On‐chip assessment of human primary cardiac fibroblasts proliferative responses to uniaxial cyclic mechanical strain

Abstract

Cardiac cell function is substantially influenced by the nature and intensity of the mechanical loads the cells experience. Cardiac fibroblasts (CFs) are primarily involved in myocardial tissue remodeling: at the onset of specific pathological conditions, CFs activate, proliferate, differentiate, and critically alter the amount of myocardial extra-cellular matrix with important consequences for myocardial functioning. While cyclic mechanical strain has been shown to increase matrix synthesis of CFs in vitro, the role of mechanical cues in CFs proliferation is unclear. We here developed a multi-chamber cell straining microdevice for cell cultures under uniform, uniaxial cyclic strain. After careful characterization of the strain field, we extracted human heart-derived CFs and performed cyclic strain experiments. We subjected cells to 2% or 8% cyclic strain for 24 h or 72 h, using immunofluorescence to investigate markers of cell morphology, cell proliferation (Ki67, EdU, phospho-Histone-H3) and subcellular localization of the mechanotransduction-associated transcription factor YAP. Cell morphology was affected by cyclic strain in terms of cell area, cell and nuclear shape and cellular alignment. We additionally observed a strain intensity-dependent control of cell growth: a significant proliferation increase occurred at 2% cyclic strain, while time-dependent effects took place upon 8% cyclic strain. The YAP-dependent mechano-transduction pathway was similarly activated in both strain conditions. These results demonstrate a differential effect of cyclic strain intensity on human CFs proliferation control and provide insights into the YAP-dependent mechano-sensing machinery of human CFs.

Human cardiac fibroblasts adaptive responses to controlled combined mechanical strain and oxygen changes in vitro

“…Mechanical strain significantly impacted cell proliferation within the PX culture condition: culturing CFs at PX and subjecting them to 2% strain induced a striking increase in mitotic cells compared to no strain control and 8% strain cultures. This strain intensity-dependent effect is in line with our previous investigations of CF proliferation under mechanical strain in NX conditions (Ugolini et al, 2016). After 12 hr of stimulation, the proliferative increase with HX is not statistically significant, while we report a significant synergistic decrease of mitotic cells induced by the combination of 8% strain and HX (Figure 2—figure supplement 1).…”

“…To explore these effects we employed low cyclic strain mimicking reduced contractility (2% strain), physiological cyclic strain (8% strain) and a static control. These values were based on cardiac imaging studies performed shortly after myocardial injury (Dandel et al, 2009; Vartdal et al, 2007) and were previously reported to elicit strain-dependent effects in CFs (Ugolini et al, 2016). …”

“…In an attempt to provide in vitro models of cardiac disease, CFs have been widely studied under relevant physico-chemical stimulation such as mechanical stress (Schroer and Merryman, 2015; Tomasek et al, 2002), oxygen deprivation (Clancy et al, 2007; Tamamori et al, 1997) and biochemical stimulation with pro-fibrotic cytokines (Edgley et al, 2012; Lijnen et al, 2000; Petrov et al, 2002). The application of mechanical stress has been shown to have the following effects on CFs: increased ECM protein synthesis (Carver et al, 1991); controversial proliferative behavior (Atance et al, 2004; Butt and Bishop, 1997; Dalla Costa et al, 2010; Liao et al, 2004), with recent reports of strain intensity-dependent effects (Ugolini et al, 2016); increased production of pro-fibrotic and inflammatory cytokines such as TGF-β (transforming growth factor-beta) (Leask, 2007) and TNF-α (tumor necrosis factor-alpha) (Yokoyama et al, 1999). …”

“…To address this need, we here report the improvement of a previously described multi-chamber microdevice dedicated to the application of cyclic strain to cell monolayers (Ugolini et al, 2016). By adding a system for controlling oxygen changes, we significantly expanded the experimental complexity and mimicking capabilities of the device.…”

“…less than 3% in ischemic regions versus approximately 10% in healthy regions [Dandel et al, 2009; Vartdal et al, 2007]). Within this range and in line with previous in vitro observations of CFs behavior (Ugolini et al, 2016), we selected 2% strain as indicative of reduced contractility and 8% strain as representative of full myocardial contractility. These environmental changes and the following initial cellular responses happen in a timeframe of hours, as shown in vitro (van Nieuwenhoven et al, 2013; Turner et al, 2007, 2009) as well as in vivo (Guillén et al, 1995; Morishita et al, 2015).…”