Fig. 7.

PGAM5 deficiency-mediated mitochondrial protection is abolished by transfection of a phosphorylation-defective PHB2 mutant. Primary neonatal cardiomyocytes were isolated from Pgam5^CKO and Pgam5^f/f mice, transfected with HA-PHB2^S91A or HA-PHB2^S91D mutant constructs, and exposed to PBS or hyperglycemia. (A) Detection of ΔΨm in cardiomyocytes loaded with the JC-1 probe. (B) The red-to-green fluorescence ratio was used for the semiquantitative analysis of changes in ΔΨm. (C) Analysis of mtROS generation in cardiomyocytes loaded with MitoSOX Red. (D) The levels of mtROS were normalized to those of the control group. (E and F) ELISA-based determination of the activity of mitochondrial respiratory complex I (E) and complex V (F). (G) Analysis of mitophagy in primary cardiomyocytes expressing the mito-Keima probe. (H) Presentative pictures of cardiomyocyte transfected with mito-Keima. (I and J) Analysis of Pgc1α (I) and Nrf2 (J) transcription levels in cultured cardiomyocytes. Values are presented as mean ± SEM from 4 independent experiments. #P < 0.05.