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. 2020 Apr 15:8:209.
doi: 10.3389/fcell.2020.00209. eCollection 2020.

The Small GTPases Rab27b Regulates Mitochondrial Fatty Acid Oxidative Metabolism of Cardiac Mesenchymal Stem Cells

Yue Jin  1 , Yan Shen  1 , Xuan Su  1 , Jingwen Cai  2 , Yutao Liu  2 , Neal L Weintraub  1 , Yaoliang Tang  1
Affiliations

The Small GTPases Rab27b Regulates Mitochondrial Fatty Acid Oxidative Metabolism of Cardiac Mesenchymal Stem Cells

Yue Jin et al. Front Cell Dev Biol. .

Abstract

Cardiac mesenchymal stem cells (C-MSCs) are endogenous cardiac stromal cells that play a crucial role in maintaining normal cardiac function. Rab27b is a member of the small GTPase Rab family that controls membrane trafficking and the secretion of exosomes. However, its role in regulating energy metabolism in C-MSC is unclear. In this study, we analyzed mitochondrial oxidative phosphorylation by quantifying cellular oxygen consumption rate (OCR) and quantified the extracellular acidification rate (ECAR) in C-MSC with/without Rab27b knockdown. Knockdown of Rab27b increased glycolysis, but significantly reduced mitochondrial oxidative phosphorylation (OXPHOS) with loss of mitochondrial membrane potential in C-MSC. Furthermore, knockdown of Rab27b reduced H3k4me3 expression in C-MSC and selectively decreased the expression of the essential genes involved in β-oxidation, tricarboxylic acid cycle (TCA), and electron transport chain (ETC). Taken together, our findings highlight a novel role for Rab27b in maintaining fatty acid oxidation in C-MSCs.

Keywords: Rab27b; cardiac mesenchymal stem cells; exosome; fatty acid oxidation; mitochondrial oxidative metabolism.

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Figures

FIGURE 1
FIGURE 1
Phenotypic characterization of C-MSCs. (A) Cultured C-MSCs at passage 10, scale bar = 1000 μm. (B) Immunofluorescent staining of GATA4, a marker for early cardiac transcription factor (red); cell nuclei were counterstained with DAPI (blue) (scale bar = 20 μm). (C) Flow cytometric analyses of C-MSCs for the profile of the cell surface markers CD105 and CD140b.
FIGURE 2
FIGURE 2
Knockdown of Rab27b in C-MSCs via lentiviral shRNAs vector transduction. (A) qRT-PCR analysis of Rab27b in C-MSCs cells infected with lentiviral vectors encoding shRNA targeting Rab27b mRNA (sh-Rab27b-1, sh-Rab27b-2, sh-Rab27b-3 and sh-Rab27b-4) or negative control (sh-NC). β-Actin was used as endogenous control. Results are shown as mean ± SD (*p < 0.05 vs. sh-NC, n = 3). (B) qRT-PCR analysis of Rab27a and Rab27b in C-MSCs infected with sh-Rab27b1 + 2 (lentiviral vectors encoding sh-Rab27b1 and lentiviral vectors encoding sh-Rab27b2) or negative control (sh-NC). GAPDH was used as endogenous control. Results are shown as mean ± SD (****p < 0.0001 vs. sh-NC, n = 3). (C) Western blotting of Rab27b and Rab27a protein using β-actin as a loading control. (D) Immunofluorescent staining of Rab27b and Rab27a (red) in C-MSCsh–NC and C-MSCsh–Rab27b1+2; cell nuclei were counterstained with DAPI (blue) (scale bar = 20 μm).
FIGURE 3
FIGURE 3
Characterization of exosomes derived from C-MSCsh–NC and C-MSCsh–Rab27b1+2. Particle concentration and size distribution in purified particles are consistent with the size range of exosomes (average size, 140–150 nm), as measured by ZetaView® Particle Tracking Analyzer.
FIGURE 4
FIGURE 4
Assessment of oxygen consumption rate (OCR) in C-MSCsh–NC and C-MSCsh–Rab27b. (A) Cells were exposed sequentially to oligomycin, FCCP, and rotenone/antimycin A. Vertical lines indicate time of addition of mitochondrial inhibitors. Oxygen consumption rate was measured over time using a Seahorse XFe96 Analyzer. Cell mito stress test was performed according to manufacturer’s protocol, results are normalized to total cellular protein (n = 7–8). (B) C-MSCsh–NC were seeded in media with Exo-depleted FBS overnight, and primed cells with 25-μg exosomes from wild-type C-MSC for 1 h before OCR assay using a Seahorse XF24 analyzer (n = 5).(C) C-MSCsh–Rab27b were seeded in media with Exo-depleted FBS overnight, and primed cells with 25-μg exosomes from wild-type C-MSC for 1 h before OCR assay using a Seahorse XF24 analyzer (n = 5). Results are presented as mean ± SD (*p < 0.05; **p < 0.01; ****p < 0.0001).
FIGURE 5
FIGURE 5
Assessment of extracellular acidification rate (ECAR) in C-MSCsh–NC and C-MSCsh–Rab27b. Cells were exposed sequentially to 0.5 or 1 μM rotenone/antimycin A and 2-DG. Extracellular acidification rate (ECAR) was measured over time using a Seahorse XFe24 Analyzer. Measurements of basal glycolysis,% PER from glycolysis and compensatory glycolysis were calculated from the ECAR data. Results are presented as mean ± SD (*p < 0.05; **p < 0.01; ***p < 0.001, n = 5).
FIGURE 6
FIGURE 6
(A) qRT-PCR analysis of genes related to mitochondrial fatty acid oxidation; TCA, tricarboxylic acid cycle; ETC, electron transport chain. The amount of mRNA was normalized using GAPDH. (n = 3–4). (B) Western blotting of H3k4me3 protein using TBP as a loading control (n = 3). (C) Mitochondrial membrane potential was measured by JC-1 staining and the images were obtained by fluorescent microscopy. Scale bars = 100 μm. The deltapsim is expressed as the ratio of red fluorescence to green fluorescence (n = 5–6). Results are shown as mean ± SD (NS, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
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