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. 2022 Oct 17;31(6):1688–1704. doi: 10.1016/j.ymthe.2022.10.005

Figure 4.

Figure 4

Transgenic expression of circSlc8a1 improved heart function against transverse aortic constriction-induced pressure overload

(A) The circSlc8a1 plasmids were transfected into HL-1 cells followed by RT-qPCR. circSlc8a1 was amplified by divergent and convergent primers in cDNA, and by convergent primers only in gDNA. (B) The circSlc8a1 plasmids were transfected into human HEK293T cells. The RNAs from vector or circSlc8a1-transfected cells were treated with RNase R or RNase A followed by reverse transcription. The expression of circSlc8a1 was validated by using mouse-specific primers against circSlc8a1. n = 4. (C) Representative images of circSlc8a1 expression by using random or oligo(dT) primers for reverse transcription. The circSlc8a1 plasmids were transfected into human HEK293T cells. The RNAs from vector or circSlc8a1-transfected cells were reverse transcribed using random or oligo(dT) primers. The expression of circSlc8a1 was validated by using mouse-specific primers against circSlc8a1. n = 6. (D) The back-splice junction of the ectopic mouse circSlc8a1 construct was confirmed by Sanger sequencing. The circSlc8a1 expression construct was used to generate transgenic mice. (E) The levels of circSlc8a1 were significantly decreased in the mouse heart tissues at 8–12 weeks after transverse aortic constriction (TAC) surgery. n = 6; ∗p < 0.05, ∗∗p < 0.01. (F) The levels of circSlc8a1 were retained at the similar levels as those in the sham group in the hearts of circSlc8a1(+)-transgenic mice 12 weeks post TAC surgery, while the levels of circSlc8a1 in the litter-matched negative mice (circNeg) were significantly decreased at the same time point after TAC surgery. n = 15; ∗∗p < 0.01. (G) Representative photographs of Masson trichrome, Sirius red, and WGA staining showed that cardiac fibrosis and cardiac hypertrophy were induced by TAC surgery in the circSlc8a1 negative (circNeg) mice. Such fibrosis and hypertrophy could be prevented in the transgenic mice overexpressing circSlc8a1. (H) The representative echocardiography of circSlc8a1-transgenic mice subjected to TAC surgery. (I and J) The circSlc8a1(+) mice subjected to TAC prevented the impairment of the heart function that was validated by (I) left ventricular ejection fraction (LVEF) and (J) left ventricular fractional shortening (LVFS). n = 10; ∗∗p < 0.01.