Introduction
The heart consists of cardiomyocytes, fibroblasts, and other cardiac cells that are supported by a complex meshwork of fibers, the cardiac extracellular matrix (ECM). In case of cardiac disease, the heart is subject to changes in composition and structure, often leading to cardiac fibrosis. While at first, fibrogenesis is an effective mechanism of tissue repair, ongoing adverse remodeling will lead to a decrease in cardiac functionality and eventually to the development of heart failure [
1]. To halt or reverse this process, anti-fibrotic therapies are being developed [
2], so far with limited success, due to the fact that the extent and distribution of fibrosis vary according to the underlying pathology. Furthermore, there is a lack of knowledge about the effects of fibrosis at the cellular level [
3]. Engineered cardiac tissues are excellent models to mimic and study normal and diseased cardiac development and physiology and therefore open new avenues for therapy assessment. The tissues can be cultured under highly controlled conditions and give insights into the responses of cells and ECM on isolated biochemical and biophysical stimuli, which would be impossible to study in vivo. To apply such tissue models as therapy screening platforms, it is important that the models accurately resemble the in vivo characteristics of the disease and that in vitro results correlate with in vivo outcomes [
4].
To our knowledge, no engineered tissue model of cardiac fibrosis has been reported that takes into account this in vivo to in vitro translation. Therefore, we aimed to develop an engineered microtissue platform that mimics various aspects of cardiac fibrosis based on the analysis of two well-established mouse models of cardiac fibrosis having either a genetic or an acquired cause of cardiac disease. By comparing the in vitro outcomes to the native situation, strengths and weaknesses of such disease models could be identified.
Current engineered cardiac disease models mainly incorporate genetically affected cells to capture a cardiac disorder [
5,
6]. These models primarily focus on intrinsic problems of the cardiomyocytes, while for cardiac fibrosis, the ECM and the cardiac fibroblasts are of critical importance. To date, only few in vitro models of cardiac fibrosis have been developed [
7‐
9]. Unfortunately, most of these models are 2D co-cultures and thus lack the important 3D environment of a fibrotic ECM [
7,
8]. For cardiac cells, it has been shown that 3D cultures more closely mimic natural tissue environments compared to 2D cultures, resulting in different outcomes of proliferation, attachment to the ECM and maturation in 2D compared to 3D cultures [
10]. Galie et al. [
9] cultured cardiac fibroblasts in a 3D collagen gel to investigate the paracrine effect of mesenchymal stem cell injection in a scar tissue. Although ECM was incorporated in this model of fibrosis, cardiomyocytes were lacking. Our engineered microtissues include cardiomyocytes, cardiac fibroblasts, and ECM, since all are affected by fibrosis.
Cardiac fibrosis is generally referred to as an increase in the number of fibroblasts in the heart which lead to the excessive production and deposition of several extracellular matrix proteins, mainly collagen type I. Yet, different forms of fibrosis can be recognized, which all have their own characteristics.
Reactive fibrosis, for instance, has mostly been described in patients with hypertension and diabetes mellitus, but is also present in the aging heart and in hearts suffering from pressure overload. An important characteristic of this type of fibrosis is the interstitial increase in ECM content and absence of cell loss [
11,
12]. Replacement or scarring fibrosis on the contrary corresponds to the local replacement of cardiomyocytes by fibrosis after cell death, for example after myocardial infarction [
11,
12].
Due to this diversity, cardiac fibrosis is such a complex condition that the relative importance of all different aspects that play a role cannot be studied at once. Therefore, our microtissue platform allows for the independent analysis of different aspects of fibrosis, hence mimicking a variety of cardiac fibrotic pathologies.
Mechanical properties, as well as composition of the ECM of the two mouse models for cardiac pathologies accompanied with fibrosis, were analyzed to obtain detailed in vivo aspects. Although multiple mouse models for cardiac disease are available in this study, the mdx and TAC model were chosen because the origin and development of fibrosis in these two models is different. Secondly, both mouse models are frequently described in literature and often used to test anti-fibrotic therapies [
12‐
16]. The
mdx mouse was chosen as a model of genetic cardiac disease [
17,
18], while a mouse model with transverse aortic constriction (TAC) was chosen to represent acquired heart disease [
19]. Both mouse models are known to develop cardiac fibrosis, based on general histological methods for collagen assessment [
15,
17]. Mechanical properties as well as localization, spatial distribution, and composition of the fibrotic areas in the heart were determined and used as input for the in vitro tissue model. Secondly, since activation and proliferation of fibroblasts is a well-known contributor of cardiac fibrosis, especially after TAC [
20,
21], increase in fibroblast number was also incorporated as aspect of fibrosis. To this end, mouse neonatal cardiac cells were cultured in engineered cardiac microtissues [
22]. To mimic various aspects of fibrosis, either the collagen content or the number of cardiac fibroblasts was systematically increased. Mechanical properties and matrix composition of the in vitro microtissues were quantified and compared to the obtained in vivo dataset. Contraction force and beating frequency of the cardiac microtissues were determined to study the correlation between fibrosis and cardiac contractile function.
In this tunable microtissue platform based on in vivo aspects of fibrosis, both the increase of collagen content and fibroblast number that occur with development of cardiac fibrosis were mimicked independently. The in vitro results showed that while increasing collagen content had little effect on microtissue contraction, increasing fibroblast density caused a significant reduction in contraction force. In addition, the beating frequency dropped significantly in tissues consisting of 50% cardiac fibroblasts or higher. Hereby, we were able to show that increased fibroblast density has a more detrimental effect on cardiomyocyte contractile function than accumulation of collagen. Furthermore, this model system can be easily adapted to mimic different stages and forms of cardiac fibrosis and thereby opens new opportunities for development of effective anti-fibrotic treatments.