Computational protocol: Adeno-associated virus-mediated CASQ2 delivery rescues phenotypic alterations in a patient-specific model of recessive catecholaminergic polymorphic ventricular tachycardia

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Protocol publication

[…] Ca2+ transients and Ca2+ sparks were acquired using a TrimScope II upright two-photon microscope (LaVision Biotec, Germany) with a 60 × LUMPFL NA 1.1 (Olympus, Japan) water immersion objective, as previously described. iPSC-CMs were seeded onto 35 mm dishes and the next day loaded with 5 μM Fluo-4 AM dye (Thermo Scientific) for 20 min. During acquisition, cells were kept under physiological condition with a perfusion pump in a buffer heated at 37°C. Ti : Sa infrared laser was tuned at 810 nm, and fluorescence light was separated from excitation light with a SP700 filter, then collected with a 525/50 emission filter and a non-descanned GaAsP detector (H7422-40, Hamamatsu Photonics, Japan) . After a first 512-pixel-wide XY image for each cell, we drew line ROIs and acquired 5000 XT line scans per ROI with a 0.375 um pixel size and 300 Hz time resolution, during which some calcium transients and some sparks occurred. Calcium transients were processed with Fiji/ImageJ (http://fiji.sc/) to generate table intensity data over time, then R statistical software (R Foundation for Statistical Computing, Vienna, Austria) was used to isolate single transients and measure normalized transient amplitudes. Minimum five transients per condition were analyzed. Calcium sparks were analyzed with SparkMaster ImageJ plugin (https://sites.google.com/site/sparkmasterhome/). Statistical analysis of both transient and sparks measurements was conducted in Graphpad Prism 4.03 (Graphpad Software Inc, La Jolla, CA, USA), One-Way ANOVA and Tukey multiple post-test (*P<0.05, **P<0.01, ***P<0.001).200–580 sparks were analyzed over a minimum of 25 cells per condition. […]

Pipeline specifications

Software tools ImageJ, SparkMaster
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens, Adeno-associated virus
Diseases Disease Models, Animal, Genetic Diseases, Inborn
Chemicals Calcium