Nanoscale cues regulate the structure and function of macroscopic cardiac tissue constructs

Abstract

Heart tissue possesses complex structural organization on multiple scales, from macro- to nano-, but nanoscale control of cardiac function has not been extensively analyzed. Inspired by ultrastructural analysis of the native tissue, we constructed a scalable, nanotopographically controlled model of myocardium mimicking the in vivo ventricular organization. Guided by nanoscale mechanical cues provided by the underlying hydrogel, the tissue constructs displayed anisotropic action potential propagation and contractility characteristic of the native tissue. Surprisingly, cell geometry, action potential conduction velocity, and the expression of a cell-cell coupling protein were exquisitely sensitive to differences in the substratum nanoscale features of the surrounding extracellular matrix. We propose that controlling cell-material interactions on the nanoscale can stipulate structure and function on the tissue level and yield novel insights into in vivo tissue physiology, while providing materials for tissue repair.

Fig. 1.

Rational design and fabrication of nanopatterned substratum of PEG hydrogels. (A–C) SEM images of ex vivo myocardium of adult rat heart. Side view (A) and top view (B) show well-aligned myocardium. The Inset in (B) and the magnified view in (C) demonstrate that the structural organization of the myocardium correlates with matrix fibers aligned in parallel beneath. (D) Photograph of a large-area (∼3.5 cm²) ANFS on a glass coverslip. (E) Cross-sectional SEM image of the ANFS. [Scale bar: 5 μm in (A); 10 μm in (B) and (C).]

Fig. 2.

Formation of confluent monolayers with controlled macroscopic alignment on the ANFS. Phase contrast imaging of NRVMs was performed to show cell growth at 32, 48, and 130 hr on the (A) unpatterned substrata and (B) ANFS (in this and other figures, the ANFS is unless otherwise specified). (C) SEM image of NRVMs cultured on the ANFS showing anisotropic nanopatterns, aligned cardiomyocytes, and intercellular junctional structures. The Inset in (C) shows the transverse interconnectivity of the cells. (D) Quantification of cell orientation on the ANFS vs. unpatterned substrata. Each dot represents a single cell, and an orientation of 0° represents perfect alignment with the ridge/groove direction. Quantification of the (E) myocyte length and (F) width on the ANFS vs. the unpatterned substratum. Quantitative comparison of the (G) cell projected area and (H) perimeter between two different ridge/groove ratios ( vs. , n = 33 for each group). Error bars in (E–H) represent the SD about the means. ^∗p < 0.005; ^∗∗p < 0.0005. [Scale bar: 5 μm in (A–C).]

Fig. 3.

Cell and cytoskeleton alignment and striations. (A) Immunofluorescent images of sarcomeric α-actinin (in red) of NRVMs cultured on the ANFS. Cell nuclei are shown in blue. (B) Cross-sectional TEM images of the engineered myocardial tissue grown on the ANFS showing aligned Mf with elongated sarcomeres. Double-headed arrows in (A) and (B) denote the direction of anisotropic nanopatterns consisting of ridges and grooves. (C) An enlarged view of actin bundles (white arrows) and focal adhesions (dark and thick lines indicated by white arrowheads) preferentially formed in parallel to the individual ridges and grooves of the ANFS. (D–E) Representative cross-sectional view of the PEG sidewalls showing the lower extent of cell protrusion into (D) a 400-nm-wide groove than of that into (E) an 800-nm-wide groove. [Scale bar: 10 μm in (A); 1 μm in (B); 200 nm in (C–E).]

Fig. 4.

Spatial regulation of GJ formation. (A) Western blot analysis of Cx43 expression on the ANFS with different widths. (B) Immunofluorescent images of the stained Cx43 of NRVMs on the ANFS. (C) TEM image of GJs aligned with the nanopatterns. The Inset in (C) denotes the GJs at the longitudinal end of two adjacent cells indicated by white arrow. [Scale bar: 20 μm in (B); 200 nm in (C).]

Fig. 5.

Contraction analysis of NRVM monolayers cultured on (A, B) unpatterned substrata and (C, D) the ANFS. A map of the contraction direction for (A) unpatterned substrata and (C) the ANFS is shown. The direction of contraction at each spot (A, C) is indicated by the color [coded to the histograms in (B) and (D)] as well as by the overlaid vector field. Semicircular histograms of contraction angles (B, D) indicate the overall directionality of contracting NRVMs on (B) the unpatterned substrata and (D) the ANFS. The black line in the center of the histogram indicates the average contraction angle, and its length indicates the degree to which the distribution is aligned with the average.

Fig. 6.

Electrophysiological characteristics of engineered cardiac tissue construct. AP propagation across monolayers cultured on (A) unpatterned substrata and (B) the ANFS. Optical maps are 17 mm in diameter. Point stimulation (3 Hz) was applied in the center of the NRVM monolayer at 0 ms (indicated by white arrows). The green arrow indicates the direction of NRVM alignment on the ANFS. Isochrone maps spaced at 5-ms intervals. Increasing the width showed a trend toward increasing the (C) LCV and (D) TCV (n = 6 for the unpatterned group, n = 4 for each patterned group, ^∗p < 0.05 compared with the unpatterned group). In addition, for NRVM monolayers on the ANFS, the LCV was significantly faster than the TCV (n = 4 per group, ^∗p < 0.05 between the LCV and TCV for and above). In contrast, NRVM monolayers on unpatterned substrata showed no directional differences in the CV (LCV = 19 ± 7 cm/ sec, TCV = 19 ± 7 cm/ sec, n = 6). (E) The ratio of longitudinal to transverse conduction velocity (LCV/TCV) was more than doubled for NRVM monolayers seeded onto patterned substrata (ridge-to-groove ratios ranging from to ), as compared with the ratio for unpatterned control substrata (n = 4 for patterned groups and n = 6 for the unpatterned control group, ^∗p < 0.05 compared with the unpatterned group). Error bars in (C–E) represent the SEM.

Fig. 7.

Model of the sensitivity of structural and functional properties of the cardiac constructs to the details of the underlying nanostructured substrata. Areas of direct plasma membrane–substratum contact are highlighted in green, and gap junctional complexes are shown in red. Areas of the cell–substratum interfaces are magnified for clarity.