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Fig. 3 | Biomaterials Research

Fig. 3

From: Identification of cell-biologic mechanisms of coronary artery spasm and its ex vivo diagnosis using peripheral blood-derived iPSCs

Fig. 3

The mechanism of hyperreactivity of iPSC-derived VSMCs from VSA patient: the enhanced sarco/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) activity. (A, B) 10 μg of whole protein lysates from each of the samples were loaded with a 10% SDS-PAGE gel, transferred to nitrocellulose membranes, and probed with anti-SERCA2a (1:1000) and anti-sarcomeric actin antibodies (1:1000). Western blot indicates that the amount of endogenous SERCA2a protein was greater in iPSC-derived VSMCs from VSA patients than from normal subjects (n = 3 per group). All samples in Western blot were obtained from individual subjects (three different normal subjects and three different VSA patients). (C, D) Immunofluorescence staining for SERCA2a shows higher intensity in iPSC-derived VSMCs from VSA patients than from normal subjects (n = 8–10 per group; scale bar, 20 μm). Each group of cells was stained with human anti-SERCA2a (1:100). SERCA2a was detected using the Alexa Fluor 488-conjugated anti-mouse secondary antibody (1:100). (E, F) Calcium-dependent ATPase activity assay indicates that the activity of SERCA2a in the VSA group was greater than in the control group (n = 6 per group). 10 μg of each cell lysate was mixed with the reaction buffer and pre-incubated for 10 min before being treated with 1 mM adenosine triphosphate (ATP). The ATPase activity was measured at 360 nm using a microplate reader. (G, H) After knocking down SERCA2a expression in iPSC-derived VSMCs from VSA patients using a lentiviral vector and induced with 250 μM carbachol, intracellular calcium efflux was downregulated to the levels of control cells (Mock n = 3; shSERCA2a n = 4). Mock indicates control lentiviral vector transfection. (I, J) In contrast, the overexpression of SERCA2a in iPSC-derived VSMCs from normal subjects using a lentiviral vector induced with 250 μM carbachol increased the level of intracellular calcium efflux and generated the second peak of calcium efflux (n = 6 per group)

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