Myocyte shape and myofibrillar assembly respond differently to collagen I and fibronectin(Fn)-coated PAA gels of varying matrix rigidity
Previous studies have extensively used PAA gels with collagen I (Engler et al., 2008
) or mixtures of collagen I and fibronectin (Chopra et al., 2011
) to culture cardiac myocytes, and the differential mechanical response between the two ligand types has not been evaluated. The response of neonatal rat cardiomyocytes (NVRM) to changes in substrate stiffness when cultured on polyacrylamide gels laminated with either fibronectin or collagen I is shown in . Both of these integrin ligands allow adhesion of the cell to the otherwise non-adhesive gels, but the typical cardiomyocyte morphology characterized by a large aspect ratio and actin fibers with striated appearance of alpha actinin staining occurs only when the substrate shear modulus is in the range from 5 to 10 kPa. Fibronectin and collagen engage different sets of integrins, and the morphologies are similar but not identical on each level of substrate stiffness. Myocytes cultured on collagen and fibronectin gels of 100Pa or 300 Pa (soft) displayed a rounded morphology, scarce F-actin assembly and little or no visible striation (). As the stiffness of the PAA gel was increased to 5-10 kPa (intermediate/physiological), myocytes on both ECM ligands increased their spread area and took on an elongated shape ().
Figure 1 NVRM myofibrillar assembly is sensitive to matrix rigidity and adhesive ligand type. Cardiac myocytes cultured on PAA gels of varying rigidity coated with Fn (a-e) and collagen (f-j), cultured for 48 hours and stained for f-actin (red), α-actinin (more ...)
Myocytes cultured on fibronectin coated PAA gels developed well-organized and polarized myofibrillar assemblies whereas on collagen this response was highly attenuated ( and ). On stiffer fibronectin coated PAA substrates (>30 kPa, shear modulus) approximating the pathologic heart, cells increased their spread area and displayed prominent F-actin stress fiber-like filaments but lacked organized myofibrillar assembly or an elongated shape (). In contrast, on stiff collagen-coated PAA substrates the cells did not further increase their spread area and continued to show an attenuated myofibrillar assembly (). These results indicate that the myocyte cytoskeleton remodels in response to substrate rigidity and is strongly influenced by the adhesive ligand to which it binds.
Cardiac myocyte myofibrillar assembly is sensitive to ECM ligand type.
Hyaluronan (HA) gels enhance myofibrillar assembly on soft substrates
When polyacrylamide is replaced by hyaluronic acid as the scaffold on which integrin ligands are attached, the dependence of myocyte morphology on substrate stiffness is radically altered. Hyaluronan gels of varying rigidity were made by the incorporation of different amounts of the crosslinker polyethylene glycol diacrylate to create gels with shear moduli of 50-350 Pa. To achieve a higher stiffness of 1.8 kPa, polyethylene glycol tetra-acrylate was incorporated in the HA solution. To test whether myocytes bind directly to hyaluronan under our culture conditions, HA gels without incorporated adhesive ligands were made, but myocytes and accompanying cardiac fibroblasts cells did not bind these substrates. Therefore, HA by itself is an inert substrate for myocyte attachment. In contrast, when adhesive ligands such as fibronectin, collagen I, gelatin, or fibrinogen were covalently incorporated within the HA gels these composite gels were excellent adhesive substrates for both myocytes and fibroblasts.
Rheological measurements showed that crosslinked HA gels had shear moduli typically in the range of 100-350 Pa, depending on their extent of crosslinking and partly on their age, as disulfide bonds can form very slowly within the networks. These values of shear modulus are in agreement with those measured previously for similar crosslinked HA gels (Vanderhooft et al., 2009
). The shear modulus also depended only weakly on frequency (3A) and strain amplitude (3B), consistent with expectations for gels formed by crosslinked flexible polymers (Chen et al., 2010
). In particular, they did not exhibit the large degree of strain stiffening seen in gels formed by filamentous biopolymers such as collagen or fibrin. Incorporation of proteins into the HA gels and prolonged incubation with cells and culture media also did not significantly alter gel stiffness ().
Myocytes cultured on fibronectin-incorporated soft HA gels of varying rigidity (50-1800 Pa) displayed a remarkably different response when compared to Fn-coated PAA gels of similar rigidity (). On soft Fn-HA gels (<300Pa) myocytes spread to areas that were comparable to or higher than those seen on even rigid (>30kPa) PAA or glass (GPa) substrates. The increased spreading of myocytes on HA gels was not accompanied by the stress fiber assembly and disorganized myofibrillar assembly that occurs when cells spread on stiff Fn-PAA gels; rather, the myocytes displayed striated F-actin assembly and organized myofibrils ()., Altering the rigidity of HA substrates beyond 1 kPa did not appreciably affect the myofibrillar assembly suggesting that stiffness responses are either overridden or saturated at this range of stiffness on hyaluronan. To examine myocyte response to relatively stiffer HA environments we performed additional experiments by plating cells on 10 μm thin gels. The response to glass like stiffness of myocyte cytoskeletal organization was disrupted and consistent with the results on stiff PAA gels/ tissue culture plastic (supplemental Figure 3
). Myocytes cultured on HA gels with covalently incorporated collagen I were more comparable to those seen on collagen I-coated PAA and did not show the same myofibrillar assembly as cells on Fn-HA (). Incorporation of gelatin, the denatured form of collagen I-HA gels resulted in a myofibril assembly response comparative to that seen on fibronectin, but not intact collagen I. These results are consistent with other findings that gelatin engages the fibronectin receptor and suggests that the ability of hyaluronan to activate myofibril formation and organization is integrin type specific.
Figure 4 Cardiac myocytes plated on fibronectin/collagen-containing HA gels of varying stiffness. (I.) Myocyte myofibrillar assembly on soft 50 Pa-1800 Pa hyaluronan gels and its sensitivity to the incorporated ECM ligand type. Cardiac myocytes plated on Fn-HA (more ...)
Long term culture of cardiac myocytes and fibroblasts
Cardiac myocytes generally atrophy or differentiate to an altered phenotype when cultured for a long period on rigid substrates. Moreover myocytes either undergo apoptosis or their cultures are predominantly taken over by fibroblasts. On HA gels, however, myocytes cultured for 7 days showed an even higher spread area and hypertrophied appearance (~3300 μm2) with remarkably well formed sarcomeres and organized myofibrils (). This result suggests that the engagement of hyaluronan enhances myocyte growth without altering its phenotype.
Figure 5 Myocytes hypertrophy is progressive showing enhanced myofibrillar assembly on HA matrices when cultured for prolonged periods. (I.) Cardiac myocytes cultured for 7 days on 300 Pa HA-Fn (a), HA-fibrinogen (b), HA-gelatin (c) displaying well formed sarcomeres (more ...)
The hypertrophic phenotype and similar myocyte cytoskeleton response was observed for HA gels with incorporated fibronectin, fibrinogen or gelatin, all of which are ligands for similar integrins e.g. α5β1, but was not observed when intact collagen I was the only adhesive anchor.
Non-muscle cells like fibroblasts present in these cultures also displayed a similar response to soft hyaluronan gels. Fibroblasts on soft PAA gels (300Pa) were devoid of stress fibers and had a low spread area (). On HA gels coated with fibronectin, fibroblasts increased their spread area and displayed prominent F-actin fibers (). These results suggest that hyaluronan also alters the phenotype of non-muscle cells bound to integrin ligands.
Hyaluronan increases the spread area of myocytes
The magnitude of spread area for myocytes on PAA gels coated with fibronectin was higher than that seen on collagen I gels or the mixture of the two (). On fibronectin, myocyte area increased with increasing substrate rigidity, whereas for collagen the magnitude of cell spreading plateaued after 10 kPa. These results suggest that a change in cell spreading behavior is a function of substrate rigidity and is in part specific to the integrin type mediating this phenomenon.
On soft hyaluronan gels, the spread area response of myocytes was remarkably altered. Compared to soft PAA gels the magnitude of cell spreading on gelatin HA gels was much higher; as the stiffness of HA gels were increased from 50Pa to 300Pa the magnitude of cell spreading also increased and plateaued thereafter (). Changing the ECM ligand type to fibronectin (Fn) on hyaluronan (HA) resulted in a greater magnitude of spreading on soft HA gels, even on the softest Fn-HA gel (50Pa) the spread area magnitude was higher than that seen for the most rigid (30kPa) PAA gel (). Cell spread area for myocytes on Fn-HA gels increased initially with substrate rigidity, but it plateaued after 300 Pa. This result indicates a reprogramming of cell spreading response to substrate rigidity on HA gels towards the softer end.
The altered spreading response of myocytes to soft HA gels was specific to the ECM protein incorporated in these gels. As mentioned previously, the magnitude of spreading was much higher for 300 Pa Fn-HA (~2500 μm2) compared to 300 Pa Fn-PAA (~500 μm2), however no difference in the magnitude of myocyte spreading was observed between PAA and HA gels when collagen type I was incorporated. This difference in spread area between HA and PAA was in part rescued by incorporating a mixture of fibronectin and collagen-I to HA gels. Therefore spreading on HA gels is in part dependent on the integrin type receptor engaged and the magnitude appears to be enhanced specifically with Fn-related integrin receptors.
Hyaluronan enhances the beating population of single myocyte cultures
The percentage of beating cells was also affected by HA in the matrix. The beating percentage of myocyte populations was higher (p<0.05, determined using Fisher’s LSD) on 300 Pa Fn-HA gels as compared to 300 Pa and 10 kPa FN-coated PAA gels. The fraction of total cell area displaced during a contractile cycle was also higher for 300 Pa Fn-HA gels. This result reflects the enhanced sarcomere assembly on Fn-HA gels. As expected, the percentage area changes for soft PAA and HA gels (~16%) were higher than those on 10 kPa gels (~6%), indicating that the imposed load/resistance experienced by myocytes on soft HA gels is exceedingly low.
The contractile strain (є) of myocytes was estimated by measuring the change in the major axis of the cell fitting an ellipse to phase contrast images of myocytes during a contractile cycle.
Contractile work per volume was computed as the elastic potential energy density
, where Y is the Young’s modulus of the matrix. On soft PAA and HA substrates the contractile work done was much lower () when compared to 10 kPa PAA gels (~83 J/m3
). These results confirm that myocytes on HA gels experience a low contractile load resistance. Remarkably, even at these low elastic loads myocytes can reassemble highly robust sarcomeres and myofibrillar structures. The contractile work done by myocytes on Fn-HA gels was minimally increased when compared to Fn-PAA gels of the same rigidity despite the noted large differences in the cell contractile cytoskeleton apparatus.
Figure 7 Percentage of observed beating cells on 300Pa HA and PAA gels (±S.E. for n>10 cells); Myocyte contractile work measured as potential elastic energy J/m3 on 300 Pa HA and PAA gels (±S.E. n>14 cells); Percentage change in (more ...)