Fibrosis has no detrimental effect on beating frequency in native-based cardiac microtissue models

Abstract

Introduction Fibrosis is a hallmark of cardiac disease leading to changes in myocardial architecture that may hamper ventricular function. However, the influence of changes in the cardiac microenvironment on cardiomyocyte contractility remains unclear. Here, we describe the characterization of the cardiac microenvironment related to fibrosis in mouse hearts suffering from dystrophinopathies. The in vivo characteristics were compared to matrix organization in novel engineered in vitro cardiac microtissues. This high throughput cardiac model system allows investigation of the effect of fibrosis on tissue contraction in healthy and fibrotic microenvironments. Materials and Methods Mdx mice are surrogates for Duchenne muscular dystrophy and suffer from heart disease at 10 months of age. Changes in structure and composition of cardiac ECM mdx hearts were investigated and compared to matrix organization in age-matched controls. Microfabricated tissue gauses (µTUGs) with flexible microposts with uniaxial or biaxial constraints to manipulate matrix organization were fabricated using soft lithography. Neonatal mouse cardiomyocytes (CMs) and fibroblasts (cFBs) were resuspended in collagen/matrigel and seeded in the µTUGs to form microtissues to recapitulate the in vivo composition. Dynamic contractile behavior of the tissues was monitored for 7 days using deflection of the microposts. ECM distribution was determined at culture day 7 with immunofluorescence. Results Mdx mouse hearts were characterized by induction of patchy fibrosis in the left ventricle which caused disorganization of the ECM. The fibrotic areas were composed of collagen I, III and fibronectin and decreased the left ventricular myocardial stiffness. Using the µTUG system we obtained aligned (anisotropic) and disorganized (isotropic) ECM organization. In vitro distribution of collagens and fibronectin was homogenous, and not patchy as in the native tissue. Although CMs in anisotropic microtissues were more aligned, no higher contractile forces were generated. However, strain analysis showed that contraction was much more homogenous in anisotropic tissue compared to isotropic. Beating frequency was not affected by ECM organization but only by the percentage of cFBs in the microtissues. Discussion and Conclusion In this study characteristics of the healthy vs diseased cellular microenvironment were implemented in a novel in vitro cardiac tissue model. Our data indicate that ECM organization does not disturb the frequency of contraction but the number of cFBs does. Furthermore, we showed that these models are suitable to study contractility in cellular and pathophysiological processes that occur during heart disease and may facilitate in the optimization of new therapies for cardiac disease