Rictor/mTORC2 involves mitochondrial function in ES cells derived cardiomyocytes via mitochondrial Connexin 43

Fig. 1. Knockdown of Rictor inhibited cardiomyocyte differentiation and impaired the mitochondrial structure and function in ESC-CMs. a The expression of Rictor/mTORC2 during cardiomyocyte differentiation. b The cell viability of ES cells after transfection with shRNA lentivirus. c The expression of Rictor and Oct4 after transfection with shRNA lentivirus. d Proportions of α-actinin-positive cells in the shRNA-Con and shRNA-Rictor groups on d 5 + 3 determined by flow cytometry. e The protein expression of α-actinin was evaluated in EBs on d 5 + 3. f The proportions of c-TNT-positive cells in the shRNA-Con and shRNA-Rictor groups on d 5+3 and d 5+5, determined by flow cytometry. g The protein expression of c-TNT was evaluated in EBs on d 5 + 3 and d 5 + 5. h The ultrastructure of ESC-CMs after Rictor knockdown. (m: mitochondrion, ER: endoplasmic reticulum). i The ΔΨm of ESC-CMs was evaluated with JC-1 staining. j The ΔΨm of ESC-CMs was further assessed by flow cytometry. k Intracellular ATP production by ESC-CMs. l The expression levels of cytochrome c in the mitochondria and cytoplasm were no different between the two groups. n ≥ 3. *P < 0.05, **P < 0.01 vs shRNA-Con. Bars = 0.1 μm, 0.25…

Fig. 2. MtCx43 is involved in the shRNA-Rictor-induced damage to mitochondrial function. a The protein expression levels of Cx43 in the total cell lysate and mitochondria. b ESC-CMs were double-stained for Cx43 (green) and mitochondria (red). c The proportion of α-actinin-positive cells in EBs on d 5 + 3 was determined by flow cytometry analysis. d The proportions of c-TNT-positive cells in EBs on d 5 + 5 were determined by flow cytometry analysis. e The ΔΨm in ESC-CMs were detected by JC-1 staining and flow cytometry. f Intracellular ATP production was assessed in ESC-CMs at d 5 + 3. n ≥ 3. **P < 0.01 vs shRNA-Con; #P < 0.05, ##P < 0.01 vs shRNA-Rictor. Bars = 25 μm and 50 μm.

Fig. 3. Rictor-mediated regulation of the mitochondrial respiratory chain via mtCx43. The activities of enzymes in complexes I (a), III (b), IV (c), and V (d) in Rictor-knockdown ESC-CMs were determined. n ≥ 3. *P < 0.05, **P < 0.01 vs shRNA-Con; #P < 0.05 vs shRNA-Rictor.

Fig. 4. The mechanism by which Rictor regulates the translocation of Cx43 to mitochondria. a The interaction of Cx43 with the mitochondrial protein import system (Hsp90, TOM20) in ESC-CMs was decreased after knockdown of Rictor. b The protein expression level of HDAC6 in ESC-CMs. c Knockdown of Rictor increased the acetylation of Hsp90 in ESC-CMs. d Knockdown of Rictor decreased the expression of SIN1, p-AktSer473, and p-mTORSer2481 in ESC-CMs. n ≥ 3. *P < 0.05, **P < 0.01 vs shRNA-Con.

Fig. 5. Schematic representation of the mechanisms by which Rictor regulates mitochondrial function in ESC-CMs.