Assessment of mitophagy

Mice transduced with AAV9 particles coding for CMV promoter–driven mitoKeima were generated by the Hope Center viral vectors core at Washington University at a dose of 3.5 × 10¹¹ viral particles per mouse. Mitophagy was assessed with expression of mitoKeima as previously described.16^,32 Briefly, live imaging of freshly harvested cardiac slices were performed with a Zeiss LSM5 Pascal confocal microscope. Two sequential excitations were performed at wavelengths of 458 and 543 nm, followed by imaging with a 560-nm longpass filter for assessment of the mitophagy signal. Laser power was individualized for each condition to optimize Keima imaging. Murine embryonic fibroblasts (MEFs) were transfected with pcDNA3.1 encoding for mitoKeima construct using GenJet transfection reagent (Sinagen) following the manufacturer’s protocols, and mitophagy was assessed with sequential excitation at wavelengths at 458 and 561 nm, followed by emission at 580 nm using a Nikon A1R confocal system at the Washington University Center for Cellular Imaging. Adenoviruses expressing Cre and rat PARKIN were generated as previously described.26 The images were quantitated using ImageJ software (NIH). Mitophagy was also assessed after injection of AAV9-cTnT-TRAF2 or AAV9-cTnT-null viral particles (with 3.5 × 10¹¹ viral particles per mouse) in young adult mitoQC mice. Mitophagy index was calculated as a ratio of the average intensity of the red/green signal using ImageJ software.