Substrate Stiffness Affects the Functional Maturation of Neonatal Rat Ventricular Myocytes

Deformable polyacrylamide cell substrate preparation and tensile modulus measurement

The deformable polyacrylamide substrates used in cell culture were made as in previous studies (26,27,33). Briefly, 3%–7% acrylamide and a 1:20 ratio of bisacrylamide with 5% fluorescent beads (Polysciences, Warrington, PA) were polymerized with 0.1% ammonium persulfate and 0.5% N,N,N′,N′-tetramethylethylenediamine. This solution was pipetted onto 18-mm glass coverslips activated with 3-aminopropyltrimethoxysilane and 0.8% glutaraldehyde. Gels were coated with 0.5 mg/mL of rat tail type I collagen bound through the heterobifunctional crosslinker N-sulfosuccinimidyl-6-[4′-azido-2′-nitrophenylamino] hexanoate (sulfo-SANPAH, Pierce Biotechnology, Rockville, IL).

The tensile elastic moduli of these gels were measured as in previous studies (26,27) by calculating the slope of the stress-strain curve found by hanging weights from a 15-mm thick cylinder of gel.

NRVM cell isolation, culture, and plating

Cardiac myocytes were harvested from freshly dissected ventricles of 1- to 3-day-old Sprague-Dawley rats using an isolation kit (Cellutron, Highland Park, NJ). Cells were plated and cultured as described previously (34) in high-serum plating media (Dulbecco modified Eagle media, 17% M199, 10% horse serum, 5% fetal bovine serum, 100 U/mL penicillin and 50 mg/mL streptomycin) at 10,000 cells/cm². Approximately 18 h later, cells were transferred to low serum maintenance media (Dulbecco modified Eagle media, 18.5% M199, 5% horse serum, 1% fetal bovine serum, and antibiotics). Cell cultures were maintained at 37°C and 5% CO₂ and fresh maintenance media was added every 2–3 days. For hydroxyfasudil treatment studies, cells were cultured in 1 μmol/L hydroxyfasudil. For C3 toxin treatment studies, cells were cultured in 5 μg/mL exoenzyme C3 from Clostridium botulinum (Calbiochem, San Diego, CA). All culture media was purchased from Invitrogen, Carlsbad, CA and sera was purchased from Gemini BioProducts, West Sacramento, CA. Animal studies were in accordance with University of California guidelines.

Morphology of NRVMs on gels of varying stiffness

To evaluate sarcomeric definition and organization, NRVMs were fixed and stained at 7 days after initial plating. Cells were fixed in 0.75% glutaraldehyde on ice for 20 min, permeabilized with 0.1% Triton X-100 for 5 min and stained with anti-α-actinin (Sigma), diluted in 1% bovine serum albumin (Gemini BioProducts) for 60 min. Secondary antibodies of tetra-rhodamine isothiocyanate-conjugated goat anti-rabbit (Jackson ImmunoResearch, West Grove, PA) in 1% bovine serum albumin were added for 30 min. Cells were imaged using a Nikon Eclipse TE300 inverted microscope with a Photometrics Cascade 512X CCD camera and acquired using Metamorph software (Molecular Devices, Sunnyvale, CA).

NRVMs used in confocal imaging experiments were stained with Vibrant CM-DiI (Invitrogen), then fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) on ice for 20 min and permeabilized with 0.5% Triton X-100. Cells were stained with FITC-phalloidin and covered with gelvatol mounting medium and a coverslip. Cells were imaged on a spinning disk confocal microscope (Olympus, Center Valley, PA).

In addition, cell area and aspect ratio (long axis/short axis) were computed for all myocytes evaluated for histology, traction force, and calcium concentration by manually outlining cells in ImageJ (National Institutes of Health, Bethesda, MD).

Dynamic traction force microscopy

A complete traction force map of shear stresses at the surface of the gel was obtained through traction force microscopy, in which fluorescent beads embedded in the gel were tracked during cell contraction (24,25). Individual NRVMs at 7 days post-isolation were stimulated with an 8 ms pulse at 0.5 Hz. Voltage was increased from 0.1 V until the cell began beating. Cells that were in contact with other cells and those that did not beat were excluded. The deformation field and corresponding traction field were computed with custom-written MATLAB 7.0 code (The MathWorks, Natick, MA) as described in the Expanded Materials and Methods in Supplementary Material, Data S1.

Calcium transient and caffeine-induced calcium release measurement with Fura-2 and Fluo-4

NRVMs at 7 days post-isolation were loaded with 2 μmol/L Fura-2 or Fluo-4 (Molecular Probes) in high-calcium Tyrodes buffer (130 mmol/L NaCl, 5.4 mmol/L KCl, 2 mmol/L CaCl, 1 mmol/L MgCl₂, 0.3 mmol/L Na₂HPO₄, 10 mmol/L HEPES, 5.5 mmol/L glucose at pH 7.4). Cells were stimulated as detailed above and imaged using a Nikon Eclipse TE3000 inverted microscope with a Lambda DG-4 filter changer (Sutter Instruments, Novato, CA) and a Photometrics Cascade 512X CCD camera, acquired using Metamorph software (Molecular Devices) and analyzed as detailed in the Expanded Materials and Methods in Data S1.

The amount of calcium stored in the sarcoplasmic reticulum of NRVMs was quantified by adding 10 mmol/L caffeine and measuring the resulting change in fluorescence as described above. Caffeine-induced calcium release was then reported as the plateau of fluorescence intensity after the addition of caffeine divided by the baseline fluorescence.

Western blot analysis of contractile and calcium handling proteins

Western blot analysis was used to assess the amount of actin, myosin, sarcoplasmic/endoplasmic reticular calcium ATPase (SERCA), phospholamban (PLN), and L-type calcium channel protein. At 7 days after plating, a lysate buffer (1% Triton X-100, 5 mmol/L EDTA, 5 mmol/L EGTA, 150 mmol/L NaCl, 20 mmol/L TRIS, 1 μmol/L PMSF, 2 μmol/L Na₃VO₄, 1 ng/mL leupeptin, and 1 ng/mL aprotinin) was added on ice for 30 min. As controls, freshly isolated NRVMs (10⁶ cells) and cardiomyocytes from a freshly dissected and lyophilized adult rat heart were lysed in 1 mL of the same lysate buffer solution on ice for 15 min. The lysates were centrifuged for 15 min at 4500 × g and 4°C and the supernatant was frozen at −20°C. Protein content was determined with a BCA protein assay kit (Pierce Biotechnology). Cell lysates with equal amount of total protein were separated with 4%–12% denaturing SDS PAGE and transferred to a nitrocellulose membrane. Total protein content and transfer effectiveness was assayed by staining with Ponceau Solution and myosin heavy chain (MHC) content was assessed from scans of this stain. Membranes were washed in a TBST buffer (20 mmol/L Tris, 500 mmol/L NaCl, and 0.5% Tween 20), blocked with 5% nonfat dry milk (Carnation, Nestle USA, Glendale, CA), and blotted with antibodies to cardiac α-actin (mouse monoclonal; Sigma), SERCA2a (rabbit polyclonal, a gift from Wolfgang Dillman, University of California, San Diego), PLN (mouse monoclonal, Affinity Bioreagents, Golden, CO), phosphor-serine-16-PLN (rabbit polyclonal, Millipore, Billerica, MA), calcium channel cardiac α_1C subunit (L-type calcium channel, rabbit; Sigma), myosin light chain (MLC2, rabbit polyclonal; Cell Signaling Technology, Danvers, MA), or Phospho-Ser19-MLC2 (rabbit polyclonal, Cell Signaling Technology) overnight. Secondary labeling was carried out with horse radish peroxidase-linked secondary antibodies for 30 min, developed with an ECL Detection System (GE Healthcare, Waukesha, WI) and exposed to film. Band densities were quantified using ImageJ.

Statistical analysis

Experimental differences were evaluated with ANOVA followed by post hoc Bonferroni t-tests. Comparisons with p < 0.05 were determined to be significant, although comparisons with p < 0.10 are noted. Error bars represent SD unless otherwise noted.

Tensile modulus of polyacrylamide gels

Polyacrylamide gel elastic modulus was measured using tensile tests. The tensile modulus of the gel substrates increased exponentially with the concentration of acrylamide and bisacrylamide (Fig. 1). The elastic modulus of each individual gel was linear up to at least 25% strain (data not shown). For simplicity, the elastic modulus reported here for each gel in the rest of the results and graphs was rounded to 1 kPa, 5 kPa, 10 kPa, 25 kPa, and 50 kPa for 3%, 4%, 5%, 6%, and 7% acrylamide gels, respectively.

Morphology of ventricular myocytes on different gels

Differences in the structure and organization of the actin cytoskeleton were observed in cells stained for α-actinin (Fig. 2, A–C) or F-actin (Fig. 2, D–H). Cells on the stiffest substrates appear to have a less structured cytoskeleton, with no apparent aligned sarcomeres and with actin localized both in the nucleus and around the perimeter of the cell. These cells also have stress fibers, which were not seen in cells on softer substrates or in vivo. However, cells on the stiffest substrate could still be induced to beat when a voltage stimulus was applied. Despite these apparent morphological differences, analysis of the cell spread area and the aspect ratio, calculated as the length of the long axis of the cell divided by the length of the short axis, was not significantly different between gel stiffness (Fig. 3, A and B). The circularity index, calculated as 4πA/P² where A is the area and P is the perimeter, is significantly greater for the cells on the stiffest gels compared to the cells on the 10 kPa gels (Fig. 3 C). The circularity index would be exactly 1 for a circular cell and decreases as the cell becomes less circular and develops a spindle, myocytes-like morphology.

Dynamic traction force microscopy and shortening velocity

An analysis of the percentage of cells that contracted when electrically stimulated shows a negative correlation with elastic modulus, as 85%, 65%, 55%, 55%, and 45% of cells (n = 20) on 1, 5, 10, 25, and 50 kPa gels, respectively, contracted when stimulated. All reported values in this study only include cells that contracted when stimulated.

Images of fluorescent beads captured at 66 images/s were compared to the cell-free reference image to generate a displacement map (Fig. 4 A) and analyzed based on continuum equations describing gel mechanics to generate a traction map (Fig. 4 B). Tractions (in units of force/area) were then integrated over area, resolved into their x and y components and projected onto cell axes to evaluate force versus time (Fig. 4 C). Neonatal rat cardiac myocytes cultured for 7 days after isolation generated an average of 720 ± 140 nN of axial force on mid-range gels of 10 kPa. However, cells plated on softer 1 kPa or stiffer 50 kPa substrates generated significantly less force during contraction (Fig. 5 A). Myocytes on intermediate gels of 5 kPa or 25 kPa elastic modulus generated intermediate forces.

The velocity of shortening of the major axis of the cardiomyocyte decreased as the extracellular substrate modulus increased (Fig. 5 B). In addition, NRVMs on 50 kPa gels had sarcomeres of 2.23 ± 0.09 μm (n = 5) and contracted 0.8 ± 0.2% (n = 15) of the cell length at 5 ± 2 μm/s (n = 15). NRVMs on 1 kPa gels had sarcomeres of 1.69 ± 0.15 μm (n = 5) and contracted 23 ± 0.4% (n = 15) of the cell length at 10 ± 1 μm/s (n = 15).

Calcium transients and caffeine-induced calcium release

The magnitudes of calcium transients were measured in stimulated NRVMs using Fura-2 or Fluo-4 calcium indicator (Fig. 6 A). The transient size was greatest in cardiomyocytes cultured on 10 kPa gels (computed as 84 ± 21 nmol/L from Fura-2 data, n = 6) and lower in cells cultured on substrates with a higher or lower elastic modulus. Note that the shape of the relationship between the calcium transient and substrate stiffness mirrored that of axial force and substrate stiffness (from Fig. 5). The magnitude of the caffeine-induced calcium release again was highest on the 10 kPa substrates and progressively lower on softer or stiffer substrates (Fig. 6 B).

Western blot analysis of contractile and calcium handling proteins

Western blots of cardiac α-actin and MHC expression (Fig. 7, A and B) show no significant differences between cardiomyocytes on any stiffness gel, suggesting the above observations are functional differences, as opposed to contractile protein expression differences. Western blot analysis of SERCA2a expression (Fig. 7, C and D) showed a substrate stiffness-dependent expression that mirrors calcium transients (Fig. 6) and axial force (Fig. 5), with the maximal SERCA2a expression occurring in cardiomyocytes cultured on gels of 10 kPa. SERCA2a expression in day-0 myocytes was four times the amount of the expression in cells on 10 kPa gels and significantly greater than any day-7 conditions (Fig. 7 C). Western blots showed no differences in abundance of PLN and the L-type calcium channel had no differences in expression between cells cultured on gels of different stiffnesses (not shown). Phospho-(Ser-16)-PLN was present in adult whole heart lysate but not in day-0 or day-7 NRVMs (not shown).

Contractile force and calcium handling in hydroxyfasudil-treated cells

Seven-day treatment of cell cultures with 1 μM hydroxyfasudil, a ROCK inhibitor (Fig. 8 A), or C3 toxin, a RhoA inhibitor (Fig. 8 B), resulted in a positive correlation between force and elastic modulus (Fig. 8). NRVMs cultured on both 25 kPa and 50 kPa gels and treated with hydroxyfasudil exhibited significantly greater traction force than control cells (p < 0.05). NRVMs treated cultured on 50 kPa gels and treated with C3 toxin also exhibited significantly greater traction force than control cells (p < 0.05). These results suggest that the effects of substrate stiffness on NRVM maturation, at least on substrates stiffer than the native myocardium, are regulated through the RhoA/ROCK pathway. Though Western blots could detect phosphorylated myosin light chain in adult rat whole heart lysate controls, phosphorylated myosin light chain levels in NRVMs were too low to detect on any gel stiffness (results not shown). However, immunostaining of fixed cells shows very low levels of phospho-(ser19)-MLC in NRVMs on soft gels and phospho-(ser19)-MLC associated with cytoskeletal fibers on gels of 25 kPa and higher stiffness (results not shown).