Figure 4.

Characterization of mitochondrial function and autophagy/mitophagy in hypertrophied neonatal rat cardiomyocytes (NCMs). (a) Ultrastructure of PBS- and Iso-treated NCMs (magnification ×7000 with a scale bar of 10 µm (left images) and ×12,000 with a scale bar of 2 µm (right images)) and quantification of mitochondria number, length (µm), width (µm) and area (µm²). Arrows indicate example of mitochondria for comparison. (b) Mitochondrial membrane potential was quantified in NCMs treated with Iso by fluorescence quantification of JC-1 dye for aggregates (red) and monomer (green) (from 3 independent experiments and at least 366 cells). (c) Representative images for quantification of Krebs cycle by Western blot of aconitase 2 and mitophagy/autophagy by Western blot of parkin, LC3II/LC3I ratio, ubiquitin and beclin-1 in NCMs treated with Iso Data were normalized to actin. (d) Representative images of parkin (red) and LC3 (green) localized in mitochondria (white) of untreated (PBS) and Iso-treated NCMs. Colocalization appeared in merge images. Nuclei were stained by Dapi (blue). Only significant p values are indicated from at least 3 independent experiments. Images were selected to represent the mean values of each condition.