Dynamic loading of human engineered heart tissue enhances contractile function and drives a desmosome-linked disease phenotype

The role that mechanical forces play in shaping the structure and function of the heart is critical to understanding heart formation and the etiology of disease but is challenging to study in patients. Engineered heart tissues (EHTs) incorporating human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have the potential to provide insight into these adaptive and maladaptive changes. However, most EHT systems cannot model both preload (stretch during chamber filling) and afterload (pressure the heart must work against to eject blood). Here, we have developed a new dynamic EHT (dyn-EHT) model that enables us to tune preload and have unconstrained contractile shortening of >10%. To do this, three-dimensional (3D) EHTs were integrated with an elastic polydimethylsiloxane strip providing mechanical preload and afterload in addition to enabling contractile force measurements based on strip bending. Our results demonstrated that dynamic loading improves the function of wild-type EHTs on the basis of the magnitude of the applied force, leading to improved alignment, conduction velocity, and contractility. For disease modeling, we used hiPSC-derived cardiomyocytes from a patient with arrhythmogenic cardiomyopathy due to mutations in the desmoplakin gene. We demonstrated that manifestation of this desmosome-linked disease state required dyn-EHT conditioning and that it could not be induced using 2D or standard 3D EHT approaches. Thus, a dynamic loading strategy is necessary to provoke the disease phenotype of diastolic lengthening, reduction of desmosome counts, and reduced contractility, which are related to primary end points of clinical disease, such as chamber thinning and reduced cardiac output

Development of polydimethylsiloxane substrates with tunable elastic modulus to study cell mechanobiology in muscle and nerve.

<p>Mechanics is an important component in the regulation of cell shape, proliferation, migration and differentiation during normal homeostasis and disease states. Biomaterials that match the elastic modulus of soft tissues have been effective for studying this cell mechanobiology, but improvements are needed in order to investigate a wider range of physicochemical properties in a controlled manner. We hypothesized that polydimethylsiloxane (PDMS) blends could be used as the basis of a tunable system where the elastic modulus could be adjusted to match most types of soft tissue. To test this we formulated blends of two commercially available PDMS types, Sylgard 527 and Sylgard 184, which enabled us to fabricate substrates with an elastic modulus anywhere from 5 kPa up to 1.72 MPa. This is a three order-of-magnitude range of tunability, exceeding what is possible with other hydrogel and PDMS systems. Uniquely, the elastic modulus can be controlled independently of other materials properties including surface roughness, surface energy and the ability to functionalize the surface by protein adsorption and microcontact printing. For biological validation, PC12 (neuronal inducible-pheochromocytoma cell line) and C2C12 (muscle cell line) were used to demonstrate that these PDMS formulations support cell attachment and growth and that these substrates can be used to probe the mechanosensitivity of various cellular processes including neurite extension and muscle differentiation.</p

Development of Polydimethylsiloxane Substrates with Tunable Elastic Modulus to Study Cell Mechanobiology in Muscle and Nerve

<div><p>Mechanics is an important component in the regulation of cell shape, proliferation, migration and differentiation during normal homeostasis and disease states. Biomaterials that match the elastic modulus of soft tissues have been effective for studying this cell mechanobiology, but improvements are needed in order to investigate a wider range of physicochemical properties in a controlled manner. We hypothesized that polydimethylsiloxane (PDMS) blends could be used as the basis of a tunable system where the elastic modulus could be adjusted to match most types of soft tissue. To test this we formulated blends of two commercially available PDMS types, Sylgard 527 and Sylgard 184, which enabled us to fabricate substrates with an elastic modulus anywhere from 5 kPa up to 1.72 MPa. This is a three order-of-magnitude range of tunability, exceeding what is possible with other hydrogel and PDMS systems. Uniquely, the elastic modulus can be controlled independently of other materials properties including surface roughness, surface energy and the ability to functionalize the surface by protein adsorption and microcontact printing. For biological validation, PC12 (neuronal inducible-pheochromocytoma cell line) and C2C12 (muscle cell line) were used to demonstrate that these PDMS formulations support cell attachment and growth and that these substrates can be used to probe the mechanosensitivity of various cellular processes including neurite extension and muscle differentiation.</p> </div

Quantification of neurite length for PC12 cells cultured on two different PDMS formulations.

<p>PC12 cells were cultured on 1.72 MPa (•, black circles) and 5 kPa (○, white circles) PDMS and evaluated at days 3, 5, 7 and 14. At days 3 and 5, neurite length on 1.72 MPa PDMS was significantly greater compared to neurite length on 5 kPa PDMS. On days 7 and 14 the neurite length was equivalent on both PDMS types. This suggests that PC12 neurites initially grow faster on stiffer PDMS substrates (up to 5 days), but by 7 days the growth rate has slowed on the stiffer PDMS and accelerated on the softer PDMS such that neurite lengths are equivalent. Data represented as mean ± standard error of the mean. Statistical significance at each time point determined by a Mann-Whitney Rank Sum Test, *indicates p≤0.001.</p

Representative AFM scans of the surface topography for the different PDMS formulations.

<p>These images show that all PDMS formulations have similar morphological appearance and total variation in height of ∼4 nm over a 20 µm scan area. The different scans are for (A) 1.72 MPa, (B) 1.34 MPa, (C) 830 kPa, (D) 130 kPa, (E) 50 kPa and (F) 5 kPa elastic modulus PDMS formulations.</p

Representative fluorescent images of C2C12 cells differentiated into myotubes on different PDMS formulations.

<p>C2C12 cells cultured and differentiated on PDMS (A) 1.72MPa, (B) 830 kPa, (C) 130 kPa, (D) 50 kPa and (E) 5 kPa formulations. All cells were stained for the nucleus with DAPI (blue) and differentiated myotubes were stained for myosin heavy chain (green). Cells cultured on the stiffer PDMS substrates (A–C) formed longer myotubes, whereas cells cultured on the softer substrates (D and E) formed shorter myotubes and tended to organize into cell clusters. Scale bars are 200 µm.</p

Representative examples of fluorescently labeled fibronectin micropatterned on the different PDMS formulations.

<p>The protein pattern is transferred with high fidelity on all the PDMS formulations indicating the substrates exhibit similar protein adsorption from the PDMS stamps (made from Sylgard 184) used for microcontact printing. The different images are for (A) 1.72 MPa, (B) 1.34 MPa, (C) 830 kPa, (D) 130 kPa, (E) 50 kPa and (F) 5 kPa elastic modulus PDMS formulations. Scale bars are 50 µm.</p

RMS roughness of the six PDMS formulations as a function of weight percent Sylgard 184.

<p>As the percentage of Sylgard 184 increases, the RMS roughness also increases, ranging from approximately 200 to 800 pm. While there are significant difference in roughness between formulations, all have an RMS roughness of <1 nm, smaller than what cells can typically differentiate. Thus, we consider all the PDMS formulations to have equivalent surface roughness in terms of what a cell can sense and respond to. The relationship between RMS roughness and weight percent Sylgard 184 is fit by a linear regression (solid line, y = 273.25 + 4.94x, R² = 0.9745). Data represented as mean ± standard deviation, statistical significance determined by one-way ANOVA on the ranks with Tukey post hoc test (n = 9) where (*) was significantly different from 0, 9.09 and 16.67%, (#) was significantly different from 0 and 9.90% and (†) was significantly different from 0% Sylgard 184 formulations (p<0.05).</p

Quantification of cell density, myotube length and myotube clustering performed as a function of the PDMS elastic modulus.

<p>(A) Average cell density of the different PDMS formulations shows no difference as a function of substrate elastic modulus. (B) Average number of myotube clusters per mm² on the different PDMS formulations (n = 9). The cells cultured on the 5 and 50 kPa substrates formed significantly more myotube clusters compared to the other substrates (*indicates p<0.001). (C) Average length of myosin heavy chain positive myotubes on the different PDMS formulations (5 kPa, n = 706; 50 kPa, n = 739; 130 kPa, n = 662; 830 kPa, n = 769; 1.72 MPa, n = 760). Cells cultured on the stiffer 1.72 MPa and 830 kPa substrates formed significantly longer myotubes compared to those formed on the softer 130, 50 and 5 kPa substrates (*indicates p<0.001). Cells cultured on the 130 kPa substrate also formed longer myotubes compared to those formed on the 5 kPa substrate (# indicates p<0.001). Data represented as mean ± standard error of the mean, statistical analysis by Kruskal Wallis ANOVA on the ranks with p<0.05 Dunn’s method for pairwise comparison.</p

Representative phase contrast images show single neurites extending from the cell body of PC12 cells.

<p>The PC12 cells were differentiated into neuron-like cells and cultured on 5 kPa and 1.72 MPa PDMS (Sylgard 527 and Sylgard 184, respectively). Laminin was micropatterned as 20 µm wide, 20 µm spaced lines to direct the linear extension of neurites, which were imaged at 3, 5 7 and 14 days. The neuron length increased with culture time and was qualitatively similar between the two PDMS types. Scale bar is 50 µm.</p