219 Fatty Acid Oxidation in PLN R14del Cardiomyopathy 8 Figure 5: Lipid accumulation and impaired mitochondria in PLN-R14del cardiac tissues and hiPSC-CMs. A) Left: Examples of Nile red staining (red, indicated by black arrows) in control and PLN-R14del paraffin-embedded cardiac tissues using immunohistochemical staining (IHC). Images were taken at 40x magnification. Right: Examples of Nile red staining (red) in control and PLN-R14del snap-frozen cardiac tissues using immunofluorescence staining (IF). Confocal images were taken at 63x magnification. Nuclei were stained by DAPI (blue) and sarcomeres were stained by TNNI3 (green). B) Bar graphs showing impaired detected mitochondria in PLN-R14del versus control hearts using transmission electron microscopy (TEM). Each dot represents an individual mitochondrial from control and PLN hearts. Representative TEM image of the control heart (bottom left) showing regular myofilaments arranged in sarcomeres (brackets), variable-sized mitochondria (M) arranged in rows in between myofibrils, lipid droplets (yellow arrows) and profiles of the intercalated disc with a large mixed-type junction (area composita, AC) in between cardiomyocytes. Whereas, representative TEM image of the PLN-R14del heart (bottom right) shows irregular myofilaments arranged in sarcomeres (brackets), variable-sized mitochondria (M) located around the myofibrils, lipid droplets (yellow arrows) and profiles of the intercalated disc with a large mixed-type junction (AC) in between cardiomyocytes. Multiple shape descriptors were determined for manually traced lipid droplets and mitochondria. C) Bar graphs showing higher coverage by lipid droplets and bigger lipid droplets in PLN-R14del versus control hearts using TEM. Representative TEM images (below) showing manually traced lipid droplets (indicated by yellow arrows) in the control and PLN-R14del hearts. Each dot represents an individual lipid droplet from control and PLN hearts. TEM images were collected from 3 healthy controls (67 traced lipid droplets and 128 traced mitochondria) and 1-2 PLN-R14del hearts (177 traced lipid droplets and 129 traced mitochondria). Data are expressed as mean ±SD. Unpaired T-test was used, *P<0.05, ****P<0.0001. Scale bar: 2 µM. D) Examples of Nile red staining to demonstrate the lipid accumulation in long-term cultured (160 days) PLN-R14del and control hiPSC-CMs in three different media. Bar graph showing a higher lipid accumulation (red) in PLN-R14del versus control hiPSC-CMs in three different media.
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