Fig. 2

Hyperreactivity of iPSC-derived VSMCs from VSA patients was triggered by an abnormal increase of intracellular calcium efflux (A, B) Intracellular calcium efflux in response to carbachol was measured with Fluo-4 (a calcium fluorescent dye. Fluo-4 treated cells were monitored through time-lapse confocal microscopy over 10 min after the addition of 250 μM of carbachol, an inducer of contraction. The iPSC-derived VSMCs from VSA patients exhibited much higher intensity of the calcium efflux peak than the cells from control subjects. Moreover, secondary or tertiary peaks of calcium efflux were observed only in the VSA group (primary human VSMCs n = 7; NC n = 13; VA n = 15). Primary human VSMCs indicate commercially available human coronary artery vascular smooth muscle cells. The orange circle indicates the secondary peak. (C-F) The addition of 250 μM ergonovine and 250 μM acetylcholine in primary human VSMCs, NC- and VSA- iPSC-derived VSMCs produced similar results to treated carbachol (Ergonovine; primary human VSMCs n = 4; NC n = 13; VA n = 15) (Acetylcholine; primary human VSMCs n = 3; NC n = 11; VA n = 13). The orange circle indicates the secondary peak