Cardiac myocytes respond differentially and synergistically to matrix stiffness and topography

Abstract

During cardiac disease progression, myocytes undergo molecular, functional and structural changes, including increases in cell size and shape, decreased myocyte alignment and contractility. The heart often increases extracellular matrix production and stiffness, which affect myocytes. The order and hierarchy of these events remain unclear as available in vitro cell culture systems do not adequately model both physiologic and pathologic environments. Traditional cell culture substrates are 5-6 orders of magnitude stiffer than even diseased native cardiac tissue. Studies that do account for substrate stiffness often do not consider intercellular alignment and vice versa. We developed a cardiac myocyte culture platform that better recapitulates native tissue stiffness while simultaneously introducing topographical cues that promote cellular alignment. We show that stiffness and topography impact myocyte molecular and functional properties. We used a spatiotemporally-tunable, photolabile hydrogel platform to generate a range of stiffness and micron-scale topographical patterns to guide neonatal rat ventricular myocyte morphology. Importantly, these substrate patterns were of subcellular dimensions to test whether cells would spontaneously respond to topographical cues rather than an imposed geometry. Cellular contractility was highest and the gene expression profile was most physiologic on gels with healthy cardiac tissue stiffness. Surprisingly, while elongated patterns in stiff gels yielded the greatest cellular alignment, the cells actually had more pathologic functional and molecular profiles. These results highlight that morphological measurements alone are not a surrogate for overall cellular health as many studies assume. In general, substrate stiffness and micropatterning synergistically affect cardiac myocyte phenotype to recreate physiologic and pathologic microenvironments.Significance StatementHeart disease is accompanied by organ- and cellular-level remodeling, and deconvoluting their interplay is complex. Cellular-level change is best studied in vitro due to greater control and uniformity of cell types compared to animals. One common metric is degree of cellular alignment as misalignment of myocytes is a hallmark of disease. However, most studies utilize featureless culture surfaces that are orders of magnitude stiffer than, and do not mimic the scaffolding of, the heart. We developed a hydrogel platform with tunable stiffness and patterns providing topographical alignment cues. We cultured heart cells on and characterized multifactorial responses to these dynamic surfaces. Interestingly, conditions that yielded greatest alignment did not yield the healthiest functional and molecular state. Thus, morphology alone is not an indicator of overall cellular health.

Asymmetric Hapln1a drives regionalized cardiac ECM expansion and promotes heart morphogenesis in zebrafish development

“…Alternatively, Hapln1a-mediated cross-linking may modulate regional stiffness of the cardiac ECM, and differential matrix stiffness has been shown to regulate cardiomyocyte form and function. 44–47 Importantly, we do not see defects in heart rate in hapln1a mutants ( Supplementary material online, Figure S5 ), suggesting this regionalized ECM is not regulating cardiac function. In addition to provision of mechanical cues to the surrounding cells, the ECM also modulates diffusion and availability of extracellular signalling molecules.…”

Biological responses to physicochemical properties of biomaterial surface

“…A roughness gradient in both micro-and nanoscale can improve interactions between various cell or protein types on the surface. 217 (ix) The synergistic effects of roughness with other physical and chemical properties, which are different depending on the 218 The same roughness value in two different types of materials can affect cell responses differently, which can be owing to the differences in their chemistry. [219][220][221][222] Fukuda et al 220 investigated the osseointegration ability of a poly(ether ether ketone) implant by enhancing its surface roughness and/or surface chemistry ( Fig.…”

Tools for studying and modulating (cardiac muscle) cell mechanics and mechanosensing across the scales

“…Early studies by the Discher lab established a clear correlation between cell differentiation and function with the substrate elasticity of polyacrylamide gels, whereby myogenic differentiation of MSCs and optimal work of quail cardiomyocytes were observed at a stiffness found in the native heart (~10 kPa) (Engler et al 2004(Engler et al , 2008. Subsequently, similar results were obtained with a range of other materials and fabrication techniques, including PDMS, poly-e-caprolactone or PEG (Forte et al 2012;Pandey et al 2018;Wan et al 2019), whereby the latter was also used to pattern the surface at the same time and found that patterning and stiffness together affected gene expression. Similarly, micropatterning was used to look at both healthy and fibrotic elastic moduli in combination with cardiomyocyte shape (i.e.…”