A differentiated Ca²⁺ signalling phenotype has minimal impact on myocardin expression in an automated differentiation...

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.