Fig 1.
Micropatterned cardiac tissues on soft gels for traction force microscopy.
(A) We cast PA gels (i) sandwiching the pre-polymer solution between activated and non-activated glass (ii) before stamping fibronectin (iii) to promote cell adhesion on the gel surface (iv). With this versatile method, we engineered neonate rat ventricular myocytes (NRVM) into diamond-shaped mini tissues (B) featuring cells aligned along the major axis of the diamond (C) thanks to a micro-contact printed brick-wall pattern of fibronectin (D). By tracking the displacement of fluorescent beads embedded in the in the soft gel during cell relaxation (E) and contraction (F), we used traction force microscopy to obtain displacement (G), and stress (H) maps at the tissue level. Importantly, since we could electrically stimulate the diamond-shaped tissues, we could measure displacement, stress, and contractile work as a function of beating frequency (I). Scale bars: 100 μm.
Fig 2.
Contractile structure and function of cardiac tissues engineered on gels of varying stiffness.
Using α-actinin immunographs from NRVM engineered on soft (1 kPa, A-i), normal (13 kPa, A-ii), and stiff (90 kPa, A-iii) gels we assessed differences in sarcomere length (SL) and sarcomere packing density (SPD, B). Scale bar 50 μm. Further, we performed TFM on diamond-shaped NRVM tissues engineered on soft (C-i), normal (C-ii), and stiff (C-iii) gels (peak systolic stress shown) to calculate (D) maximum deformation, peak stress, and total contractile work (strain energy). Results are given as mean ± s.e.m. and N = 3, 4, and 7 for soft, normal, and stiff gels respectively. The symbol * implies significant differences (p < 0.05) compared with the group of the same color.
Fig 3.
Metabolic differences in engineered cardiac tissues on gels of different stiffness.
(A) We measured oxygen consumption rates of NRVM cultured on soft (1 kPa), normal (13 kPa), and stiff (90 kPa) gels during a standard mitochondria stress test. We assessed basal respiration rate (B), ATP production (C), spare respiratory capacity (D), and nonmitochondrial respiration (E) for each substrate. Results are given as mean ± s.e.m. with sample size equal to 12, 15, and 22 for soft, normal, and stiff gels, respectively. The symbol * implies significant differences (p < 0.05) compared with the group of the same color.
Fig 4.
Cardiomyocytes operate optimally on substrates of physiological stiffness possibly due to a link between metabolism and contractile structure and function.
Based on our data, we speculated that substrate stiffness might regulate the balance of energy production and utilization in cardiac tissues as follows. Cardiomyocytes on soft gels (A) need abundant ATP (white spheres) derived by mitochondria (orange organelles) to promote the contraction of sarcomeres (Z-disks in black) via cross-bridge cycling (brown lines). This might lead to very inefficient mechanical work as only a small stress (red arrows) is required to deform the soft gel substantially. Conversely, NRVM cultured on normal (B) and stiff (C) substrates have well organized contractile cytoskeletons and need a limited amount of ATP to fuel sarcomere contraction. Since the same amount of contractile force causes a smaller displacement of stiffer gels, the product between force and displacement (work) is maximum on gels of physiological stiffness.