Cardiac myocyte force development during differentiation and maturation

Abstract

The maturation of cardiac myocytes during the immediate prenatal period coincides with changes in the mechanical properties of the extracellular matrix. We investigated the effects of extracellular stiffness on cardiomyocyte maturation in neonatal rat ventricular myocytes grown on collagen-coated gels. Cells on 10-kPa substrates developed aligned sarcomeres, while cells on stiffer substrates had unaligned sarcomeres and stress fibers. Cells generated greater mechanical force on gels with stiffness similar to that of the native myocardium than on stiffer or softer substrates. To investigate the differentiation of myocyte progenitors, we used clonal expansion of engineered human embryonic stem cells. Puromycin-selected cardiomyocytes exhibited a gene expression profile similar to that of adult human cardiomyocytes and generated force and action potentials consistent with normal fetal cardiomyocytes. These results suggest that extracellular stiffness significantly affects maturation and differentiation of immature ventricular myocytes.

Figure 1

The elastic moduli of polyacrylamide gels can be controlled by varying the concentration of monomer (%PAAm) up to 7% with a 1:20 monomer:crosslinker ratio. Shear moduli shown here in the range 1 to 16 kPa correspond to Young’s moduli for tensile stiffness of 2 to 50 kPa for this gel material. These values span the range of developing and adult resting myocardium at low strain.

Figure 2

Dynamic traction force microscopy of isolated neonatal rat ventricular myocytes grown on polyacrylamide gels coated with type I collagen. Phase contrast images define the cell outline. Fluorescence microscopy reveals microbeads embedded in the gel just below the surface. Automated image processing with an optical flow algorithm using the cross-correlation between a reference image and subsequent frames during stimulated cell contractions was used to compute displacement vector fields. The solution of a Boussinesq problem for deformation of a linearly elastic half-space was used to compute corresponding traction vector fields that are integrated over the cell area to give resultant axial and transverse cell forces during each cell twitch. See Jacot et al for detailed methods.

Figure 3

NRVMs on polyacrylamide gels and labeled for α-actin have poorly defined striations on soft 1 kPa substrates, well defined and aligned striations on 10 kPa substrates and unaligned striations with long, large stress fibers on stiff 50 kPa gels. Modified from Jacot et al with permission.

Figure 4

Calcium transients (insert) were measured as peak fluorescence divided by baseline fluorescence, in Fura-2 or Fluo-4 labeled NRVMs. The magnitude of calcium transients (bar graph) on 10 kPa gels was significantly greater than transients on 1 kPa and 50 kPa gels (P < 0.05). See Jacot et al for details.

Figure 5

Cardiospheres were isolated at day 12-13.5 and cultured until day 48 when they were dispersed and deposited onto gelatin-functionalized surfaces of polyacrylamide cast with fluorescent beads and analyzed for force generation at day 50. Human embryonic stem cell-derived cardiomyocytes generated axial and total traction forces on 4 kPa gels are comparable in magnitude to those generated on similar soft gels by neonatal rat ventricular myocytes, but significantly lower than the maximal forces seen in neonatal myocytes cultured on stiffer gels. Corresponding average contractile peak stresses were 220 ± 70 Pa. Modified with permission from Kita-Matsuo et al .

Figure 6

Human embryonic stem cell-derived cardiomyocytes purified at day 12 with puromycin for αMHC positive cells were plated on polyacrylamide substrates at day 16 post-differentiation and fixed on day 23. Immunostaining was performed for cardiac-specific α-actinin (Green) and nuclear DAPI (Blue) to image cardiomyocytes (40×) and compare cellular morphology as a function of substrate stiffness: (A) 1 kPa gel, (B) 10 kPa gel, (C) 50 k Pa gel, and (D) glass. With increasing stiffness, the cells become more spread and exhibit signs of stress fiber formation.