Recent studies have shown that circRNAs (circRNAs) have a very important role in cardiovascular disease. Therefore, elucidating the regulatory mechanisms associated with circRNAs in cardiovascular disease is essential for cardiovascular disease prevention and **.
February 2023,The team of Professor Yang Bohua from the University of Toronto, CanadaInresearch（if=11.0) Published the article "A novel circular RNA circitga9 predominantly generated in human heart disease induces cardiac remodeling and fibrosis", in which the authors found 32,034 previously undiscovered circRNAs with different cardiac expression patterns. Notably, circrna derived from integrin-9 circitga9 showed significant upregulation in patients with myocardial hypertrophy. CircitGA9 can regulate and induce actin polymerization by binding to tropomyosin 3 (TPM3), thereby affecting cardiac tissue fibrosis. Finally, the authors developed a blocking oligomer targeting CircitGA9 that inhibits CircitGA9 and TPM3 interactions to improve cardiac function and reduce fibrosis, and the oligomer can be used for further preclinical and translational development. In summary, elevated levels of circitga9 drive cardiac remodeling and fibrosis. By identifying circitga9 as the first target, the authors provide new ideas for innovative interventions to alleviate cardiac remodeling and fibrosis.
First, the authors identified a total of 45,284 circRNAs in sequencing and analysis of heart tissue from patients with heart disease or normal humans, of which 13,250 have been reported in circbase, while the remaining 32,034 circRNAs have not been reported before. The GO analysis showed that 13 of the 2,900 functions involving a total of 11,243 genes reached significant levels. The number of genes involved in these 13 functions is 295. Among the 295 genes, only the gene ID ensg00000144668 for integrin-9 appeared in all 13 functional traits. KEGG pathway analysis showed that ENSG00000144668 was involved in three pathways, including hypertrophic cardiomyopathy, dilated cardiomyopathy, and cell adhesion molecule, which are important in cardiac remodeling. Interestingly, among the upregulated circRNAs, HG38 CIRC 0030600 (circitga9) was significantly upregulated, and its sequence was derived from the 14th and 15th exons of integrin-9. When siRNA silencing CircitGa9 was used, changes in fibrosis-related markers including fibroblast adhesion, proliferation, decreased expression of transforming growth factor (TGF-) and collagen deposition were most significant, but cardiomyocyte viability was increased. In addition, the level of circitga9 in 73 patients with hypertrophy and 25 normal cardiac specimens was significantly higher than in the control samples. Digital droplet PCR results showed a significant increase in the copy number of CircitGa9 in patient specimens compared to control samplesThis suggests that circitga9 has a very important role in cardiac disease-induced fibrosis.
fig 1.Expression of circRNA in the tissues of patients with hypertrophy of the heart.
To further the effect of circitga9 on cardiac function, the authors treated mice by coarctation of the aorta (TAC). The results showed that endogenous circitga9 levels increased most in cardiac tissue after TAC, followed by aorta and brain tissue. To study the role of CircitGa9 in cardiac function, the authors constructed an overexpression vector of human CircitGa9 and a linear plasmid lacking a cyclization sequence, and subsequently injected nanoparticle-bound human CircitGA9 expression plasmid and linear plasmid into the peritoneum of TAC mice. The experimental results showed that the expression levels of endogenous mouse circitga9 and human circitga9 in the heart were significantly increased after 8 weeks of treatment. Cardiac function measurements using the VEVO 2100 imaging system found that overexpression of CircitGa9 resulted in a significant increase in left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) in TAC mice compared to the control group, while LVESD-LVEDD showed a significant decrease. In addition, the authors observed a reduction in left ventricular pressure (DP dt), left ventricular ejection fraction (LVEF), and left ventricular fraction (LVFS). The M-type plot shows that the ventricular contraction of TAC is attenuated after injection of the Circitga9 expression plasmid, indicating that cardiac function is decreased in the Circitga9 overexpression group compared to the control group.
The authors then designed 2 siRNAs targeting the circitga9 ligation sequence. After conjugating siRNA to gold nanoparticles and sending them into TAC mice, it was found that although the level of circitga9 in the heart was significantly increased 8 weeks after TAC, the siRNA still decreased the level of circitga9. In cardiac function measurements of TAC mice silencing Circitga9, the authors detected a significant decrease in LVESD and LVEDD, but LVESD-LVEDD showed a significant increase. The authors also found significant elevations in left ventricular pressure, LVEF, LVEF, and LVFS. M-mode ultrasound showed that silencing CircitGa9 prevented TA-induced reduction in left ventricular contraction.
fig 2.Overexpression of circitga9 decreases cardiac function.
Since cardiac fibrosis is one of the main outcomes closely related to cardiac hypertrophic remodeling, the authors investigated the role of circitga9 in cardiac fibrosis by observing collagen deposition by staining cardiac tissue with Masson tricolor and Sirius red. Quantitative analysis showed that the staining of Masson tricolor and Sirius red in the hearts of TAC mice was significantly increased compared to the control group, while the delivery of overexpressing CircitGA9 by injection further increased the collagen deposition of TAC mice. The qPCR results showed that the expression of collagen I and collagen III in TAC mice was significantly up-regulated, indicating that the overexpression of Circitga9 further increased cardiac collagen levels, resulting in enhanced fibrosis. After silencing circitga9, the levels of marson tricolor and Sirius red in heart tissue were significantly reduced, and the level of collagen deposition was reduced. In addition, qPCR results showed a significant reduction in collagen I and collagen III in these heart tissues. In patient specimens, collagen I levels were positively correlated with circitga9 expression, while circitga9 expression was negatively correlated with cardiac function.
fig 3.Effect of circitga9 on cardiac fibrosis under pressure overload.
In order to reveal the downstream molecules that may be involved in mediating the function of circitga9 in cardiac fibrosis, the authors detected that tropomyosin 3 (TPM3) can bind to circitga9 through co-immunoprecipitation and protein profiling. To validate the results of mass spectrometry analysis, the authors transfected the CircitGa9 expression construct and control vector into MCF cells and fished CircitGa9 with TPM3 antibody. The results showed that TPM3 antibody was significantly enriched to CircitGa9 in the CircitGa9 overexpression group compared to the linear vector. In addition, TPM3 protein was detected by using the CircitGa9 probe for pulldown of the binding protein, and by Western blot detection of the pulldown product by TPM3 antibodyThe results show that CircitGA9 can specifically bind to TPM3.
fig 4.Binding of CircitGA9 to TPM3.
Tropomyocoin plays a crucial role in stabilizing actin filaments, and the conformational change between tropomyosin and actin leads to actin polymerization. Actin polymerization plays an important role in regulating cellular activity and is closely related to tissue fibrosis. Therefore, the authors examined whether the binding of circitga9 and TPM3 affects cardiac fibrosis by regulating actin polymerization. Experimental results showed that overexpression from CircitGA9 promoted increased actin polymerization in MCF cells. To further elucidate the mechanisms associated with CircitGa9 function, HEK-293T cells were transfected with CircitGa9 and co-immunoprecipitation assays were performed using TPM3 and actin antibodies. The results showed that the expression of CircitGa9 inhibited the interaction between TPM3 and -Actin. In situ hybridization and immunofluorescence staining of human heart tissue, the authors observed elevated levels of CircitGa9 and F-Actin in hypertrophic heart tissue compared with normal heart tissue, and colocalization of Circitga9 and TPM3 in cells.
In order to further confirm the binding activity of CircitGa9 to TPM3, the authors performed site-directed mutagenesis at the binding site, and the mutation of the binding site did not affect the circularization of the mutant CircitGa9. After co-immunoprecipitation experiments, it was found that the binding site mutation inhibited the enrichment of CircitGA9 by TPM3 antibody. Conversely, when the mutant is transfected into MCF cells, the CircitGa9 probe can no longer be called to TPM3. Subsequently, the authors performed cardiac function tests on TAC mice injected with ** mutant vector, and detected a significant decrease in LVESD and LVEDD, but a significant increase in LVESD-LVEDD. The authors also found significant increases in left ventricular pressure, LVEF (Fig. 7f), and LVFS. M-mode sonography showed that the effect of CircitGa9 on cardiac function in TAC mice was weakened relative to the CircitGa9 group. In summaryCircitga9 regulates actin polymerization by binding to tropomyosin 3, thereby influencing cardiac fibrosis.
fig 5.CircitGA9 affects cardiac function by binding TPM3.
To explore the potential of developing the best method, the authors synthesized blocking oligos to block the binding of circitga9 to TPM3. qPCR analysis showed that blocking the CircitGa9 binding site significantly reduced the binding of CircitGa9 to TPM3. The M-pattern plot showed that the left ventricular systolic function of the TAC heart was enhanced after blocking the interaction of CircitGA9 with TPM3. In cardiac function measurements in TAC mice injected with blocking oligomers, the authors observed a significant decrease in LVEDD and LVESD, and a significant increase in LVESD-LVEDD. The authors also detected a significant increase in left ventricular pressure and a significant increase in LVFS and LVEF, suggesting improved cardiac function in TAC mice.
In addition, in TAC mouse heart sections administered blocking oligomers, Marson tricolor and Sirius red staining showed reduced staining levels and collagen deposition levels. qPCR testing showed a significant decrease in collagen I and collagen III in heart tissue. In cell viability assays, the authors found that transfection-blocking oligomers reduced the survival, adhesion, and migration of primary-isolated cardiac fibroblasts.
fig 6.Blocks the action of circitga9.
In summary, in this paper, the authors demonstrate that circitga9 forms a complex with TPM3 and -actin, which promotes actin polymerization and thus regulates cardiac fibrosis.