Reprogramming cardiomyocyte mechanosensing by crosstalk between integrins and hyaluronic acid receptors

Abstract

The elastic modulus of bioengineered materials has a strong influence on the phenotype of many cells including cardiomyocytes. On polyacrylamide (PAA) gels that are laminated with ligands for integrins, cardiac myocytes develop well organized sarcomeres only when cultured on substrates with elastic moduli in the range 10 kPa-30 kPa, near those of the healthy tissue. On stiffer substrates (>60 kPa) approximating the damaged heart, myocytes form stress fiber-like filament bundles but lack organized sarcomeres or an elongated shape. On soft (<1 kPa) PAA gels myocytes exhibit disorganized actin networks and sarcomeres. However, when the polyacrylamide matrix is replaced by hyaluronic acid (HA) as the gel network to which integrin ligands are attached, robust development of functional neonatal rat ventricular myocytes occurs on gels with elastic moduli of 200 Pa, a stiffness far below that of the neonatal heart and on which myocytes would be amorphous and dysfunctional when cultured on polyacrylamide-based gels. The HA matrix by itself is not adhesive for myocytes, and the myocyte phenotype depends on the type of integrin ligand that is incorporated within the HA gel, with fibronectin, gelatin, or fibrinogen being more effective than collagen I. These results show that HA alters the integrin-dependent stiffness response of cells in vitro and suggests that expression of HA within the extracellular matrix (ECM) in vivo might similarly alter the response of cells that bind the ECM through integrins. The integration of HA with integrin-specific ECM signaling proteins provides a rationale for engineering a new class of soft hybrid hydrogels that can be used in therapeutic strategies to reverse the remodeling of the injured myocardium.

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 (green) and nucleus (blue). Myocytes on intermediate Fn gels reassembled myofibrils whereas cells on relatively soft and hard matrices exhibited lower myofibril assembly. Myocytes on collagen I matrices were less efficient at reassembling myofibril. Scale bar indicates 20μm

Figure 2

Cardiac myocyte myofibrillar assembly is sensitive to ECM ligand type. (I.) Fourier transform spectra of single myocytes cultured for 48 hours on 1 kPa Col-I (a,b) and Fn (c,d) coated PAA gels. F-actin staining was used to identify repeating patterns/striated myofibrils. Myocytes on collagen-PAA gels (b) did not show patterned Fourier transform spectra when compared to Fn (d). (II.) Percentage of aligned fibers and estimated striated area was calculated for myocytes on fibronectin and collagen-I coated 10kPa PAA gels; myofibrils within 15° from the long axis of the cell were considered to be ‘aligned’. Myocyte striation area having sarcomeres of ~ 1.7 to 1.8 μm were used to estimate the total ‘striation area’(± S.E. for >15 cells).

Figure 3

Shear moduli of derivitized crosslinked HA gels. A. Frequency dependence of shear modulus of PEG diacrylate-crosslinked HA (Hystem) measured at 2% maximal strain. B. Strain dependence of similar Hystem sample measured at 10 rad/s. C. Shear moduli measured at 5% strain and 1 rad/s of HA gels covalently modified with Fn or collagen and incubated with myocytes for 1 weeks in culture before measurement. Crosslinker concentration was 2% except for the HA-Fn gel with 0.5% crosslinker, as shown.

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 gels (a-f) and col-I-HA (g-j) of varying matrix rigidity, stained for F-actin (red), α-actinin (green) and nucleus (blue) (a,c,e,g,i) and F-actin only (b,d,f,h,j). Myocytes on Fn-HA matrices reassemble myofibrils whereas on collagen myofibrillar assembly is attenuated. Scale bar 20μm. (II.) Percentage of aligned fibers and estimated striated area was calculated for myocytes on fibronectin and collagen-I coated 300Pa HA gels; myofibrils within 15° from the long axis of the cell were considered to be aligned. Myocyte striation areas having sarcomeres of ~ 1.7 to 1.8 μm were used to estimate the total ‘striation area’(± S.E. for >15 cells).

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 stained for α-actinin (green). Scale bar 20μm (II.) Fibroblasts present in NVRM cultures plated on 300 Pa Fn-PAA gels (a) and Fn-HA gels (b), imaged after 48 hours and stained for f-actin (red). Scale Bar 10 μm. Fibroblasts on HA gels increase their spread area and display prominent actin fiber bundles when compared to PAA gels of the same rigidity.

Figure 6

Myocyte spreading is sensitive to both substrate rigidity and ligand type. NVRM were cultured for 48 hours on HA or PAA hydrogels of different rigidity that were derivitized with various adhesion proteins as shown. Myocytes cultured on Fn-bound HA gels showed the highest spreading ( ± S.E. for >150 cells).

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 area/area strain of contractile myocytes on soft HA and PAA gels (±S.E. n>14 cells).