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

Abstract

Rictor is a key component of the mammalian target of rapamycin complex 2 (mTORC2) and is required for Akt phosphorylation (Ser473). Our previous study shows that knockdown of Rictor prevents cardiomyocyte differentiation from mouse embryonic stem (ES) cells and induces abnormal electrophysiology of ES cell-derived cardiomyocytes (ESC-CMs). Besides, knockdown of Rictor causes down-expression of connexin 43 (Cx43), the predominant gap junction protein, that is located in both the sarcolemma and mitochondria in cardiomyocytes. Mitochondrial Cx43 (mtCx43) plays a crucial role in mitochondrial function. In this study, we used the model of cardiomyocyte differentiation from mouse ES cells to elucidate the mechanisms for the mitochondrial damage in ESC-CMs after knockdown of Rictor. We showed swollen and ruptured mitochondria were observed after knockdown of Rictor under transmission electron microscope. ATP production and mitochondrial transmembrane potential were significantly decreased in Rictor-knockdown cells. Furthermore, knockdown of Rictor inhibited the activities of mitochondrial respiratory chain complex. The above-mentioned changes were linked to inhibiting the translocation of Cx43 into mitochondria by knockdown of Rictor. We revealed that knockdown of Rictor inactivated the mTOR/Akt signalling pathway and subsequently decreased HDAC6 expression, resulted in Hsp90 hyper-acetylation caused by HDAC6 inhibition, thus, inhibited the formation of Hsp90-Cx43-TOM20 complex. In conclusion, the mitochondrial Cx43 participates in shRNA-Rictor-induced mitochondrial function damage in the ESC-CMs.

Keywords: Connexin43; Rictor/mTORC2; cardiomyocyte differentiation; embryonic stem cell; mitochondria.

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 μm, 0.5 μm, 25 μm, and 50 μm.

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-Akt^Ser473, and p-mTOR^Ser2481 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.