miR-29b-3p regulates cardiomyocytes pyroptosis in CVB3-induced myocarditis through targeting DNMT3A

Abstract

Background: Viral myocarditis (VMC) is a disease resulting from viral infection, which manifests as inflammation of myocardial cells. Until now, the treatment of VMC is still a great challenge for clinicians. Increasing studies indicate the participation of miR-29b-3p in various diseases. According to the transcriptome sequencing analysis, miR-29b-3p was markedly upregulated in the viral myocarditis model. The purpose of this study was to investigate the role of miR-29b-3p in the progression of VMC.

Methods: We used CVB3 to induce primary cardiomyocytes and mice to establish a model of viral myocarditis. The purity of primary cardiomyocytes was identified by immunofluorescence. The cardiac function of mice was detected by Vevo770 imaging system. The area of inflammatory infiltration in heart tissue was shown by hematoxylin and eosin (H&E) staining. The expression of miR-29b-3p and DNMT3A was detected by quantitative real time polymerase chain reaction (qRT-PCR). The expression of a series of pyroptosis-related proteins was detected by western blot. The role of miR-29b-3p/DNMT3A in CVB3-induced pyroptosis of cardiomyocytes was studied in this research.

Results: Our data showed that the expression of miR-29b-3p was upregulated in CVB3-induced cardiomyocytes and heart tissues in mice. To explore the function of miR-29b-3p in CVB3-induced VMC, we conducted in vivo experiments by knocking down the expression of miR-29b-3p using antagomir. We then assessed the effects on mice body weight, histopathology changes, myocardial function, and cell pyroptosis in heart tissues. Additionally, we performed gain/loss-of-function experiments in vitro to measure the levels of pyroptosis in primary cardiomyocytes. Through bioinformatic analysis, we identified DNA methyltransferases 3A (DNMT3A) as a potential target gene of miR-29b-3p. Furthermore, we found that the expression of DNMT3A can be modulated by miR-29b-3p during CVB3 infection.

Conclusions: Our results demonstrate a correlation between the expression of DNMT3A and CVB3-induced pyroptosis in cardiomyocytes. These findings unveil a previously unidentified mechanism by which CVB3 induces cardiac injury through the regulation of miR-29b-3p/DNMT3A-mediated pyroptosis.

Keywords: CVB3; DNMT3A; Pyroptosis; Viral myocarditis; miR-29b-3p.

Fig. 1

CVB3 induced VMC in mice. The male BALB/c mice were treated with 1 × 10⁴ TCID50 CVB3 and then sacrificed on day 7 p.i. A A schematic diagram of the viral myocarditis modeling process induced by CVB3. B The body weight change of mice after CVB3 infection. C The survival rate of mice following CVB3 infection. D The appearance of mice on day 7 p.i. E M-mode images of heart and F The cardiac function was evaluated using echocardiography. G Heart appearance of mice on day 7 post CVB3 infection. H H&E staining of the heart tissue sections; the magnifications were 1.25 × , 10 × , 40 × , respectively, and the scale bar was 100 μm. I Quantification of inflammatory areas based on the images shown in H. N = 3, *p < 0.05, **p < 0.01, and ****p < 0.0001

Fig. 2

Pyroptosis increased in both in vivo and in vitro VMC models induced by CVB3. A The expressions of pyroptosis related proteins in heart tissue. B Primary cardiomyocytes isolated from SD rats were identified by immunofluorescence staining. The primary antibody of anti-cTnT was applied. The scale bar was 100 μm. C The total RNA was extracted from the primary cardiomyocytes exposed to varying concentrations of CVB3 and the expressions of inflammatory cytokines IL-1β and IL-18 were evaluated by qRT–PCR. D The cell viability was detected by CCK8. ####p < 0.0001 represents the comparison with control at 48 h; ****p < 0.0001 represents the comparison with control at 72 h. E, F The expressions of pyroptosis-related proteins in CVB3 treated primary cardiomyocytes and HL-1 cells were measured using western blotting. N = 3, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

Fig. 3

MiR-29b-3p was significantly upregulated in VMC models. The total RNA, extracted from VMC mice and the control, was applied to transcriptome sequencing (N = 3). A Compared with the control group, there were 1444 miRNAs increased and 1138 miRNAs decreased in VMC mice heart tissues. B The most differentially expressed miRNAs were displayed through clustering heat map. C The Venn diagram indicated that five miRNAs have consistent expression trends in all samples. D The expression levels of miR-29b-3p in HL-1 cells, primary cardiomyocytes, and heart tissue were evaluated by qRT–PCR. N = 3, **p < 0.01, ***p < 0.001, and ****p < 0.0001

Fig. 4

The role of miR-29b-3p in CVB3-induced cardiomyocyte pyroptosis. Primary cardiomyocytes were transfected with 20 μM miR-29b-3p inhibitor/mimic or the negative control (inhibitor/mimic n.c.), respectively, after CVB3 treatment. A, CThe relative expression of miR-29b-3p was measured by qRT–PCR. U6 served as the internal reference. B, D The expressions of pyroptosis associated proteins were assessed by western blotting. N = 3, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

Fig. 5

Downregulation of miR-29b-3p-ameliorated, CVB3-induced VMC through decreasing pyroptosis in vivo. A A schematic diagram of miR-29b-3p antagomir treatment in VMC model induced by CVB3. The mice were inoculated with CVB3 on day 0 and then treated with indicated concentrations of miR-29b-3p antagomir (5, 10, 20 nM) on days 1, 3, and 5, respectively. B The body weight change of VMC mice after miR-29b-3p antagomir treatment. C M-mode images of heart and D–G the parameters of cardiac function were evaluated using echocardiography. H Heart appearance of VMC mice treated with miR-29b-3p antagomir on day 7. I H&E staining of the heart tissue sections; the magnifications were 1.25 × , 10 × , and 40 × , respectively, and the scale bar was 100 μm. J The inflammatory lesions were quantified using Image-Pro Plus 6.0 software. K Total RNA was extracted from heart tissue and the expression of miR-29b-3p was evaluated by qRT–PCR. L Total proteins were extracted from heart tissue and the expressions of pyroptosis proteins were analyzed by western blotting. N = 3, #p < 0.05, ##p < 0.01, ###p < 0.001, and ####p < 0.0001; ns significance is annotated as not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 6

DNMT3A acted as a target gene of miR-29b-3p in CVB3-induced myocarditis and reduced pyroptosis. A Bioinformatic analysis predicted the potential targets of miR-29b-3p. B The seed region of miR-29b-3p matches the sequence (nucleotides 825–831) on 3′ UTR of DNMT3A. C, D Total RNA and protein were extracted from heart tissues to detect the expression levels of DNMT3A. E, F The primary cardiomyocytes were transiently transfected with 20 μM miR-29b-3p inhibitor or mimic. A total of 48 h later, total protein was extracted and subjected to western blotting. The relative expression of DNMT3A was represented as a histogram and normalized using GAPDH. G, H Primary cardiomyocytes were transfected with 2.5 μg plasmid (phage-DNMT3A or phage-vector) for 48 h, and then total RNA and protein were extracted to evaluate DNMT3A expression. I CVB3-infected cardiomyocytes were transfected with 2.5 μg plasmid as indicated. After 48 h, pyroptosis level was evaluated by western blotting. N = 3, ####p < 0.0001,** p < 0.01, ***p < 0.001, and ****p < 0.0001

Fig. 7

miR-29b-3p regulated cardiomyocyte pyroptosis via DNMT3A. The primary cardiomyocytes were exposed to CVB3 for 6 h, then co-transfected with phage-DNMT3A or phage-vector and miR-29b-3p inhibitor for another 48 h. A The expression levels of proteins related to pyroptosis. B, C Primary cardiomyocytes were transfected with 20 μM of si-DNMT3A or si n.c. for 48 h, then the expressions of DNMT3A mRNA and protein were measured. D The CVB3-infected cells were co-transfected with si-DNMT3A or si n.c. and miR-29b-3p inhibitor. A total of 48 h later, pyroptosis level of cardiomyocytes was evaluated by western blotting. N = 3, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001

Fig. 8

Schematic representation of CVB3-induced miR-29b-3p-mediated pyroptosis in cardiomyocytes. Infection with CVB3 leads to an upregulation in the expression of cellular miR-29b-3p, which subsequently downregulates the expression of DNMT3A by binding to the 3′-UTR of DNMT3A. This reduction in DNMT3A levels activates the NLRP3 inflammasome, resulting in the cleavage of pro-caspase-1 into its active form. The activated cleaved caspase-1 then cleaves GSDMD, generating N-terminal fragments that form pores in the cell membrane and trigger pyroptosis. Additionally, cleaved caspase-1 also activates pro-IL-1β and pro-IL-18, promoting their maturation and release. This process exacerbates the inflammatory damage to myocardial cells, leading to myocardial damage and a decline in cardiac function