BS46 Calcium signalling modulates VSMC response to matrix stiffness in ageing and disease

IntroductionThe aorta is the largest blood vessel in the body. Healthy aortae are flexible and can stretch and recoil cyclically in response to blood flow. Decreased aortic compliance is a major risk factor for cardiovascular diseases including atherosclerosis and hypertension. Decreased compliance is primarily driven by loss of elastic extracellular matrix (ECM) components and increased deposition of non-elastic collagen-I. In response, mechanosensitive vascular smooth muscle cells (VSMCs) generate enhanced actomyosin-derived forces which further contribute to vessel stiffening. The mechanisms driving this response are unknown.Ca2+ signalling can initiate contraction in two distinct ways. Firstly, via activation of membrane bound GPCRs by contractile agonists such as Angiotensin II (Ang II). GPCR activation leads to production of IP3 which bind to and activates IP3 receptors (IP3R) on the sarcoplasmic reticulum (SR). Activation of IP3R initiates Ca2+ release from the SR into the cytosol. The second pathway involves the flow of extracellular Ca2+ ions into the cell, through stretch activated ion channels such as Piezo1.We have shown that contractile agonist stimulation of VSMCs triggers contraction on pliable hydrogels and hypertrophy on stiff hydrogels. We therefore hypothesised that Ca2+ signalling regulates VSMC contractility in response to arterial stiffening. Specifically, that intracellular Ca2+ regulates contraction and extracellular Ca2+ induces VSMC dysfunction and hypertrophy.MethodsPrimary human aortic VSMCs were cultured on polyacrylamide hydrogels of physiological and pathophysiological stiffnesses. Concentration responses of pharmacological inhibitors of Ca2+ signalling were performed on angiotensin II (Ang II) stimulated cells. VSMC area was used as a measure of VSMC contractility or hypertrophy.ResultsOur results show that pharmacological inhibition of the IP3R (Xestospongin C) or ryanodine receptors (Dantrolene) prevents both VSMC contraction and hypertrophy on pliable and stiff substrate, respectively. Meanwhile, blockade of membrane bound piezo1 (GsMTx-4) or TRPC1/4/5 channels (Pico145) prevented VSMC hypertrophy on stiff substrates only.ConclusionOur data suggests that release of Ca2+ from intracellular stores is important for cell contraction and hypertrophy, whereas influx of Ca2+ from the extracellular environment is involved in VSMC hypertrophy and dysfunction on stiff substrates. Blocking the influx of extracellular Ca2+ prevents V