Mitofusin 1 and optic atrophy 1 shift metabolism to mitochondrial respiration during aging

Summary Replicative and chronological lifespan are two different modes of cellular aging. Chronological lifespan is defined as the duration during which quiescent normal cells retain their capacity to re‐enter the proliferative cycle. This study investigated whether changes in metabolism occur during aging of quiescent normal human fibroblasts (NHFs) and the mechanisms that regulate these changes. Bioenergetics measurements were taken in quiescent NHFs from younger (newborn, 3‐day, 5‐month, and 1‐year) and older (58‐, 61‐, 63‐, 68‐, and 70‐year) healthy donors as well as NHFs from the same individual at different ages (29, 36, and 46 years). Results show significant changes in cellular metabolism during aging of quiescent NHFs: Old NHFs exhibit a significant decrease in glycolytic flux and lactate levels, and increase in oxygen consumption rate (OCR) and ATP levels compared to young NHFs. Results from the Seahorse XF Cell Mito Stress Test show that old NHFs with a lower Bioenergetic Health Index (BHI) are more prone to oxidative stress compared to young NHFs with a higher BHI. The increase in OCR in old NHFs is associated with a shift in mitochondrial dynamics more toward fusion. Genetic knockdown of mitofusin 1 (MFN1) and optic atrophy 1 (OPA1) in old NHFs decreased OCR and shifted metabolism more toward glycolysis. Downregulation of MFN1 and OPA1 also suppressed the radiation‐induced increase in doubling time of NHFs. In summary, results show that a metabolic shift from glycolysis in young to mitochondrial respiration in old NHFs occurs during chronological lifespan, and MFN1 and OPA1 regulate this process.


Metabolic flux analysis
Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured using Seahorse XF96 Extracellular Flux Analyzer (Seahorse Bioscience). Contact inhibited quiescent cultures of NHFs were plated in Seahorse XF96 cell culture microplates at a density of 1.2 × 10 4 cells per well. Pre-warmed Seahorse XF assay media supplemented with 2 mM Glutamax pH 7.4 at 37 °C was used in the Glycolytic Stress Test and Seahorse XF assay media supplemented with 2 mM Glutamax, 1 mM sodium pyruvate, and 25 mM glucose pH 7.4 at 37 °C was used in the Cell Mito Stress test. The buffer capacity was determined before the experiment at 37 °C and pH 7.4 in both the Glycolysis Stress Test and the Cell Mito Stress Test (Divakaruni et al. 2014). Seahorse media addition were: glucose 10 mM, oligomycin 2.5 μM, 2-deoxyglucose (2-DG) 100 mM, carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) 0.5 μM, antimycin A 10 μM, and rotenone 10 μM final concentration. Cell number in individual wells at the end of the measurements was determined using a hemocytometer. ECAR is calculated as npH cell -1 s -1 (pH units x 10 -9 cell -1 s -1 ), and OCR results are presented as attomoles O2 cell -1 s -1 (abbreviation = amol O2 cell -1 s -1 ). Lactate levels were measured using a Lactate Colorimetric/Fluorometric assay kit (BioVision Technologies). A CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega) was used to measure cellular ATP content. Results are presented as femtomoles ATP per cell (fmol ATP cell -1 ).

Imaging and quantitative analysis of mitochondria dynamics
Quiescent monolayer cultures of NHFs were incubated with Hanks buffered salt solution (HBSS) containing 0.5 µM MitoTracker Green (Invitrogen), and fluorescence was visualized (488 nm excitation laser and 530 nm emission filter) by using a Zeiss 510 confocal microscope (The University of Iowa Central Microscopy Research Core Facility). Images for single cells were captured using the Z stack function of 12 layered images to compose a 3-dimensional image of the cells. Images were then processed and analyzed using Imaris Scientific 3D/4D Image Processing and Analysis Software (Bitplane Scientific Software). The Software creates a 3D image of the Z stack and then processes it to create a virtual model of the mitochondria network. The model was used to calculate the volume and number of compartments of the mitochondria network based on the connection and disconnection within the network. Results are plotted as total volume vs. compartment number ± SEM.

Senescence assay
Senescence-associated β-galactosidase Activity Assay (BioVision Technologies) was performed to measure cellular senescence. Senescence status was also examined by quantitative RT-PCR measurements of mRNA levels of cyclin dependent kinase inhibitors, p16 and p21.

PFK activity assay
PFK catalytic activity was measured using the Phophofructokinase Activity Colorimetric Assay Kit (BioVision Technologies). In presence of ATP, PFK converts fructose-6phosphate to fructose-diphosphate and ADP. ADP is then converted to AMP and NADH. Measurements of NADH at 450 nm represent PFK activity.

MnSOD and CuZnSOD activity assay
Ten micrograms of total protein extract prepared from quiescent cultures of young and old NHFs were separated by native polyacrylamide gel electrophoresis (Darby Weydert et al. 2003). Gels were stained with nitro blue tetrazolium (NBT) and riboflavin-TEMED solution for 30 min at room temperature. The bands were visualized and quantified with a computerized digital imaging system interfaced with AlphaImager 2000 software (Alpha Innotech.). Results are representative of two or more experiments.

Complex II activity assay
A cell-based assay was used to measure Complex II activity (Zhang et al. 2013). NHFs were air-dried and incubated with 0.55 mM NBT and 0.05 M disodium succinate at 37 °C overnight. Cells were stained with Hoechst to visualize nuclei. Olympus CKX41 microscope (Olympus) and ImageJ software (National Institutes of Health) were used to quantitate results. The integrated density of each image was normalized to the number of nuclei in that field. Two thousand nuclei in each treatment group were analyzed, and fold change in complex II activity was calculated relative to complex II activity of 3-d NHFs.

Cell survival assay
A clonogenic assay was used to measure cell survival. Lethally irradiated (30 Gy) V79 Chinese Hamster Fibroblasts were used as a feeder layer for the clonogenic assay. Single cell suspension of control and irradiated NHFs were plated on top of the feeder layer and cultured for 14 d followed by fixation in ethanol and staining with 0.8% Coomassie Brilliant Blue G-250 in 50% methanol and 20% acetic acid. Surviving fraction was calculated using the following formula: surviving fraction (SF) = (number of colonies counted) / (number of cells seeded × plating efficiency).

Statistical analysis
Statistical analyses were performed using GraphPad Prism software 6.0 (GraphPad Software Inc.). Statistical analysis was determined using the one-way analysis of variance (ANOVA) followed by Dunnett's post test; the Student's t test was used for experiments with less than three groups. Homogeneity of variance was assumed at 95% confidence interval. Results from at least three independent experiments with p < 0.05 were considered statistically significant.

Fig. S1. An age-related shift in metabolism from glycolysis in young to mitochondrial respiration in old NHFs is associated with: a decrease in lactate levels; an increase in ATP levels; an increase in cellular ROS levels; and a decrease in superoxide dismutase (SOD) and complex II activities. (A)
A colorimetric assay (BioVision) was used to measure lactate levels. (B) CellTiter-Glo kit (Promega) was used to measure ATP levels. (C) Flow cytometry measurements of dihydroethidium (DHE) oxidation was used to assess cellular reactive oxygen species (ROS) levels. Asterisks represent statistical significance compared to 3-d NHFs; n = 3, p < 0.05. (D) A native gel electrophoresis assay was used to measure activities of MnSOD and CuZnSOD: representative nitroblue tetrazolium (NBT) stained gels are shown on left and quantitation of results are shown in the right. Results from the 3-d, 5m, and 1-y NHFs were pooled as the Young group, and 58-y, 61-y, 63-y, 68-y, and 70-y NHFs were pooled as the old group. (E) A quantitative RT-PCR assay was used to measure mRNA levels of complex II subunits: succinate dehydrogenase A, B, C, and D. Asterisks represent statistical significance compared to mRNA levels of 3-d NHFs; n = 3, p < 0.05. (F) An immunoblotting assay was used to measure protein levels of complex II subunits. (G) A cell-based assay (Zhang et al. 2013) was used to measure complex II activity: representative microscopy images of cells showing complex II activity (purple color; magnification: x400) are shown on the left; cells with purple color were scored and normalized to the number of nuclei in each image using ImageJ software. A total of 2,000 nuclei in images from six different dishes were scored. Asterisks represent statistical significance compared to complex II activity of 3-d NHFs; n = 6, p < 0.05.