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. 2019 Jul;15(7):1182-1198.
doi: 10.1080/15548627.2019.1580095. Epub 2019 Feb 22.

BNIP3L/NIX and FUNDC1-mediated mitophagy is required for mitochondrial network remodeling during cardiac progenitor cell differentiation

Mark A Lampert  1 , Amabel M Orogo  1 , Rita H Najor  1 , Babette C Hammerling  1 , Leonardo J Leon  1 , Bingyan J Wang  2 , Taeyong Kim  2 , Mark A Sussman  2 , Åsa B Gustafsson  1
Affiliations

BNIP3L/NIX and FUNDC1-mediated mitophagy is required for mitochondrial network remodeling during cardiac progenitor cell differentiation

Mark A Lampert et al. Autophagy. 2019 Jul.

Abstract

Cell-based therapies represent a very promising strategy to repair and regenerate the injured heart to prevent progression to heart failure. To date, these therapies have had limited success due to a lack of survival and retention of the infused cells. Therefore, it is important to increase our understanding of the biology of these cells and utilize this information to enhance their survival and function in the injured heart. Mitochondria are critical for progenitor cell function and survival. Here, we demonstrate the importance of mitochondrial autophagy, or mitophagy, in the differentiation process in adult cardiac progenitor cells (CPCs). We found that mitophagy was rapidly induced upon initiation of differentiation in CPCs. We also found that mitophagy was mediated by mitophagy receptors, rather than the PINK1-PRKN/PARKIN pathway. Mitophagy mediated by BNIP3L/NIX and FUNDC1 was not involved in regulating progenitor cell fate determination, mitochondrial biogenesis, or reprogramming. Instead, mitophagy facilitated the CPCs to undergo proper mitochondrial network reorganization during differentiation. Abrogating BNIP3L- and FUNDC1-mediated mitophagy during differentiation led to sustained mitochondrial fission and formation of donut-shaped impaired mitochondria. It also resulted in increased susceptibility to cell death and failure to survive the infarcted heart. Finally, aging is associated with accumulation of mitochondrial DNA (mtDNA) damage in cells and we found that acquiring mtDNA mutations selectively disrupted the differentiation-activated mitophagy program in CPCs. These findings demonstrate the importance of BNIP3L- and FUNDC1-mediated mitophagy as a critical regulator of mitochondrial network formation during differentiation, as well as the consequences of accumulating mtDNA mutations. Abbreviations: Baf: bafilomycin A1; BCL2L13: BCL2 like 13; BNIP3: BCL2 interacting protein 3; BNIP3L: BCL2 interacting protein 3 like; CPCs: cardiac progenitor cells; DM: differentiation media; DNM1L: dynamin 1 like; EPCs: endothelial progenitor cells; FCCP: carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; FUNDC1: FUN14 domain containing 1; HSCs: hematopoietic stem cells; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MFN1/2: mitofusin 1/2; MSCs: mesenchymal stem cells; mtDNA: mitochondrial DNA; OXPHOS: oxidative phosphorylation; PPARGC1A: PPARG coactivator 1 alpha; PHB2: prohibitin 2; POLG: DNA polymerase gamma, catalytic subunit; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TMRM: tetramethylrhodamine methyl ester.

Keywords: Autophagy; differentiation; heart failure; mitochondria; mitophagy; stem cells.

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Figures

Figure 1.
Figure 1.
POLG CPCs have reduced mitophagy upon induction of differentiation. (a) Representative western blots of LC3-II and ACTA1 in WT and POLG CPCs after incubation in differentiation medium (DM). To assess flux, cells were incubated with 50 nM bafilomycin A1 (Baf) for 4 h before harvesting cells. (b) Quantification of LC3-II:ACTA1 in WT CPCs and POLG CPCs (n = 3). (c) Quantification of LC3-II:ACTA1 ± Baf in WT and POLG CPCs (n = 3). (d) Representative western blots of BECN1 and SQSTM1 in WT and POLG CPCs. Cells were incubated in DM for 7 d. (e) Quantitation of BECN1:ACTA1 and SQSTM1:ACTA1 in WT and POLG CPCs (n = 3). (f) Representative fluorescent images of WT and POLG CPCs overexpressing GFP-LC3. Quantitation of GFP-LC3 and TOMM20 colocalization in WT and POLG CPCs (n = 3). Cells were incubated in DM for the indicated time, fixed, and stained with anti-TOMM20 to label mitochondria. Scale bar: 20 μm. (g) Representative fluorescent images of WT and POLG CPCs overexpressing COX8-EGFP-mCherry at D0 and D3. Quantification of acidic (red-only) mitochondria in WT and POLG CPCs (n = 3). Data are mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n.s., not significant.
Figure 2.
Figure 2.
Mitophagy of depolarized mitochondria is functional in POLG CPCs. Cells were infected with β-Gal or mCherry-PRKN prior to treatment with 25 μM FCCP for 24 h. (a) Representative western blots of LC3-II and GAPDH in WT and POLG CPCs. (b) Quantification of LC3-II:GAPDH in WT (n = 4) and POLG CPCs (n = 3). (c) Representative western blots of the mitochondrial protein TIMM23 and GAPDH in WT and POLG CPCs. (d) Quantitation of TIMM23:GAPDH in WT (n = 4) and POLG CPCs (n = 3). Data are mean ± SEM. *p < 0.05; **p < 0.01; ****p < 0.0001.
Figure 3.
Figure 3.
PRKN is not required for mitophagy in CPCs. (a) Representative western blots of PRKN and GAPDH in mouse CPCs and adult hearts. (b) Real-time PCR analysis of Prkn transcript levels in CPCs and heart tissue (n = 3). (c) Representative western blots of TIMM23 and ACTA1 in WT and prkn−/- CPCs after treatment with 25 μM FCCP for 24 h. (d) Quantification of TIMM23:ACTA1 in WT and prkn−/- CPCs (n = 3). (e) The number and percentage of cells with mRNA detected by single-cell RNA sequencing for Prkn and mitophagy genes in mouse CPCs at passage 0 (fresh) or passage 5 (cultured). Violin plots display gene expression of mitophagy genes in mouse CPCs. (f) The number and percentage of cells with mRNA detected by single-cell RNA sequencing for PRKN and mitophagy receptors in human CPCs at passage 5 (cultured). Violin plots display gene expression of mitophagy genes in human CPCs. Data are mean ± SEM. ***p < 0.001; n.s., not significant.
Figure 4.
Figure 4.
Mitophagy receptors are upregulated in WT CPCs upon differentiation. Real-time PCR analysis of mitophagy receptors (a) Fundc1, (b) Bnip3l and (c) Bnip3 transcript levels in WT and POLG CPCs at baseline and after incubation in DM (n = 4). (d) Representative western blots and quantification of FUNDC1:ACTA1 in WT and POLG CPCs (n = 4). (e) Representative western blots and quantification of BNIP3L:ACTA1 in WT and POLG CPCs (n = 4). Data are mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; n.s., not significant.
Figure 5.
Figure 5.
Knockdown of Bnip3l and Fundc1 decreases mitophagy in WT CPCs. (a) Representative western blots of BNIP3L and FUNDC1 protein levels after Bnip3l or Fundc1 siRNA co-transfection. FUNDC1 protein increased upon Bnip3l knockdown, while BNIP3L protein increased upon Fundc1 knockdown. (b) Quantification of BNIP3L and FUNDC1 protein levels after siRNA knockdown (n = 3). (c) Real-time PCR analysis of Bnip3l and Fundc1 transcript levels (n = 3). Representative western blots of BNIP3L and FUNDC1 after Bnip3l+Fundc1 knockdown (n = 4). (d) Representative fluorescence images of CPCs overexpressing COX8-EGFP-mCherry. Cells were transfected with siRNA against Bnip3l, Fundc1, or Bnip3l+Fundc1, and incubated in DM for 3 d. Scale bar: 20 μm. (e) Quantification of acidic (red-only) mitochondria in CPCs (n = 3). (f) Representative western blots of TIMM23 after siRNA knockdown of Bnip3l+Fundc1 and treatment with 25 µM FCCP for 24 h. (g) Quantification of TIMM23:ACTA1 in CPCs (n = 3). Data are mean ± SEM; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n.s., not significant.
Figure 6.
Figure 6.
Mitophagy is dispensable for lineage commitment and mitochondrial biogenesis in CPCs. (a) Real-time PCR analysis of Gata4, Gata6, and Mef2c transcripts in CPCs (n = 4). (b) Real-time PCR analysis of Ppargc1a transcript levels in CPCs (n = 4). (c) Representative western blots of mitochondrial OXPHOS subunit proteins ATP5F1A, UQCRC2, MT-CO1, and SDHB in CPCs. (d) Quantitation of mitochondrial OXPHOS subunit protein levels (n = 4). (e) Real-time PCR analysis of secretome-related Ccl2, Il6, and Wnt5a transcripts (n = 3). (f) Representative images and quantitation of endothelial tube formation (complete circles) after incubation in conditioned CPC media for 14 h (n = 3). Data are mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; n.s., not significant.
Figure 7.
Figure 7.
Knockdown of mitophagy receptors leads to formation of a fragmented mitochondrial network. (a) Representative fluorescence images of mitochondrial network in CPCs before and after incubation in DM. Scale bar: 20 μm. (b) Quantification of CPCs with fragmented mitochondria (n = 3). (c) Representative electron microscopy images of mitochondria in CPCs before and after incubation in DM. Arrows point to donut-shaped mitochondria. Scale bar: 2 μm. (d) Representative western blots of DNM1L, MFN1 and MFN2. (e) Quantification of DNM1L (n = 5), MFN1 (n = 3) and MFN2 (n = 3) protein levels. (f) Representative western blots of p-DNM1L (Ser 616) and DNM1L. (g) Quantification of p-DNM1L (n = 3) and DNM1L (n = 3) protein levels. Data are mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; n.s. not significant.
Figure 8.
Figure 8.
Knockdown of mitophagy receptors leads to reduced mitochondrial function and increased susceptibility to cell death. (a) Representative images of TMRM fluorescence in CPCs. Scale bar: 20 μm. (b) Quantification of TMRM fluorescence (n = 3). (c) Mitochondrial respiration in CPCs at D7. Oxygen consumption rate (OCR) was normalized to cell number (n = 4). (d) Quantification of cell death in differentiating CPCs (D7). Cells were treated with 200 μM H2O2 for 8 h in serum-free DM in the presence or absence of 10 μM Mdivi-1 (n = 3). (e) Representative scans of mouse hearts for retention of GFP-positive CPCs. Hearts from animals injected with either control CPCs or Bnip3l and Fundc1 knockdown CPCs were immunostained with GFP to detect surviving CPCs at 5 d after infarction. Dil dye was utilized to track the injections through the heart. GFP-positive control CPCs were detected in the border zone of infarcted hearts (n = 2), while GFP-positive Bnip3l and Fundc1 knockdown CPCs were not present in the infarcted hearts (n = 5). Scale bar: 1 mm and 10 μm, respectively. Data are mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. n.s., not significant.
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