HIF-1α-regulated lncRNA-TUG1 promotes mitochondrial dysfunction and pyroptosis by directly binding to FUS in myocardial infarction

Abstract

Myocardial infarction (MI) is a fatal heart disease that affects millions of lives worldwide each year. This study investigated the roles of HIF-1α/lncRNA-TUG1 in mitochondrial dysfunction and pyroptosis in MI. CCK-8, DHE, lactate dehydrogenase (LDH) assays, and JC-1 staining were performed to measure proliferation, reactive oxygen species (ROS), LDH leakage, and mitochondrial damage in hypoxia/reoxygenation (H/R)-treated cardiomyocytes. Enzyme-linked immunoassay (ELISA) and flow cytometry were used to detect LDH, creatine kinase (CK), and its isoenzyme (CK-MB) levels and caspase-1 activity. Chromatin immunoprecipitation (ChIP), luciferase assay, and RNA-immunoprecipitation (RIP) were used to assess the interaction between HIF-1α, TUG1, and FUS. Quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemistry were used to measure HIF-1α, TUG1 and pyroptosis-related molecules. Hematoxylin and eosin (HE), 2,3,5-triphenyltetrazolium chloride (TTC), and terminal deoxynucleotidyl transferase dUTP risk end labelling (TUNEL) staining were employed to examine the morphology, infarction area, and myocardial injury in the MI mouse model. Mitochondrial dysfunction and pyroptosis were induced in H/R-treated cardiomyocytes, accompanied by an increase in the expression of HIF-α and TUG1. HIF-1α promoted TUG1 expression by directly binding to the TUG1 promoter. TUG1 silencing inhibited H/R-induced ROS production, mitochondrial injury and the expression of the pyroptosis-related proteins NLRP3, caspase-1 and GSDMD. Additionally, H/R elevated FUS levels in cardiomyocytes, which were directly inhibited by TUG1 silencing. Fused in sarcoma (FUS) overexpression reversed the effect of TUG1 silencing on mitochondrial damage and caspase-1 activation. However, the ROS inhibitor N-acetylcysteine (NAC) promoted the protective effect of TUG1 knockdown on H/R-induced cardiomyocyte damage. The in vivo MI model showed increased infarction, myocardial injury, ROS levels and pyroptosis, which were inhibited by TUG1 silencing. HIF-1α targeting upregulated TUG1 promotes mitochondrial damage and cardiomyocyte pyroptosis by combining with FUS, thereby promoting the occurrence of MI. HIF-1α/TUG1/FUS may serve as a potential treatment target for MI.

Fig. 1. H/R treatment led to mitochondrial dysfunction and pyroptosis in cardiomyocytes.

HL-1 and primary mouse cardiomyocytes were treated with H/R for 24 h. A Cell proliferation ability was measured using a CCK-8 assay. B ROS levels were measured using a DHE assay. C LDH levels were measured using an LDH assay kit. D, E Mitochondrial membrane potential was evaluated using JC-1 staining. F IL-1β and G IL-18 mRNA levels were assessed using qRT-PCR. H, I Western blot analysis determined the expression levels of pyroptosis-related markers. *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig. 2. HIF-1α increased the expression of TUG1 in cardiomyocytes by binding to its promoter region.

qRT-PCR measurement of (A) HIF-1α and (B) TUG1 expression in cardiomyocytes treated with H/R. C qRT-PCR and D, E western blot measurement of HIF-1α expression in cardiomyocytes transfected with shHIF-1α, shNC, HIF-1α, or empty vector. F qRT-PCR measurement of TUG1 levels in cardiomyocytes. G Dual luciferase reporter gene assay on the regulation of TUG1 expression by HIF-1α in cardiomyocytes. H ChIP assay on the presence of TUG1 after pulldown by HIF-1α antibody in cardiomyocytes. *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig. 3. Silencing TUG1 inhibited H/R treatment-induced mitochondrial dysfunction and pyroptosis in cardiomyocytes.

A qRT-PCR measurement of TUG1 levels in cardiomyocytes transfected with shTUG1 or cshNC. HL-1 and primary mouse cardiomyocytes were transfected with shNC or shTUG1 and then treated with H/R. B ROS levels were measured using a DHE assay. C, D Mitochondrial membrane potential was evaluated using JC-1 staining. E, F Caspase-1 activity was measured using flow cytometry. G IL-1β and H IL-18 expression was measured using qRT-PCR. I, J The expression of pyroptosis-related markers was measured using western blotting. *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig. 4. TUG1 promoted FUS expression by directly binding to the molecules, and overexpression of FUS reversed the effects of TUG1 silencing on H/R-induced mitochondrial dysfunction in cardiomyocytes.

A qRT-PCR measurement of FUS levels in H/R-treated cardiomyocytes transfected with shTUG1 or shNC. B RIP assay on the presence of TUG1 in cardiomyocyte samples pulled down by antiFUS antibody or control IgG. C Western blotting measurement of FUS expression in the cardiomyocytes cytoplasm, mitochondrial fraction. The D mRNA and E, F protein levels of FUS were measured using qRT-PCR and western blotting in cardiomyocytes transfected with shFUS, shNC, vector expressing FUS, or empty vector. G ROS levels were measured using a DHE assay. H, I Mitochondrial membrane potential was evaluated using JC-1 staining. *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig. 5. Overexpression of FUS reversed the effects of TUG1 silencing on H/R-induced pyroptosis in cardiomyocytes.

A, B Caspase-1 activity was measured using flow cytometry. C IL-1β and D IL-18 expression was measured using qRT-PCR. E, F The expression of NLRP3, ASC, and other downstream pyroptosis-related factors was measured using western blotting. *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig. 6. Silencing TUG1 inhibited H/R-induced pyroptosis of cardiomyocytes by decreasing the generation of ROS.

HL-1 and primary mouse cardiomyocytes were transfected with shTUG1 or treated with the ROS inhibitor NAC and then subjected to H/R. A, B Caspase-1 activity was measured using flow cytometry. C IL-1β, and D IL-18 expression was measured using qRT-PCR. E, F The expression of NLRP3, ASC, and other downstream pyroptosis-related factors was measured using western blotting. *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig. 7. Silencing TUG1 reduced I/R-induced myocardial damage in a mouse model.

A, B TTC staining was used to show the region of MI in the myocardial tissue of I/R mice. C Histological changes in the myocardial tissue were examined using H&E staining. The serum levels of D LDH, E CK, and F CK-MB were determined using ELISA. G, H Cell death was determined using TUNEL staining. *P < 0.05, **P < 0.01 and ***P < 0.001.

Fig. 8. Silencing TUG1 reduced FUS expression and FUS-induced mitochondrial dysfunction and pyroptosis in a mouse model.

The expression of A TUG1 and B FUS was measured using qRT-PCR in the myocardial tissue of I/R mice. C FUS expression in myocardial tissue was stained using immunohistochemistry. D ROS levels were measured using a DHE assay. The mRNA expression of E IL-1β and F IL-18 was measured using qRT-PCR. G, H The protein levels of FUS, NLRP3, ASC, and other downstream pyroptosis-related factors were measured using western blotting. *P < 0.05, **P < 0.01 and ***P < 0.001.