Dual-labeling of RV Cardiomyocytes and Tissue

For details on fixation and labelling of isolated cardiomyocytes and tissue sections, refer to the Supplementary Data file. Confocal and STED images of RV sections dual-labelled with translocator of the outer mitochondrial membrane/ryanodine receptors (TOM20/RyR2) were obtained with an Olympus IX83 Abberior Facility Line STED microscope using a 60× oil immersion objective lens (NA 1.42). To show a larger portion of the tissue being analysed, confocal images were first captured with a 70 µm × 70 µm frame size at 80 nm pixel resolution. Then, both confocal and STED images were captured from a smaller portion of the tissue section (15 µm × 15 µm frame size) at a 15 nm pixel resolution with 594 nm and 640 nm lasers simultaneously at excitation laser powers 3–6% for confocal and STED images. Power for the STED depletion laser, emitted at 775 nm, was between 6% and 10%. Furthermore, confocal images of RV sections co-labelled for F-actin (Alexa Fluor 488 Phalloidin conjugate, 1:50, A12379, Thermofisher Scientific, Waltham, MA, USA) and mitochondria (TOM20) were obtained with a Zeiss LSM800 laser-scanning confocal microscope using a 63× oil-immersion objective lens (NA of 1.4). Images were captured at a 50 nm pixel resolution with 488 nm and 594 nm lasers simultaneously at 0.4% laser power.