Substrate stiffness-dependent metabolic reprogramming of iPSC-derived cardiomyocytes on physiological PDMS polymers

Many cardiac pathologies are characterised by increased stiffness of the myocardium, due to excess deposition of extracellular matrix (ECM) proteins and structural remodelling, impacting the behaviour of cardiomyocytes (CMs). Metabolism of CMs shifts in cardiac pathologies, with the healthy heart primarily utilising fatty acids as its source of energy production, whilst the diseased heart switches to utilise glucose. The shift in metabolic source with stiffness of the myocardium has not been investigated. To investigate the effect of ECM stiffnesses on iPSC-CM metabolism, iPSC-CMs were cultured on polydimethylsiloxane (PDMS) substrates of healthy and fibrotic stiffness (20 kPa and 130 kPa respectively) and plastic. Cellular metabolism of iPSC-CMs was assessed through isotope-labelled mass spectrometry with central carbon tracing as well as real-time cellular bioenergetics using extracellular flux analysis. Key metabolic genes were investigated at transcript and protein level, with proteomics analysis conducted to identify protein profiles on substrate stiffnesses. Mass spectrometry data revealed greater utilisation of glucose in iPSC-CMs cultured on plastic compared to softer PDMS substrates, indicating greater glycolytic activity. Extracellular flux analysis demonstrated greater lactic acid efflux from iPSC-CMs cultured on plastic substrates, reflective of increased glycolytic flux and a shift towards aerobic glycolysis as the primary source of ATP synthesis. This study revealed culture of iPSC-CMs on traditional cell culture plastics or glass coverslips displaying pathological metabolism, highlighting the use of physiological substrates for metabolic investigation.