Pyroptosis is involved in DCM through NLRP3-caspase-1 pathway in vitro
We treated neonatal rat cardiomyocytes (PNRC) and cardiomyocyte cell line H9c2 with glucose at the concentration of 5.5 mmol/L (normal control) or 50 mmol/L (high glucose, HG) to imitate the hyperglycemic condition. As shown in Figure 1A, due to the HG treatment, H9c2 cells exhibited characteristic morphological changes, such as swelling changes and rupture of cell membrane, suggesting that pyroptosis was directly involved in DCM process. Then we performed Calcein-AM staining analysis and found that HG treatment caused an increased damage of cell membrane when compared with control treatment (Figure 1B). Moreover, the protein expression of pyroptosis markers, including NLRP3, cleaved caspase-1, and GSDMD-N, were significantly upregulated in HG-treated cardiomyocytes (Figure 1C). Importantly, treatment with MCC950, a well-known NLRP3 inhibitor, dramatically reversed pyroptosis of cells treated with HG (Figure 1C). We also evaluated the effect of HG treatment on cell apoptosis by performing TUNEL staining. As shown, apoptosis was activated by upon HG treatment, however, this activation was not restored by MCC treatment (Figure 1D). These data showed that HG treatment resulted in pyroptosis of cardiomyocytes in an NLRP3-dependent manner.
Pyroptosis was activated in cardiomyocyte of DCM rats
To further verify the essential role of pyroptosis in HG-induced toxicity of cardiomyocytes, we constructed DCM model using Wistar rats by single intraperitoneal injection of streptozotocin. Echocardiography was performed to evaluate the cardiac function of DCM rats. Our data revealed a reduced LVEF and FS in rats with DCM in contrast to normal rats. The LVEF level of DCM rats was 48%, which is significantly lower than that in normal rats (68%). Moreover, the average FS of rats with DCM was also dramatically decreased compared to control rats (21% vs. 37%) (Figure 2A). Interestingly, when NLRP3 was silenced with MCC950 in DCM rats, the damaged cardiac function caused by DCM was dramatically reversed (Figure 2A). By detecting myocardial enzyme markers, such as AST, LDH and CK-MB, we identified an elevated expression in DCM rats, however, MCC950 treatment partly restored this effect. (Figure 2B). In addition, electron microscopy imaging of cardiomyocyte ultrastructure showed that DCM rats showed serious cardiomyocyte damage compared to control hearts, including increased inter-mitochondrial distance, disconnected cardiac myofibers and thinner myofibers, and this damage could be partially rescued by MCC950 (Figure 2C). Finally, the expressions of pyroptosis proteins, NLRP3 and GSDMD-N, were dramatically increased in rats of DCM compared, meanwhile, this influence was significantly relieved by MCC950 (Figure 2D). Finally, the pyroptosis in DCM was finally proved by Masson staining (Figure 2E). Taken together, we demonstrated that DCM was closely associated with cardiomyocytes pyroptosis in an NLRP3-dependent manner.
METTL14 is downregulated in DCM
To find whether METTL14 was involved in pyroptosis in DCM models, we detected the expression level of METTL14 in DCM rats. As shown in Figure 3A-B, METTL14 was significantly downregulated in heart tissue and serum samples of DCM rats compared to those of controlled rats. Moreover, a decreased METTL14 level was identified in PNRC and H9c2 cells treated with HG when compared with control cells (Figure 3C-D). In addition, serum METTL14 was also downregulated in patients with DCM than that of healthy individuals (Figure 3E). These results indicate that METTL14 may be key molecular regulator during DCM initiation and progression.
METTL14 suppresses DCM via modulating pyroptosis
To clarify the role of m6A modification in pyroptosis and DCM progression, we evaluated the effect of METTL14 in DCM progression. As expected, injection of Lv-METTL14 into DCM rats significantly increased m6A level in DCM rats (Figure 4A-B). Echocardiography suggested that overexpression of METTL14 increased LVEF, FS (Figure 4C). Meanwhile, electron microscopy revealed a relieved injury in cardiomyocyte (Figure 4D). Moreover, enhanced METTL14 inhibited pyroptosis level in myocardial tissues, including downregulation of NLRP3, caspase-1 and GSDMD-N (Figure 4E), suggesting that METTL14-mediated m6A modification may suppress DCM through modulating pyroptosis. To confirm this hypothesis in vitro, we silenced METTL14 and m6A modification level in H9c2 and PNRC cells (Figure 4F-G). Reversed morphologic changes of pyroptosis were observed upon silence of METTL14 (Figure 4H). Western blotting showed that sh-METTL14 caused upregulated NLRP3, cleaved caspase-1 and GSDMD-N (Figure 4I). Consistently, Calcein-AM staining revealed an increased membrane damage in H9c2 cells silenced with METTL14 compared to controls, however, this effect was abrogated by MCC950 (Figure 4J). Collectively, we proved that METTL14-mediated m6A modification play essential roles in DCM via regulating cardiomyocyte pyroptosis.
METTL14 suppresses pyroptosis via targeting TINCR lncRNA
It is reported that m6A process was closely associated with lncRNA processing (14). By performing GSEA analysis, we revealed that METTL14 was involved in the process and degradation of ncRNAs (Figure 5A). Based on our previous observation of lncRNA TINCR in pyroptosis, we supposed that METTL14 may regulate DCM through TINCR-mediated pyroptosis. To prove this assumption, we detected TINCR expression and found that TINCR was negatively correlated with METTL14 in patients with DCM (Figure 5B). In addition, TINCR was upregulated in HG-treated cells and DCM rats (Figure 5C-D). Next, we silenced METTL14 in PNRC and H9c2 cells, and found that TINCR was upregulated accordingly (Figure 5E), while overexpression of METTL14 leads to a decreased expression of TINCR (Figure 5F).
Then, we performed gain-or-loss functional assays by injection TINCR lentiviral vector into METTL14-overexpressing DCM rats. Intriguingly, overexpression of TINCR reversed the METTL14-induced effects on LVEF, FS, and cardiomyocyte damage in DCM rats (Figure 5G-H). In addition, enhanced TINCR abrogated METTL14-caused suppression of pyroptosis in PNRC and H9c2 cells (Figure 5I). Collectively, we proved that lncRNA TINCR was, at least partly, responsible for METTL14-induced suppression of pyroptosis and DCM.
METTL14-dependent m6A methylation downregulated expression of TINCR
Take a step further, we sought to find whether it is the METTL14-mediated m6A modification that downregulated TINCR expression. By analyzing the potential m6A binding sites with online SRAMP database (http://www.cuilab.cn/sramp), we verified 45 m6A residues located across TINCR sequence (Figure 6A), among which 11 were identified as high/very high confidence (Table 1). It is well demonstrated that METTL14 acted as an interactor with WTAP, binding to the methyltransferase to form a complex which mediates m6A methylation on RNAs. Here, sh-WATP vector was injected into METTL14-overexpressed DCM rats (Figure 6B). Suppressed TINCR caused by METTL14 was abrogated by silence of WATP (Figure 6C). Then, we performed RIP assay using m6A antibody, and found a significantly decreased methylated TINCR (site 7709) bounded by m6A upon METTL14 deletion or WTAP deletion (Figure 6D). Above results strongly suggest that METTL14-dependent m6A modification of TINCR results in its downregulation.
m6A methylation is a characterized process which needs the involvement of m6A reader proteins, including YTHDFs (15). Given that YTHDF2 participated in modulation of m6A-dependent RNA degradation (16), we therefore assumed that YTHDF2 may be essential for METTL14-mediated TINCR decay and downregulation. By performing RIP using antibody against YTHDF2, we identified the positive binding of TINCR by YTHDF2 antibody in DCM rats (Figure 6E). Consistently, RNA pulldown assay showed that YTHDF2 was significantly enriched by TINCR (Figure 6F). Then, we silenced YTHDF2 expression in cardiomyocytes (Figure 6G). The decay rate of TINCR was significantly slower in shYTHDF2-infected cells (Figure 6H), indicating that YTHDF2 mediated m6A-RNA decay of TINCR. Altogether, our results revealed that METTL14-mediated m6A modification inhibited TINCR expression via YTHDF2-regulated RNA degradation.
TINCR promotes pyroptosis and DCM via stabilizing NLRP3 mRNA
Previously, we demonstrated that TINCR regulated cardiomyocytes pyroptosis via stabilizing NRLP3 in DOX-induced cardiotoxicity (13). To verify whether this regulation mode applies in DCM, we performed gain-and-loss functional assays. As shown, MCC950 remarkedly abrogated the TINCR-regulated pyroptosis in HG-treated cardiomyocytes (Figure 7A). To further confirm the direct interaction between TINCR and NLRP3 RNA, we conducted RNA pulldown assay by generating biotinylated oligonucleotides in HG-treated H9c2 and PNRC cells. As shown, NLRP3 RNA was remarkably enriched by biotinylated TINCR (Figure 7B). By treatment with actinomycin D (ActD), a well-known inhibitor for RNA transcription, we evaluated the effect of TINCR on downstream mRNA degradation. The results revealed that knockdown of TINCR resulted in an accelerated degradation of NLRP3 mRNA in cardiomyocytes (Figure 7C). More importantly, METTL14 decreased the stability of NLRP3, and overexpression of TINCR reversed this effect (Figure 7D). Collectively, the above results which strongly supports that METTL14-mediated m6A induced suppression of TINCR, which further regulates pyroptosis via stabilizing NLRP3 mRNA (Figure 7E).