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. Author manuscript; available in PMC: 2021 Apr 14.
Published in final edited form as: Circulation. 2020 Feb 11;141(15):1249–1265. doi: 10.1161/CIRCULATIONAHA.119.043067

Figure 5. Pkm2 upregulates the anabolic enzyme G6pd, which reduces oxidative stress, ROS production and oxidative damage and leads to increased expression of cell cycle markers in postnatal CMs.

Figure 5.

A. qPCR comparisons of G6pd expression 2 days post MI and administration of cmsLuc or cmsPkm2 modRNAs with cmsihCD25, when adult CMs were isolated using magnetic beads (please see method in Suppl. Fig. 13). B. Experimental timeline used to evaluate reduced oxidative stress or ROS production post MI and delivery of cmsLuc or cmsPkm2 modRNA. C. HPLC quantification of GSH/GSSG ratio (n=6). D&E. HPLC quantification of superoxide probe dihydroethidium (DHE) in 2-hydroxyethidium (D. n=5, EOH) or ethidium (E. n=4, E) 2 days post MI and cmsLuc or cmsPkm2 modRNA injection or sham operation. F. Representative images of 8-OHG foci frequency in CMs 2 days post MI and transfection with either CMSLuc or CMSPkm2 modRNA (co-stained α-Actinin and DAPI). G. Quantitative analysis of F (n=3). H. Representative images of pATM foci frequency in CMs 2 days post MI and transfection with CMSLuc or CMSPkm2 modRNA. I. Quantitative analysis of H (n=3). J. Experimental plan for evaluating overexpression of G6pd alone or with Pkm2 modRNA and studying its effect on the expression of cell cycle genes in P3 neonatal rat CMs. K. Representative images of G6pd expression 1 day post Luc or G6pd modRNAs transfection in vitro (co-stained α-Actinin and DAPI). L&M. Quantification of cell cycle markers (Ki67, pH3) in neonatal rat CMs 3 days after transfection of Luc, Pkm2 or G6pd modRNA alone or combined Pkm2 and G6pd modRNAs (n=3). O. Experimental timeline used to evaluate the role of G6pd in CMs post Pkm2 modRNA delivery. P. Representative images of G6pd inhibition by siRNA in CMs post Pkm2 modRNA delivery. Q&R. Quantification of cell cycle markers (Ki67, pH3) in neonatal rat CMs 3 days after transfection with Luc or Pkm2 modRNA with scrambled siRNA or Pkm2 modRNA with siRNA for G6pd (n=3). One-way ANOVA, Tukey's Multiple Comparison Test for L&M, Q&R. Unpaired two-tailed t-test for A, C-E, G and I. ***, P<0.001, **, P<0.01, *, P<0.05, N.S, Not Significant. Scale bar = 5μm (D) and 25μm (G&K).