Metabolic maturation of differentiating cardiosphere-derived cells

Abstract

Cardiosphere-derived cells (CDCs) can be expanded in vitro and induced to differentiate along the cardiac lineage. To recapitulate the phenotype of an adult cardiomyocyte, differentiating progenitors need to upregulate mitochondrial glucose and fatty acid oxidation. Here we cultured and differentiated CDCs using protocols aimed to maintain stemness or to promote differentiation, including triggering fatty acid oxidation using an agonist of peroxisome proliferator-activated receptor alpha (PPARα). Metabolic changes were characterised in undifferentiated CDCs and during differentiation towards a cardiac phenotype. CDCs from rat atria were expanded on fibronectin or collagen IV via cardiosphere formation. Differentiation was assessed using flow cytometry and qPCR and substrate metabolism was quantified using radiolabelled substrates. Collagen IV promoted proliferation of CDCs whereas fibronectin primed cells for differentiation towards a cardiac phenotype. In both populations, treatment with 5-Azacytidine induced a switch towards oxidative metabolism, as shown by changes in gene expression, decreased glycolytic flux and increased oxidation of glucose and palmitate. Addition of a PPARα agonist during differentiation increased both glucose and fatty acid oxidation and expression of cardiac genes. We conclude that oxidative metabolism and cell differentiation act in partnership with increases in one driving an increase in the other.

Keywords: Cardiac progenitor cells; Cardiomyogenic differentiation; Extracellular matrix; Glycolytic metabolism; Oxidative phosphorylation.

Fig. 1

Characterization of cardiosphere-derived cells. (A) Yield of explant-derived cells and CDCs at P1 and P2 for cells cultured on FN/PDL or ColIV/HD (n = 6). (B) Proliferation of control P2-CDCs on FN or ColIV (n = 3). Data are presented as mean ± SEM and assessed using a two-way ANOVA with Tukey post hoc test ** p < 0.01, *** p < 0.001 compared with P2-CDCs on ColIV for (A) and with P2-CDCs on ColIV at the same time point for (B). (C) Representative flow cytometry dot plots and quantification of flow cytometry analysis of P2-CDCs for CD90 Data are presented as mean ± SEM (n = 3) and assessed using an unpaired t-test. **** p < 0.0001.

Fig. 2

Cardiomyogenic differentiation of passage-2 cardiosphere-derived cells. (A) Timeline of the differentiation procedure showing days of addition of 5 µM 5-Aza (yellow arrows) or 0.1 µM ascorbic acid (green arrows) to the differentiation medium, which was refreshed every 2–3 days. (B) Representative flow cytometry dot plots and (C) quantification of flow cytometry analysis of cells expressing CD90, cTnnT2, cMHC and titin in P2-CDCs differentiated on CollIV or FN. Data are presented as mean ± SEM (n = 3) and assessed using an unpaired t-test. * p < 0.05; ** p < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Expression of metabolic-related genes in control and 5-Aza treated CDCs. The relative mRNA expression of genes involved in glucose (GLUT1, GLUT4, PDK1 and PDK4) and fatty acid (CD36, CPT1B, PPARα, MCAT and ACADM) metabolism was assessed using qPCR in control and differentiated P2-CDCs created through FN/PDL or ColIV/HD. The mRNA expression was normalized to the geometric mean of Actb and β2M as reference genes and ColIV control as calibrator. Data are presented as mean ± SEM (n = 3), assessed using an ANOVA with Tukey post hoc test. Multicolour stars indicate multiple comparisons between groups and FN and ColIV control, black stars indicate comparison between control and differentiated cells. *p < 0.05; **p < 0.01; ***p < 0.001; **** p < 0.0001.

Fig. 4

Substrate utilisation by control and 5-Aza treated CDCs. (A) The rate of glycolysis and of oxidation of glucose, palmitate and acetoacetate measured in control and differentiated P2-CDCs created through FN/PDL or ColIV/HD. (B) Calculated rates of adenosine triphosphate (ATP) production in control and differentiated P2-CDCs. Data are presented as mean ± SEM (n = 3–5), assessed using an ANOVA with Tukey post hoc test. Multicolour stars indicate multiple comparisons between groups and FN and ColIV control, black stars indicate comparison between control and differentiated cells for A and as indicated for B; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Fig. 5

Differentiated and WY-14643 treated cells form organoids. (A) CDCs, expanded on ColIV were plated on FN and differentiated by treatment with 5-Azacytidine (5-Aza) for 3 days followed by addition of ascorbic acid (AA) or a combination of AA/TGFβ/WY-14643 as indicated for 30 days. (B) Representative micrographs of WY-14643-treated differentiating CDCs which started to aggregate on day 10 and formed organoid/tubular structures on day 20. Scale bar 100 µm (C) Z-stack confocal images of 3D organoids showing expression of MHC, Titin, cTnnT2, MLC, CX43 as indicated, scale bar = 300 µm, unmerged images are shown in Figure S11. (D) The relative mRNA expression of cardiac genes was assessed using qPCR. The mRNA expression was normalized to the geometric mean of Actb and β2M as reference genes and baseline as calibrator; Green stars indicate comparison between baseline and differentiated cells and black stars indicate comparison between differentiated cell groups; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Metabolic changes after differentiation with TGFβ and WY-14643. (A) The relative mRNA expression of genes involved in glucose (GLUT1, GLUT4, PDK1 and PDK4) and fatty acid (CD36, CPT1B, PPARα, MCAT and ACADM) metabolism was assessed using qPCR in control, and TGFβ differentiated CDCs with and without treatment with WY-14643. The mRNA expression was normalized to the geometric mean of Actb and β2M as reference genes and baseline as calibrator. (B) Changes in rates of glycolysis and glucose and palmitate oxidation, expressed as a fold change over baseline. Data are presented as mean ± SEM (n = 3) and assessed using an ANOVA with Tukey post hoc test. Black stars indicate comparison between baseline and differentiated cells and blue stars indicate comparison with TGFβ differentiated cells; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. (C) CDCs differentiated using both TGFβ and WY-14643, stained with Mitotracker® Red CMXRos and showing formation of mitochondrial networks. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)