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Figure 4. SRF153(A3) mmRNA transfection into neonatal rat ventricular myocytes (NRVM) induced stem cell and replication gene activity. (A) Schematic diagram shows the synthetic mmRNA transfection protocol of NRVM. RNA was isolated from SRF153(A3) mmRNA plus Lipofectamine MessengerMAX treated and controlled Lipofectamine MessengerMAX only myocytes at time points that approximated the appearance of stem cell factors at 24 h, mitosis at 32 h, DNA packaging at 40 h, and the inhibition of cardiac specified genes at 40 h. (B) NRVM were transfected with SRF153(A3) mmRNA tipped at the 5’UTR end with or without beta-globin 5’UTR. SRF153(A3) was detected on a protein blot with an anti-SRF antibody. (C) NRVM were transfected with SRF153(A3) and, after 6 h, were washed with fresh media to remove excess RNA. Cellular RNA samples were collected at 12, 24, and 48 h, and the amount of SRF mRNA was evaluated by RT-PCR assay represented by closed circles and controls represented as open circles. (D) Protein blots of extracts from NRVM transfected with SRF153(A3) mmRNA assayed after 24 h revealed the appearance of NANOG by anti-NANOG staining. (E) NRVM transfected with SRF153(A3) mmRNA showed the significant induction of NANOG and OCT4 but not KLF4 transcripts by quantitative PCR compared to the control group, as determined by two-tailed statistical analysis in which *P < 0.05. (F) NRVM transfected with SRF153(A3) mmRNA showed immuno-staining with anti-NANOG was co-stained and merged with DAPI in comparison to control myocytes (lines are 10 µm in length). (G) Stem cell factors, NANOG, and OCT4 were upregulated in the SRF153(A3) mRNA treatment group within 24 h post-transfection assayed by RNA seq (Illumina) at 24 h post-transfection. (H) Increased expression of mitotic genes such as Bub1, Bub1b, Cenpe, Ndc80, CcnB1, and Dync1 was observed by 32 h post-SRF153(A3) treatment. (I) The appearance of DNA packaging genes, which mark the S phase of the cell cycle, occurred 40 h post-SRF153(A3) treatment, including Histone 1 genes such as Hist1h1a, Hist1h1b, and Hist1h2ba and the Histone 3 gene, Hist3h2ba. Other critical DNA packaging factor genes included, Npm1, Rbb4, ANP32B, Nap1|1, Hells, and SET. (J) At the height of the S phase by 40 h post-SRF153(A3) treatment, the inhibition of cardiac-specific gene expression was well on its way, including contractile proteins ACTC1, ACTN2A, and MYH6; transcription factors MYOCD and MEF2C; kinases AKAP12, MYLK3, AND GSK3B; and signaling factor VEGFA.