Introduction:
Cardiac ageing is characterised by increased stiffness of the myocardium, due to excess deposition of extracellular matrix (ECM) proteins such as collagen. The structural remodelling of the myocardium can consequently impact the behaviour of surrounding cells, such as cardiomyocytes (CMs). Current research using induced pluripotent stem-cell derived cardiomyocytes (iPSC-CMs) may not truly reflect the physiological stiffness of the ECM in healthy or diseased conditions. In order to assess the effect of ECM stiffness on iPSC-CMs structure and metabolic function, polydimethylsiloxane (PDMS) substrates can be used to recapitulate healthy (20kPa) and fibrotic (130kPa) stiffnesses.
Aims:
To investigate differences in structure and function of iPSC-CMs on soft and stiff PDMS substrates.
To explore changes in iPSC-CM metabolism on substrate stiffnesses.
Materials and Methods:
iPSC-CMs were plated onto PDMS gels of 20kPa and 130kPa and harvested between days 20-30 of differentiation. Molecular analyses of iPSC-CMs were investigated using qPCR, western blotting and immunofluorescence to explore cardiac gene expression profiles and structural parameters such as sarcomere length and actin organisation. Contractility of iPSC-CMs on stiffnesses were assessed using MuscleMotion software, which utilised video recordings of cells from a GoPro. Isotope labelled mass spectrometry was conducted on iPSC-CMs on varying stiffnesses to explore changes in metabolic pathways, as well as metabolic gene expression changes.
Results: Softer substrates induce maturation of iPSC-CMs, with significantly higher expression of cardiac maturity markers such as MYH7:MYH6 ratios and MYL2:MYL7 ratio compared to stiffer plastic conditions. Maturation is further displayed on 20kPa PDMS, with iPSC-CMs portraying a higher level of sarcomere alignment compared to plastic and 130kPa. Contractility of iPSC-CMs is altered on softer substrates. Metabolic profiles of iPSC-CM on stiffer gels display higher glycolytic metabolites such as glucose, whereas cells on softer substrates portray TCA cycle metabolites.
Conclusions: Substrate stiffnesses can act as physiological models of healthy and diseased ECM by portraying molecular changes in iPSC-CM structure and maturity, as well as recapitulating switches in iPSC-CM metabolism.