Biohybrid thin films for measuring contractility in engineered cardiovascular muscle.

In vitro cardiovascular disease models need to recapitulate tissue-scale function in order to provide in vivo relevance. We have developed a new method for measuring the contractility of engineered cardiovascular smooth and striated muscle in vitro during electrical and pharmacological stimulation. We present a growth theory-based finite elasticity analysis for calculating the contractile stresses of a 2D anisotropic muscle tissue cultured on a flexible synthetic polymer thin film. Cardiac muscle engineered with neonatal rat ventricular myocytes and paced at 0.5 Hz generated stresses of 9.2 +/- 3.5 kPa at peak systole, similar to measurements of the contractility of papillary muscle from adult rats. Vascular tissue engineered with human umbilical arterial smooth muscle cells maintained a basal contractile tone of 13.1 +/- 2.1 kPa and generated another 5.1 +/- 0.8 kPa when stimulated with endothelin-1. These data suggest that this method may be useful in assessing the efficacy and safety of pharmacological agents on cardiovascular tissue.</p

Effects of calcium concentration in growth media on d30 hiPSC-CM force production on a substrate with modulus 9.8 kPa.

<p>Data shown as mean ± SEM. A. Total force versus calcium concentration (mean = 0.083 ± 0.013 μN, n = 15; 0.147 ± 0.02 μN, n = 14). B. Cell area versus calcium concentration (mean = 702.3 ± 63.2 μm², 1130.3 ± 86.2 μm²). C. Normalized force versus calcium concentration (mean = 13.0 ± 2.14 mN/mm2 x 10⁻⁵, 14.0 ± 2.0 mN/mm2 x 10⁻⁵). D. Rcan1.4 mRNA fold change (low Ca²⁺ group mean = 1.00 ± 0.29, n = 8; physiological Ca²⁺ mean, 1.74 ± 0.29, n = 7)(P = 0.02).</p

Effects of single cell d90 hiPSC-CM morphology on force production.

<p>A. Peak force versus cell area, R² = 0.21, P<0.03, n = 24. B. Peak force versus long axis (axis of contraction). C. Peak force versus short axis (perpendicular to axis of contraction). D. Peak force versus aspect ratio (long axis/short axis).</p

HiPSC-CMs and measurement of force by TFM.

<p>A. Representative cell, 30 days post-differentiation on a 9.8 kPa substrate. B. Heat map showing magnitudes of deformation strain of the substrate under the representative cell. C. Heat map showing magnitudes of stress of the representative cell calculated from strain of the substrate. D. Total force of a single cell over time with respect to baseline at the point t = 0 seconds, over four contractions paced at 0.5 Hz, fitted with a smoothed spline curve. E. Total force of a single cell over time, average of four contractions, fitted with a smoothed spline curve. F. Histograms showing distribution of cell geometries.</p

Effects of length of hiPSC differentiation on force production.

<p>Data shown as mean ± SEM. A. Representative hiPSC-CMs from day 14, day 30, and day 90 post-differentiation, and representative NRVM on substrate with modulus 9.8 kPa. B. Total force versus length of differentiation (mean = 0.012 ± 0.001 μN, n = 17; 0.083 ± 0.013 μN, n = 15; 0.103 ± 0.011 μN, n = 24; 0.113 ± 0.016 μN, n = 12). C. Cell area versus length of differentiation (mean = 605.7 ± 47.1 μm², 702.3 ± 63.2 μm², 898.2 ± 64.4 μm², 741.9 ± 61.4 μm²). D. Normalized force versus length of differentiation in culture (mean = 2.12 ± 0.23 mN/mm2 x 10⁻⁵, 13.0 ± 2.14 mN/mm2 x 10⁻⁵, 11.4 ± 1.4 mN/mm2 x 10⁻⁵, 15.3 ± 1.5 mN/mm2 x 10⁻⁵).</p

Controlling the contractile strength of engineered cardiac muscle by hierarchal tissue architecture.

<p>The heart is a muscular organ with a wrapping, laminar structure embedded with neural and vascular networks, collagen fibrils, fibroblasts, and cardiac myocytes that facilitate contraction. We hypothesized that these non-muscle components may have functional benefit, serving as important structural alignment cues in inter- and intra-cellular organization of cardiac myocytes. Previous studies have demonstrated that alignment of engineered myocardium enhances calcium handling, but how this impacts actual force generation remains unclear. Quantitative assays are needed to determine the effect of alignment on contractile function and muscle physiology. To test this, micropatterned surfaces were used to build 2-dimensional myocardium from neonatal rat ventricular myocytes with distinct architectures: confluent isotropic (serving as the unaligned control), confluent anisotropic, and 20 μm spaced, parallel arrays of multicellular myocardial fibers. We combined image analysis of sarcomere orientation with muscular thin film contractile force assays in order to calculate the peak sarcomere-generated stress as a function of tissue architecture. Here we report that increasing peak systolic stress in engineered cardiac tissues corresponds with increasing sarcomere alignment. This change is larger than would be anticipated from enhanced calcium handling and increased uniaxial alignment alone. These results suggest that boundary conditions (heterogeneities) encoded in the extracellular space can regulate muscle tissue function, and that structural organization and cytoskeletal alignment are critically important for maximizing peak force generation.</p