Novel thiazolidinedione mitoNEET ligand-1 acutely improves cardiac stem cell survival under oxidative stress

Abstract

Ischemic heart disease (IHD) is a leading cause of death worldwide, and regenerative therapies through exogenous stem cell delivery hold promising potential. One limitation of such therapies is the vulnerability of stem cells to the oxidative environment associated with IHD. Accordingly, manipulation of stem cell mitochondrial metabolism may be an effective strategy to improve survival of stem cells under oxidative stress. MitoNEET is a redox-sensitive, mitochondrial target of thiazolidinediones (TZDs), and influences cellular oxidative capacity. Pharmacological targeting of mitoNEET with the novel TZD, mitoNEET Ligand-1 (NL-1), improved cardiac stem cell (CSC) survival compared to vehicle (0.1% DMSO) during in vitro oxidative stress (H2O2). 10 μM NL-1 also reduced CSC maximal oxygen consumption rate (OCR) compared to vehicle. Following treatment with dexamethasone, CSC maximal OCR increased compared to baseline, but NL-1 prevented this effect. Smooth muscle α-actin expression increased significantly in CSC following differentiation compared to baseline, irrespective of NL-1 treatment. When CSCs were treated with glucose oxidase for 7 days, NL-1 significantly improved cell survival compared to vehicle (trypan blue exclusion). NL-1 treatment of cells isolated from mitoNEET knockout mice did not increase CSC survival with H2O2 treatment. Following intramyocardial injection of CSCs into Zucker obese fatty rats, NL-1 significantly improved CSC survival after 24 h, but not after 10 days. These data suggest that pharmacological targeting of mitoNEET with TZDs may acutely protect stem cells following transplantation into an oxidative environment. Continued treatment or manipulation of mitochondrial metabolism may be necessary to produce long-term benefits related to stem cell therapies.

Fig. 1

NL-1 treatment increases CSC survival under oxidative stress. a Morphology of CSC under phase microscopy. b CSC immediately following 4-h treatment with 500 μM H₂O₂. c CSC immediately following 4-h treatment with 500 μM H₂O₂ + 10 μM NL-1. d, e Represent CSC survival determined by an automated cell counter with trypan blue exclusion (d) and flow cytometry with propidium iodide exclusion (e). Relative cell survival was normalized to non-treated CSC cultured in parallel with treated groups. *p < 0.05, **p < 0.01 vs. H₂O₂. ^#p < 0.01, ^δp < 0.05 vs. H₂O₂ + vehicle. a-d N = 6, e N = 3

Fig. 2

NL-1 reduces maximal oxygen consumption rate of CSCs. a Absolute oxygen consumption rate (OCR) of CSCs with or without NL-1 treatment represented as pmol O₂/min/mg protein. b OCR of under same conditions as A represented as percent baseline. Baseline is set at 100 %, and is defined as the OCR (or ECAR) at the time point immediately preceding oligomycin administration. c Maximal OCR as measured during the first three time points following FCCP administration. d Extracellular acidification rate (ECAR) of CSCs with or without NL-1 treatment. e Maximal ECAR as measured during the first three time points following oligomycin administration. **p < 0.01 vs. both no treatment and vehicle. N = 3

Fig. 3

NL-1 reduces maximal ocr of differentiated CSCs. a Absolute oxygen consumption rate (OCR) of CSCs following a 7-day differentiation protocol under the specified treatment conditions, represented as pmol O₂/min/mg protein. b OCR and d extracellular acidification rate (ECAR) of CSC represented as percent baseline (baseline is defined as OCR or ECAR at time point immediately preceding FCCP or oligomycin administration, respectively). c Maximal OCR as measured during the first three time points after FCCP administration and represented as percent baseline. e Maximal ECAR as measured during the first three time points following oligomycin administration and represented as percent baseline. **p < 0.01 vs. undifferentiated. ^#p < 0.01 vs. both DM and DM + vehicle. *p < 0.05 vs. both DM and DM + vehicle. N = 4. DM dexamethasone-treated CSCs

Fig. 4

NL-1 treatment does not appear to influence CSC differentiation. a Quantitative PCR and b western blot analyses showing relative expression of SM α-actin normalized to undifferentiated CSCs. *p < 0.05, **p < 0.01, ***p < 0.001 vs. undifferentiated CSCs. ^#p < 0.01 vs. DM. N = 3. DM dexamethasone-treated CSCs

Fig. 5

NL-1 treatment increases CSC Survival during differentiation under chronic oxidative stress. a Cardiac stem cell (CSC) survival determined via automated cell counting (trypan blue exclusion) following 7 days' culture in differentiation medium [10⁻⁸ M dexamethasone (DM)] in the presence of 800 ng/mL of glucose oxidase. Data are presented relative to percent of viable cells receiving no glucose oxidase treatment. N = 3 separate experiments. *p < 0.05. b–g Phase-contrast microscopy of CSCs following 4 days of culture in differentiation medium under the following treatment conditions: b dexamethasone (DM) only (no glucose oxidase); c DM + glucose oxidase (GO) (800 ng/mL); d DM + DMSO (0.1 %) + GO; e DM + NL-1 (10 μM) + GO; f DM + rosiglitazone (10 μM) + GO; g DM + NL-1 + GW9662 (20 μM) + GO

Fig. 6

NL-1 treatment of mitoNEET knockout cells. Flow cytometric analysis of cell survival (propidium iodide exclusion) in cardiac cells from mitoNEET knockout or wild-type mice following treatment with 250 μM H₂O₂ and a 24-h recovery period. N = 3. *p < 0.05

Fig. 7

NL-1 treatment improves short-term, but not long-term CSC survival under in vivo oxidative stress. Real-time PCR analysis of relative CSC survival following injection into ZOF rat myocardia. Values were normalized to CSC injection without NL-1 treatment. N = 3 separate experiments. ****p < 0.0001