Differential mitochondrial bioenergetics and cellular resilience in astrocytes, hepatocytes, and fibroblasts from aging baboons

Abstract

Biological resilience, broadly defined as the ability to recover from an acute challenge and return to homeostasis, is of growing importance to the biology of aging. At the cellular level, there is variability across tissue types in resilience and these differences are likely to contribute to tissue aging rate disparities. However, there are challenges in addressing these cell-type differences at regional, tissue, and subject level. To address this question, we established primary cells from aged male and female baboons between 13.3 and 17.8 years spanning across different tissues, tissue regions, and cell types including (1) fibroblasts from skin and from the heart separated into the left ventricle (LV), right ventricle (RV), left atrium (LA), and right atrium (RA); (2) astrocytes from the prefrontal cortex and hippocampus; and (3) hepatocytes. Primary cells were characterized by their cell surface markers and their cellular respiration was assessed with Seahorse XFe96. Cellular resilience was assessed by modifying a live-cell imaging approach; we previously reported that monitors proliferation of dividing cells following response and recovery to oxidative (50 µM-H₂O₂), metabolic (1 mM-glucose), and proteostasis (0.1 µM-thapsigargin) stress. We noted significant differences even among similar cell types that are dependent on tissue source and the diversity in cellular response is stressor-specific. For example, astrocytes had a higher oxygen consumption rate and exhibited greater resilience to oxidative stress (OS) than both fibroblasts and hepatocytes. RV and RA fibroblasts were less resilient to OS compared with LV and LA, respectively. Skin fibroblasts were less impacted by proteostasis stress compared to astrocytes and cardiac fibroblasts. Future studies will test the functional relationship of these outcomes to the age and developmental status of donors as potential predictive markers.

Keywords: Astrocytes; Baboons; Bioenergetics; Fibroblasts; Hepatocytes; Resilience.

Fig. 1

Representative photomicrographs of baboon astrocyte, fibroblast, and hepatocyte cultures. A Astrocytes derived from the prefrontal cortex and hippocampus show immunoreactivity for astrocyte markers: aquaporin 4, glial fibrillary acidic protein (GFAP), and excitatory amino acid transporter 2 (EAAT2). The absence of immunoreactive signals for myelin basic protein and postsynaptic density protein 95 (PSD95) indicates that astrocyte cultures do not contain oligodendrocytes and neurons, respectively. B Expression of fibroblast marker, vimentin in fibroblasts from different heart regions such as the left ventricle, right ventricle, left atrium, and right atrium, and skin epidermal fibroblasts express fibroblast marker, vimentin. C Phase contrast microscopy of hepatocyte cultures and immunoreactive signals of epithelial cell marker, cytokeratin 8. Scale 20 µm. Images were acquired with a Zeiss LSM 880 confocal microscope using a 63 × oil immersion lens. Donor animal: male, 16.4 years old

Fig. 2

Oxygen consumption rate in baboon astrocytes, fibroblasts, and hepatocytes. Oxygen consumption rate (OCR) was measured under basal condition and in response to serial injection of mitochondrial inhibitors including Oligomycin (Oligo; 1.5 µM), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP; 0.5 µM), and rotenone and antimycin A cocktail (Rot/AA; 0.5 µM). OCR kinetics in A astrocytes derived from the Broca’s area of the prefrontal cortex (FC), B astrocytes from the hippocampus, C fibroblasts from the left ventricle of the heart, D skin fibroblasts, and E hepatocytes under standard culture condition and in response to low glucose (1 mM glucose for 2 h). F Time-course OCR of cortical astrocytes, hippocampal astrocytes, LV fibroblasts, skin fibroblasts, and hepatocytes under standard culture conditions. G Basal respiration, H ATP-linked respiration, and I maximal respiration in astrocytes derived from the FC and hippocampus (hipp) as well as left ventricle fibroblasts (LV), skin fibroblasts (skin), and hepatocytes (liver). Data from individual animal were expressed as mean ± standard error of mean, with male and female data combined. Data from left and right lobe hepatocytes were pooled for comparison with other cell types. For each primary cell line, respiration rates were from 4 to 6 replicate samples measured using the Seahorse XFe96 flux analyzer. Donor age, 13.3–17.8 years, sample size (cortical astrocytes, n = 10; hippocampal astrocytes, n = 10; LV and skin fibroblasts, n = 6, respectively; hepatocytes, n = 7). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 3

Effects of oxidative, metabolic, and proteostasis stress on proliferation of baboon astrocytes and fibroblasts. Astrocytes derived from prefrontal cortex Broca’s area 10 (FC) or the entire hippocampus (hipp) encompassing anterior, middle, and posterior hippocampus and fibroblasts from the left ventricle (LV), right ventricle (RV), and ear skin (skin) were exposed to 50 µM H₂O₂, 1 mm glucose, and 0.1 µM thapsigargin for 2 h to model oxidative, metabolic, and proteostasis stress, respectively. Time-course changes in cell confluence in response to challenge were monitored in real time with the IncuCyte live-cell imaging system housed within a cell culture incubator (3% O₂, 5% CO₂ at 37 °C). The blue line represents untreated cells (designated as control); the red line, 50 µM H₂O₂; the green, 1 mM glucose; and the purple, 0.1 µM thapsigargin. Proliferation rate (% confluence/h) calculated from the slope of the kinetic graph between 30 and 144 h was shown in response to untreated and challenged cells. Proliferation rate was analyzed using two-way ANOVA. Data expressed as mean ± standard error of mean; each data point represents 3 replicate wells for each animal; cell seeding density was 2000 cells/well; donor age was between 13.3 and 17.8 years, with male and female data combined. Sample size: FC, n = 8; hipp, n = 10; LV, n = 8; RV, n = 5; skin, n = 7. *p < 0.05, **p < 0.01, ****p < 0.0001

Fig. 4

Correlation between astrocyte maximal respiration and proliferation response to oxidative stress. The maximal oxygen consumption rates (OCR) in baboon astrocytes, induced by the mitochondrial uncoupler FCCP, show a positive correlation with their proliferation response to 50 µM H₂O₂. OCR was determined with the Seahorse XFe96 flux analyzer. Cellular proliferation was monitored with a live-cell imaging system (IncuCyte) with the proliferation rate calculated from the slope of cell kinetics over 5-day period. The correlation between maximal respiration and proliferation rate in response to H₂O₂ was determined using Pearson’s correlation coefficient. Data are from primary astrocytes obtained from the baboon prefrontal cortex and hippocampus encompassing both male and female animals, with ages ranging from 13.3 to 17.8 years (n = 17)

Fig. 5

Oxygen consumption rate of baboon cardiac fibroblasts. Time-course graph of oxygen consumption rates (OCR) measured with a Seahorse XFe96 flux analyzer using baboon cardiac fibroblasts derived from A left ventricle, LV; B right ventricle, RV; C left atrium, LA; and D right atrium, RA, under standard culture condition (designated as control) and in response to low glucose media (1 mM glucose). E Basal respiration, F ATP-linked respiration, and G maximal respiration were derived from the kinetic graph following initial measurement, in the presence of oligomycin and in response to carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), respectively. Data, expressed as mean ± standard error of mean, are from individual animal, with male and female data combined. For each primary cell line, respiration rates from 4 to 6 replicate samples were measured using the Seahorse XFe96 flux analyzer. Donor age, 13.3–17.8 years, n = 5–6 per cardiac fibroblasts from different heart regions

Fig. 6

Effects of oxidative, metabolic, and proteostasis stress on proliferation of cardiac fibroblasts from different heart regions. Fibroblasts derived from the four chambers of the baboon heart encompassing the left ventricle (LV), right ventricle (RV), left atrium (LA), and right atrium (RA) were exposed to 50 µM H₂O₂, 1 mm glucose, and 0.1 µM thapsigargin for 2 h to model oxidative, metabolic, and proteostasis stress, respectively. Time-course changes in cell confluence of cardiac fibroblasts in response to challenge compounds were monitored in real time with the IncuCyte live-cell imaging system housed within a cell culture incubator (3% O₂, 5% CO₂ at 37 °C). The blue line represents untreated cells (designated as control); red line, 50 µM H₂O₂; green, 1 mM glucose; and purple, 0.1 µM thapsigargin. Proliferation rate (% confluence/h) calculated from the slope of the kinetic graph between 30 and 144 h was shown in response to untreated and challenged cells. Proliferation rate was analyzed using two-way ANOVA. Data expressed as mean ± standard error of mean; each data point represents 3 replicate wells for each animal; cell seeding density was 2000 cells/well; donor age was between 13.3 and 17.8 years, male and female data combined; n = 8 for LV, 5 for RV, 5 for LA and 7 for RA. ***p < 0.001, ****p < 0.0001