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

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 Ca signalling in electrically coupled, differentiated cells to increased dependence on store-operated Ca entry and sarcoplasmic reticulum Ca release in synthetic cells. Conversely, an increased voltage-gated Ca entry is seen when proliferating A7r5 smooth muscle cells quiesce. We asked whether this change in Ca signalling was linked to changes in the expression of the phenotype-regulating transcriptional co-activator myocardin or α-smooth muscle actin, using correlative epifluorescence Ca 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 Ca signalling showed increasing dependence on L-type voltage-gated Ca 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 Ca 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 Ca signalling phenotype, while chronic inhibition of voltage-gated Ca entry modestly increased immunoreactivity of myocardin. Stepwise regression analyses suggested that changes in myocardin expression had a weak relationship with Ca signalling synchronicity, but not frequency or amplitude. In conclusion, we report a 96-well assay and analytical pipeline to study the link between Ca 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 Ca signalling phenotype.