Accumulation of Mitochondrial DNA Mutations Disrupts Cardiac Progenitor Cell Function and Reduces Survival

Abstract

Transfer of cardiac progenitor cells (CPCs) improves cardiac function in heart failure patients. However, CPC function is reduced with age, limiting their regenerative potential. Aging is associated with numerous changes in cells including accumulation of mitochondrial DNA (mtDNA) mutations, but it is unknown how this impacts CPC function. Here, we demonstrate that acquisition of mtDNA mutations disrupts mitochondrial function, enhances mitophagy, and reduces the replicative and regenerative capacities of the CPCs. We show that activation of differentiation in CPCs is associated with expansion of the mitochondrial network and increased mitochondrial oxidative phosphorylation. Interestingly, mutant CPCs are deficient in mitochondrial respiration and rely on glycolysis for energy. In response to differentiation, these cells fail to activate mitochondrial respiration. This inability to meet the increased energy demand leads to activation of cell death. These findings demonstrate the consequences of accumulating mtDNA mutations and the importance of mtDNA integrity in CPC homeostasis and regenerative potential.

Keywords: aging; cardiac progenitor cells; glycolysis; heart failure; mitochondria; mitochondrial DNA (mtDNA); mitophagy; stem cells.

FIGURE 1.

Activation of the differentiation program is associated with induction of mitochondrial biogenesis and expansion of the mitochondrial network in CPCs. A, mitochondrial content increased in CPCs after 7 days of incubation in DM. The CPCs with increased mitochondrial content were also positive for the cardiac myocyte lineage marker GATA-4. Scale bar = 20 μm. B, quantitation of GATA-4 positive CPCs at day 0 and day 7 (n = 4). C, real-time qPCR analysis of GATA-4 (n = 6), GATA-6 (n = 3), and PECAM1 (n = 4) gene expression at day 0 and day 7. D, phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) in CPCs after incubation in DM (n = 3). E, real-time qPCR analysis of the mitochondrial biogenesis regulator PGC-1α (n = 4) gene expression at day 0 and day 7. F, real-time qPCR of mtDNA content at day 0 and day 7 (n = 8). Data are normalized to genomic DNA. G, representative Western blot of mitochondrial OXPHOS proteins and quantitation of bands (n = 6). *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus day 0.

FIGURE 2.

No differences in cardiac and mitochondrial function in WT and POLG mice at 2 months. A, average body weight, heart weight/tibia length (HW/TL), and lung weight/tibia length (LW/TL) ratios were measured in age-matched WT and POLG litter mates (n = 10). Echocardiography measurements at 2 months of age show no significant differences in % fractional shortening (% FS) (B) and % ejection fraction (% EF), left ventricular internal dimension at diastole (LVID; d), and left ventricular internal dimension at systole (LVID; s) (n = 7) (C). D, representative Western blot of mitochondrial biogenesis and OXPHOS proteins in the heart at 2 months. E, mitochondria were isolated from the hearts of 2-month-old mice. Substrates and inhibitors specific to each respiratory complex were added, and oxygen consumption was measured using an oxygen electrode (n = 3). F, respiratory control ratio (RCR) was calculated by dividing state 3/state 4 oxygen consumption. G, transmission electron micrographs show normal mitochondrial ultrastructure in the hearts of WT and POLG mice. Scale bar = 1 μm.

FIGURE 3.

Mutant CPCs exhibit reduced proliferation and increased sensitivity to stress. A, WT and POLG CPCs were incubated in growth medium for up to 3 days, and proliferation was determined by assessing changes in cell number (n = 4) or by CyQUANT fluorescence (n = 9). WT and POLG CPCs were treated with H₂O₂ (B) and doxorubicin or sunitinib (C) for 24 h. Cell death was assessed using YO-PRO-1 staining (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 4.

Mutant CPCs have abnormal mitochondrial morphology and reduced activation of the differentiation program. A, the mitochondrial network in POLG CPCs has a fragmented appearance at both day 0 and day 7. Scale bar = 20 μm. Quantitation of fragmented mitochondria (n = 3) (B) and GATA-4-positive CPCs (n = 3) (C). D, real-time qPCR analysis of GATA-4 gene expression at day 0 and day 7 in WT and POLG CPCs (n = 4). E, representative Western blots and band quantitation of DRP1 (n = 4), MFN1 (n = 5), and MFN2 (n = 4) in WT and POLG CPCs. F, transmission electron micrographs of WT and POLG CPCs under baseline conditions and after incubation in DM. CPCs contain immature mitochondria. Mitochondrial electron density increased in WT CPCs after 7 days in DM. POLG CPCs had abnormal mitochondrial structure and reduced cristae. Scale bar = 500 nm. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant.

FIGURE 5.

POLG myoblasts generate smaller skeletal muscle fibers. A, WT and POLG myoblasts have similar morphology pre-differentiation (Pre-diff). After 4 days of differentiation, POLG myotubes had significantly smaller diameter than WT (n = 4). Scale bar = 100 μm. B, both WT and POLG myoblasts express phenotypic muscle marker MyoD pre- and post-differentiation and NCAM post-differentiation. Scale bar = 20 μm. C, Western blot of MyoD protein levels pre- and post-differentiation. **, p < 0.01 versus WT.

FIGURE 6.

Mutant CPCs have reduced levels of both nuclear and mitochondrial encoded OXPHOS proteins and reduced activation of mitochondrial biogenesis. A, representative Western blots revealed reduced levels of selective OXPHOS subunits in POLG CPCs. B, quantitation of mitochondria-encoded complex IV Subunit 1 (n = 5) and nuclear-encoded subunit 4 protein expression (n = 5). C, real-time qPCR analysis of Cox1 (complex IV Subunit 1) and Cox4i1 (complex IV subunit 4) gene expression at baseline (n = 3). D, real-time qPCR analysis of PGC-1α (n = 3) and PGC-1β (n = 3) gene expression at day 0 and day 7 in WT and POLG CPCs. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant.

FIGURE 7.

Mutant CPCs have impaired mitochondrial respiration and reduced membrane potential. A, mitochondrial respiration was measured in WT and POLG CPCs using a Seahorse XF analyzer. Maximal oxygen consumption rate (OCR) was normalized to cell number (n = 4). Cell seeding density: 10K = 10,000; 20K = 20,000. FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. B, representative fluorescent images and quantitation of tetramethylrhodamine methyl ester (TMRM) fluorescence in WT and POLG CPCs (n = 3). Scale bar = 25 μm. Units are arbitrary. C, analysis of cellular ATP content in WT and POLG CPCs (n = 3). D, l-lactate present in growth medium was measured in WT and POLG CPCs (n = 3). E, 2-deoxyglucose (10 nm) was added to the growth medium to inhibit glycolysis in WT and POLG CPCs. Cell death was assessed using YO-PRO-1 staining (n = 3). **, p < 0.01; ***, p < 0.001 versus WT; n.s., not significant.

FIGURE 8.

Activation of differentiation leads to down-regulation of cytosolic glycolytic enzymes in both WT and POLG CPCs. A, WT and POLG CPCs were incubated in differentiation medium, and cell death was assessed using YO-PRO-1 staining 4 days later (n = 3). B, representative Western blots of glycolytic enzymes after incubation in DM. C, quantitation of hexokinase I, hexokinase II, PKM1/2, and GAPDH at different time points after differentiation (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 9.

Mutant CPCs have normal autophagic flux but increased mitophagy. A, representative Western blots show similar levels of LC3II protein in both WT and POLG CPCs after treatment with 50 nm bafilomycin A1 (Baf) for 2 h. B, quantitation of LC3II protein levels (n = 3). Representative fluorescent images (C) and quantitation of LAMP2 staining in WT and POLG CPCs (n = 3) (D) are shown. Scale bar = 20 μm. A.U., arbitrary units. Representative fluorescent images (E) and quantitation of GFP-LC3 and Tom20 colocalization per cell in WT and POLG CPCs (n = 3) (F) are shown. Cells were treated with DMSO or 50 nm bafilomycin A1 for 3 h before fixation. Scale bar = 20 μm. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant.