Fig 2: Increase in intracellular glucose promotes cardiac regeneration in Glut1 transgenic heart. (a) Heart weight-body weight (HW/BW) ratio of Wild type and Glut1 transgenic (Glut1 tg) mice without injury. n = 3–7 for each group, p = n.s. (b) Representative images of hearts. (c) Heart weight-body weight (HW/BW) ratio of Wild type and Glut1 transgenic mouse post-injury. The hearts were cryoinjured at P1 and examined at P7, 14, 21 and 40. Note that the HW/BW is higher in Glut1 transgenic hearts at P7 and 14. n = 4–9 for each group, *p < 0.05. (d) Images of representative hearts post-injury. Note the ballooning of the heart at P14 in both wild type and Glut1 transgenic hearts. (e) %Fibrotic area measured by Image J capture of Picrosirius red stainings of the hearts. Hearts were cryoinjured at P1 and examined at P7, 14, 21 and 40. n = 5–8 for each group, *p < 0.05. (f) Representative images of Picrosirius red staining of wild type and Glut1 transgenic hearts. Arrowheads indicate fibrotic area. Scale bar = 200 µm. (g) PCNA staining of the sections from wild type and Glut1 transgenic hearts 7 days post-injury. Sections were stained with a cardiac marker (Tnnt2; Red), proliferation marker (PCNA; Green) and a nuclear marker (DAPI; Blue). Note that PCNA staining is more abundant in Glut1 transgenic heart. Arrowheads indicate PCNA positive cardiomyocytes. Scale bar = 50 µm and 20 µm respectively.

Fig 3: Tnnt2low cardiomyocytes are mitotically activated by the increase in intracellular glucose. (a) Representative flow cytometry profile of the ventricular cardiomyocytes from P7 Wild type and Glut1 transgenic hearts stained with Tnnt2 and Mitotracker. (b) Representative flow cytometry plot of EdU incorporation assay of Tnnt2low cardiomyocytes. Wild type and Glut1 transgenic mice were injected with EdU and the ventricular cardiomyocytes were isolated and analyzed by Tnnt2, Mitotracker, and EdU staining. (c) Mitotic index of Tnnt2high and Tnnt2low cardiomyocytes in Wild type and Glut1 transgenic heart with or without injury. n = 3–8. *p < 0.05. ***p < 0.001. Note that Tnn2low cardiomyocytes are more mitotic than Tnn2high cardiomyocytes and that the mitotic activity of Tnnt2low cardiomyocytes drastically increases in Glut1 transgenic background.

Fig 4: mRNA expression profile of Tnnt2high and Tnnt2low cardiomyocytes from Wild type and Glut1 transgenic hearts at P1. (a) Representative dot plot of flow cytometry analysis of P1 Wild type hearts for Tnnt2 (cardiac marker) and Mitotracker (mitochondrial contents). Note two populations of cardiomyocytes. (b) Flow cytometry analysis of P1 aMHC-Cre tg; R26+/YFP reporter and aMHC-hGLUT1 tg; aMHC-Cre tg; R26+/YFP reporter analysis for YFP (cardiac lineage) and Mitotracker (mitochondrial contents). Note two populations of cardiomyocytes. (c) Venn diagram of mRNA expressed in Tnnt2high and Tnnt2low cardiomyocytes from Wild type and Glut1 transgenic hearts. Bar graphs represent top three gene ontology (GO) term enriched in the genes uniquely expressed in each population. Although 4 populations are similar, G1 Tnnt2low cardiomyocytes are enriched for the genes associated with mitosis, cell cycle, and cell division. (d) GO analysis of the genes upregulated in Glut1 tg Tnnt2low vs Glut1 tg Tnnt2high. (e) Heatmap of expression level of representative cardiac, cell cycle, and mitochondrial genes differentially expressed in Glut1 tg Tnnt2low vs Glut1 tg Tnnt2high.