Pim-1 preserves mitochondrial morphology by inhibiting dynamin-related protein 1 translocation

Abstract

Mitochondrial morphological dynamics affect the outcome of ischemic heart damage and pathogenesis. Recently, mitochondrial fission protein dynamin-related protein 1 (Drp1) has been identified as a mediator of mitochondrial morphological changes and cell death during cardiac ischemic injury. In this study, we report a unique relationship between Pim-1 activity and Drp1 regulation of mitochondrial morphology in cardiomyocytes challenged by ischemic stress. Transgenic hearts overexpressing cardiac Pim-1 display reduction of total Drp1 protein levels, increased phosphorylation of Drp1-(S637), and inhibition of Drp1 localization to the mitochondria. Consistent with these findings, adenoviral-induced Pim-1 neonatal rat cardiomyocytes (NRCMs) retain a reticular mitochondrial phenotype after simulated ischemia (sI) and decreased Drp1 mitochondrial sequestration. Interestingly, adenovirus Pim-dominant negative NRCMs show increased expression of Bcl-2 homology 3 (BH3)-only protein p53 up-regulated modulator of apoptosis (PUMA), which has been previously shown to induce Drp1 accumulation at mitochondria and increase sensitivity to apoptotic stimuli. Overexpression of the p53 up-regulated modulator of apoptosis-dominant negative adenovirus attenuates localization of Drp1 to mitochondria in adenovirus Pim-dominant negative NRCMs promotes reticular mitochondrial morphology and inhibits cell death during sI. Therefore, Pim-1 activity prevents Drp1 compartmentalization to the mitochondria and preserves reticular mitochondrial morphology in response to sI.

Fig. 1.

Drp1 Localizes to the mitochondria after sI in vitro. (A) Tom20-labeled NRCMs showing the transition from reticular to punctate mitochondria phenotypes after either sI or sI/R insults. Quantification of mitochondrial phenotype in NRCMs subjected to sI or sI/R. FM, full media; GF, glucose-free media; *, significant compared with 30 min GF control; #, not significant versus sI 30. (B) Immunoblot of Drp1 after 30 min of sI versus control, full media M199 with 10% FBS NRCMs. Ctrl, control; **, significant compared with control. (C) Immunoblot of Drp1 localization in NRCMs after sI/R (30 min/30 min) versus 30 min sI. (D) Immunoblot of Drp1 localization in adult murine hearts subjected to a sham LAD procedure, 50 min LAD ligation, or 50 min/15 (120) min LAD ligation/reperfusion. **, significant compared with sham. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 2.

Drp1 Phosphorylation is regulated by Pim-1. (A–F) Immunoblot and quantification of pDrp1^S637 and total Drp1 in murine PimWT and PDN whole-heart lysates. (G–H) Immunoblot of Drp1 localization in isolated mitochondria from PimDN (PDN) and FVB nontransgenic (NTG) control hearts harvested from age- and sex-matched mice. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. 3.

Pim-1 overexpression affects Drp1 Localization in NRCMs. (A) Immunoblot of Drp1 localization in NRCMs transduced with adEGFP control virus or adEGFP-PimWT after sI. (B) Protein analysis of Drp1 compartmentalization in adEGFP- or adPDN-transduced NRCMs after fractionation. (C) Analysis and quantification of mitochondrial morphology in NRCMs infected with adPimWT or adEGFP subjected to up to 30 min of sI. *, significant to EGFP by two-way ANOVA (D) Quantification of adPDN and adEGFP NRCMs maintaining reticular mitochondria before and after 30 min of sI. **, significant compared with EGFP control. (E) Immunoprecipitation of Drp1-infected NRCMs with Pim-1 followed by immunoblot of Drp1 and Pim-1. (F) Proximity ligation assay with Pim-1 and Drp1 in cultured HeLa cells. Each red dot represents a single protein–protein interaction. (G) Phosphorylation of Drp1 by Pim-1 kinase in vitro. Recombinant Drp1 was used as substrate in an in vitro kinase assay that measures γ-³²p-ATP incorporation. His-Pim-1 (wild-type, kinase dead) was expressed in Escherichia coli and affinity-purified for use in the kinase assay. Phosphorylated proteins were separated by SDS/PAGE and visualized by autoradiography. *P < 0.05; **P < 0.01.

Fig. 4.

PUMA mediates the effect of Pim-1 on Drp1 translocation. (A) Immunoblot of PUMA in nontransgenic (NTG) and PimWT transgenic mice. Immunoblot of NRCM lysates infected with either (B) adPimWT and adEGFP or (C) adPDN and adEGFP adenovirus for PUMA. (D) Immunoblot of Drp1 protein levels at the mitochondria after rescue with adPumaDN. *, significant compared with EGFP control; #, significant compared with adPDN. (E) Drp1 protein expression in total NRCM lysates after infection with adEGFP, adPDN, adPumaDN, and adPDN + adPumaDN. (F) Morphological assessment of reticular mitochondria in the aforementioned groups by Tom20 staining. **, significant compared with EGFP; ##, significant compared with PDN. (G) Cell death analysis with propidium iodide of NRCMs subjected to simulated ischemia. *, significant compared with respective baseline control; #, significant compared with EGFP sI; $, significant compared with PDN sI. #P < 0.05; *P < 0.05; ##P < 0.01; $$P < 0.01; **P < 0.01; $$$P < 0.001; ***P < 0.001.