microscopic techniques, such as FRAP have yielded quantitative information about the processes that regulate actin polymerization in living myocytes. The methods and analysis for FRAP of actin-GFP were described by our lab (Lin et al. 2013). NRVMs were treated with a FAK inhibitor PF-573228 (30 µM) (Cat#PZ0117, Sigma USA) (Slack-Davis et al. 2007) for one hour prior to the FRAP experiment. In the present study, five myocytes were analyzed per culture, and at least three separate cultures were studied for 100 kPa, 400 kPa and glass. Stiffness affects embryonic cardiomyocyte structure and contractility (Engler et al. 2008). We also found very rapid beating of the neonatal myocytes on 10 kPa substrates, which did not permit the imaging quality and time resolution necessary for FRAP analysis.

Statistics Data are presented as mean ± SEM. Statistical significance was determined by OneWay ANOVA with Tukey’s multiple comparison tests. Significance was taken as p

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