Human cardiac fibrosis-on-a-chip model recapitulates disease hallmarks and can serve as a platform for drug testing
Graphical abstract
Introduction
Heart failure is the end-stage clinical manifestation of multiple forms of cardiovascular diseases. It is characterized by cardiac remodeling and reduced ventricular compliance as a consequence of interstitial fibrosis [1]. Heart damage caused by various insults (i.e. hypoxia during myocardial infarction, MI) triggers complex wound healing mechanisms to restore homeostasis and while collagen deposition is a normal and essential component of wound healing, it can evolve into a progressively irreversible fibrotic response. In the setting of cardiac fibrosis, fibroblast activation leads to excessive deposition of extracellular matrix components such as collagen and fibronectin, and increases tissue stiffness, which further activates cardiac fibroblasts and results in a positive feedback loop of activation and increased stiffness [2]. This can compromise heart function and increase the risk of arrhythmia, ultimately leading to heart failure.
Although a number of factors have been implicated in orchestrating the fibrotic response, tissue fibrosis is dominated by a central mediator: transforming growth factor-β (TGF-β [3]). Sustained TGF-β secretion leads to a continuous cycle of growth factor signaling and dysregulated matrix turnover. Despite abundant evidence from animal models of fibrosis and samples from human biopsies supporting a central role for TGF-β in tissue fibrosis, modulating TGF-β function to attenuate or reverse fibrosis is replete with challenges. For example, attempts to block TGF-β signaling using antagonists to the type I activin receptor-like kinase 5 (ALK5) have failed to reach clinical studies, owing mainly to safety concerns [4]. Therefore, it is necessary to improve our understanding of disease progression to uncover alternative targets and develop strategies to treat cardiac fibrosis.
Despite advances in our knowledge of fibroblast activation and fibrosis due to research performed in animal models and in vitro studies on fibroblast activation, our understanding of disease progression in humans is limited. Moreover, animal models often fail to faithfully mimic human responses, while reductionist approaches at the cell level are most commonly used in conventional 2D in vitro platforms that fail to recapitulate higher dimensionality as well as higher order intercellular interactions. This motivates the need for a human biomimetic in vitro platform to investigate the progression of fibrotic remodeling in three dimensions (3D) that allows for real-time monitoring of functional outcomes. Such near-physiological models enable the study of a range of biological processes such as cell-cell interactions and signaling that take place in the 3D environment, as well as tissue responses to drugs [5]. They also allow for controlled studies of organ-level aspects of human physiology and disease, and have been successfully used to uncover new potential therapeutic targets [6].
We and others have generated “healthy” and disease models of human cardiac tissues in vitro from hiPSC-CMs, including long-QT and Barth syndromes [[7], [8], [9], [10], [11], [12], [13], [14]]. However, cardiac fibrosis, models described to date have largely been of animal origin [15,16] requiring interspecies extrapolation. Recently emerged human tissue models of cardiac fibrosis include cardiac fibroblast tissues for assessing fibroblast conversion to myofibroblasts (*Kong et al., 2019), organoids (*Bracco Gartner et al., 2019; Lee at al., 2019), and cardiac microtissues (*Wang et al., 2019), but they are limited in the extent of their analysis.
Here, we describe for the first time and thoroughly characterize the development of a 3D model of human cardiac fibrosis (hCF-on-a-chip) using human cardiac fibroblasts together with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in a platform with live imaging capabilities and real-time assessment of contractile function. The resulting hCF-on-a-chip technology displayed the hallmark characteristics of cardiac fibrosis and associated heart failure including impaired functional capacity, increased collagen deposition and tissue stiffness, increased brain natriuretic peptide (BNP) secretion and a ‘fibrotic’ transcriptomic signature, including changes in key microRNAs secreted in extracellular vesicles and implicated in human cardiac fibrosis. Moreover, we show that standard-of-care drugs losartan and carvediolol significantly decreased BNP secretion in the 3D hCF-on-a-chip tissues, but not in 2D monolayers. In addition, pirfenidone, a commercially available drug for treatment of idiopathic pulmonary fibrosis [17] (IPF), significantly reduced tissue stiffness and BNP secretion, as well as changed the microRNA signature in tissues that had already displayed significant functional capacity loss. If performed for longer time, pirfenidone treatment also led to improvement is passive tissue tension.
Section snippets
Human cardiac fibrosis-on-a-chip (hCF) design, fabrication and characterization
To recapitulate cardiac fibrosis in vitro and concurrently facilitate force of contraction measurements, we developed and used an accessible, two-material microwell chip consisting of a cell culture compartment and two parallel flexible horizontal rods at each end of the culture compartment (Fig. 1a). The device was fabricated in poly(methylmethacrylate) (PMMA or acrylic) or polystyrene using micromilling methods in thermoplastics [18]. The two flexible rods were made from
Discussion
We describe for the first time the development of a human heart failure model that is driven by cardiac fibrosis and displays the major hallmarks of the disease, including increased collagen deposition, higher tissue stiffness, loss of contractile function, and induced BNP secretion. Moreover, we demonstrated that the mRNA and miR signatures of fibrotic tissues recapitulate those of samples from human patients with cardiac fibrosis and/or heart failure [43].
Previous methods for studying cardiac
Device manufacture
Devices were designed using Autodesk Fusion 360 and milled from poly(methylmethacrylate) (PMMA) or polystyrene using a CNC milling machine (Personal CNC 770, Tormach, WI, USA). Polydimethylsiloxane (PDMS) (Dow Corning Corporation, cat# 3,097,358-1004) was mixed at indicated ratios of curing agent to PDMS base to produce the prepolymer mixture, degassed in a vacuum chamber, and then used to fill 27-gauge syringe needles. The prepolymer mixture was cured at 80 °C for 2 h and then extracted from
Acknowledgements
This work was supported by grants from the Canadian Institutes of Health Research (CIHR), Institute of Circulatory and Respiratory Health (137352 and PJT153160) to S.S.N. A Discovery grant from the Natural Sciences and Engineering Research Council (NSERC) (RGPIN 06621-2017) to S.S.N. partially supported the following trainees: O.M., K.W. and M.K. O.M. was partially supported by a NSERC CREATE Training program in organ-on-a-chip engineering and entrepreneurship (TOeP) and NSERC Canada Graduate
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