Fig. 4

The enhanced SERCA2a activity of iPSC-derived VSMCs from VSA patient was induced by the increased small ubiquitin-related modifier (SUMO)ylation of SERCA2a. (A-C) Western blot shows that the amount of endogenous SERCA2a and SUMO1 proteins was greater in VSA patient-derived VSMCs than in normal subject-derived VSMCs (n = 4–7 per group). 50 μg of whole protein lysates from each sample were loaded with a 10% SDS-PAGE gel, transferred to nitrocellulose membranes, and probed with anti-SERCA2a (1:1000), anti-SUMO1 (1:1000), anti-UBA2 (1:1000), anti-SENP1 (1:1000), and anti-sarcomeric actin antibodies (1:1000). SUMO1 = Small Ubiquitin Like Modifier 1; UBA2 = Ubiquitin-like modifier-activating enzyme 2; SENP1 = Sentrin-specific protease 1. (D) Immunoprecipitation assay for SUMO1 and SERCA2a indicates that SUMO1 bound only to SERCA2a of VSMCs from VSA patients, not from normal subjects. (E, F) We transfected VSA-patient derived VSMCs with lentiviral vectors encoding SERCA2a with mutated lysine 480 and 585 residues. Each group was treated with Fluo-4 and monitored through time-lapse confocal microscopy over 10 min after the addition of 250 μM carbachol. The transfection with the mutant forms exhibited lower intracellular calcium efflux, indicating that these SUMOylated sites are responsible for the increased activity of SECAR2a in the VSMCs from VSA patients (n = 3 per group). (G, H) 10 μg of each cell lysate was mixed with the reaction buffer and treated with 1 mM ATP and Ginkgolic acid (10 μM), an inhibitor of SUMOylated E1 molecules. Ginkgolic acid suppressed enhanced SERCA2a activity in iPSC-derived VSMCs from VSA patients (n = 9 per group). GK Ginkgolic acid