Substrate Stiffness Modulates Gene Expression and Phenotype in Neonatal CardiomyocytesIn Vitro

Abstract

Biomaterials to be used as cell delivery systems for cardiac tissue engineering should be able to comply with cardiac muscle contractile activity, while favoring cell survival and neo-angiogenesis in a hostile environment. Biocompatible synthetic materials can be tailored to mimic cardiac tissue three-dimensional organization in the micro- and nanoscales. Nonetheless, they usually display mechanical properties that are far from those of the native myocardium and thus could affect host cell survival and activity. In the present investigation, inert poly-ε-caprolactone planar layers were manufactured to change the surface stiffness (with Young's modulus ranging from 1 to 133 MPa) without changing matrix chemistry. These substrates were challenged with neonatal murine cardiomyocytes to study the possible effect of substrate stiffness on such cell behavior without changing biological cues. Interestingly, softer substrates (0.91±0.08 and 1.53±0.16 MPa) were found to harbor mostly mature cardiomyocytes having assembled sarcomeres, as shown by the expression of alpha actinin and myosin heavy chain in typical striations and the upregulation of sarcomeric actin mRNA. On the other hand, a preferential expression of immature cardiac cell genes (Nkx-2.5) and proteins (GATA-4) in cardiac cells grown onto stiffer materials (49.67±2.56 and 133.23±8.67 MPa) was detected. This result could not be ascribed to significant differences in cell adhesion or proliferation induced by the substrates, but to the stabilization of cardiomyocyte differentiated phenotype induced by softer layers. In fact, cardiac cell electromechanical coupling was shown to be more organized on softer surfaces, as highlighted by connexin 43 distribution. Moreover, a differential regulation of genes involved in extracellular matrix remodeling was detected on soft films (0.91±0.08 MPa) as compared with the stiffest (133.23±8.67 MPa). Finally, the upregulation of a number of genes involved in inflammatory processes was detected when the stiffest polymer is used. These events highlight the differences in cell mechanosensitivity in a heterogeneous cell preparation and are likely to contribute to the differences encountered in cardiac cell phenotype induced by substrate stiffness.

Effect of Substrate Mechanics on Cardiomyocyte Maturation and Growth

“…Interestingly, CMs seeded on softer substrates in the range of 0.91 -0.08 and 1.53 -0.16 MPa were mostly mature and had assembled sarcomeres. 33 On the contrary, the CMs grown on the stiff PCL (49.67 -2.56 and 133.23 -8.67 MPa) expressed immature cardiac cell genes (Nkx-2.5) and proteins (GATA-4), in addition to a number of genes involved in inflammatory processes. This confirmed the hypertrophy that the differentiation of CM phenotypes is influenced by the substrate stiffness showing that softer substrates induce a more mature phenotype.…”

“…This confirmed the hypertrophy that the differentiation of CM phenotypes is influenced by the substrate stiffness showing that softer substrates induce a more mature phenotype. 33 Furthermore, the effect of substrate stiffness on CM beating has also been investigated, 16 by seeding CMs, derived from embryonic (day 8) chicks, on PA gel substrates of different stiffness for 1-5 days. The PA gel substrates that were considered had a stiffness of 1, 18, and 50 kPa.…”

Introduction to cell–hydrogel mechanosensing

“…They noted that neurogenic differentiation was optimal at a stiffness of 0.1-1 kPa, myogenic differentiation at 8-17 kPa and osteogenic differentiation at 25-40 kPa. Several subsequent studies have shown how substrate stiffness affects several different cell types, including neuronal cells [134,135], chondrocytes [136], cardiomyocytes [137,138], dermal fibroblasts [139] and limbal stem cells [140]. Recently, Trappmann et al [141] suggested that substrates of different stiffness have differing protein anchorage densities and configurations that regulate stem cell fate through the mechanical feedback of cells attached to anchored proteins [141].…”

Hippo Pathway Effectors Control Cardiac Progenitor Cell Fate by Acting as Dynamic Sensors of Substrate Mechanics and Nanostructure

“…28,29 The responsiveness of adult and undifferentiated cells to substrate stiffness and topology has been the subject of a number of investigations so far, showing that cell-specific responses can be triggered by the mechanics of the substrate. 12,20,30,41,42 Moreover, a specific response of tissue resident cardiac progenitor cells to substrate mechanical properties has been highlighted. 25,43 Recently, cell sensitivity to substrate composition has been associated with the ability of the Hippo pathway downstream effectors YAP and TAZ to act as transcriptional coactivators directly binding to lineage-specific effectors, thus acting as onÀoff relays in mechano-transduction.…”