A differentiated Ca2+ signalling phenotype has minimal impact on myocardin expression in an automated differentiation assay using A7r5 cells

Abstract

Vascular smooth muscle cells are unusual in that differentiated, contractile cells possess the capacity to “de-differentiate” into a synthetic phenotype that is characterized by being replicative, secretory, and migratory. One aspect of this phenotypic modulation is a shift from voltage-gated Ca2+ signalling in electrically coupled, differentiated cells to increased dependence on store-operated Ca2+ entry and sarcoplasmic reticulum Ca2+ release in synthetic cells. Conversely, an increased voltage-gated Ca2+ entry is seen when proliferating A7r5 smooth muscle cells quiesce. We asked whether this change in Ca2+ signalling was linked to changes in the expression of the phenotype-regulating transcriptional co-activator myocardin or α-smooth muscle actin, using correlative epifluorescence Ca2+ imaging and immunocytochemistry. Cells were cultured in growth media (DMEM, 10% serum, 25 mM glucose) or differentiation media (DMEM, 1% serum, 5 mM glucose). Coinciding with growth arrest, A7r5 cells became electrically coupled, and spontaneous Ca2+ signalling showed increasing dependence on L-type voltage-gated Ca2+ channels that were blocked with nifedipine (5 μM). These synchronized oscillations were modulated by ryanodine receptors, based on their sensitivity to dantrolene (5 μM). Actively growing cultures had spontaneous Ca2+ transients that were insensitive to nifedipine and dantrolene but were blocked by inhibition of the sarco-endoplasmic reticulum ATPase with cyclopiazonic acid (10 μM). In cells treated with differentiation media, myocardin and αSMA immunoreactivity increased prior to changes in the Ca2+ signalling phenotype, while chronic inhibition of voltage-gated Ca2+ entry modestly increased immunoreactivity of myocardin. Stepwise regression analyses suggested that changes in myocardin expression had a weak relationship with Ca2+ signalling synchronicity, but not frequency or amplitude. In conclusion, we report a 96-well assay and analytical pipeline to study the link between Ca2+ signalling and smooth muscle differentiation. This assay showed that changes in the expression of two molecular differentiation markers (myocardin and αSMA) tended to precede changes in the Ca2+ signalling phenotype.