CircRNA DICAR as a novel endogenous regulator for diabetic cardiomyopathy and diabetic pyroptosis of cardiomyocytes

Figures

• 

  Fig. 1. Identified circRNA expression in diabetic cardiomyopathy mouse. a Microarray identified in the hearts of db/db mice. b Select significantly regulated circRNA detected by qPCR (n = 9). c CircRNA resistance detected by RNAse. d Mm9-circ-008009 structure. e Identification of the mm9-circ-008009 back-spliced junction sites. f Identification of the hsa-circ-0131202 back-spliced junction sites. g, h Sanger sequence of mm9-circ-008009 and hsa-circ-0131202. i, j Second structure prediction of mm9-circ-008009 and hsa-circ-0131202. k FISH detection of mm9-circ-008009 in the mouse heart tissues. Data are represented as mean ± SEM. **P < 0.01 vs. WT group

• 

  Fig. 2. Effect of DICAR on the cardiac functions and remodeling. a Echocardiography images of DICAR+/− mouse. b Summary of the heart functions, including stroke volume (SV), ejection fraction (EF), left ventricular end-diastolic dimension (LVEDD), cardiac output (CO), fractional shortening (FS). c Representative images and summary data of wheat germ agglutinin (WGA) staining. d Representative images and summary data of Masson staining of DICAR+/−. e Representative IHF images and summary data of collagen III. f Representative and summary data of WB images of pyroptosis detected in the heart tissues. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 vs. WT group, n = 6–8

• 

  Fig. 3. Gain of DICAR function ameliorates T2DM-induced heart function in DICARTg mouse. a Representative echocardiography images of DICARTg mouse. b The DICAR overexpression ameliorates heart functions, including stroke volume (SV), ejection fraction (EF), left ventricular end-diastolic dimension (LVEDD), left ventricular end-diastolic volume (LVEDV), cardiac output (CO), and fractional shortening (FS). c The DICAR overexpression decreased the cardiomyocytes area detected by wheat germ agglutinin (WGA) staining. d Collagen deposition detected by Masson staining. e Collagen III expression detected by IHC. f In the DICARTg mouse, the DICAR overexpression inhibited the pyroptosis of heart tissue induced by T2DM that detected by the GSDMD, ASC, IL-1β, and NLRP3 protein expression. Data are represented as mean ± SEM. **P < 0.01 vs. Normal, ++P < 0.01 vs. Normal + T2DM, n = 8. g Adenovirus (AD)-DICAR infected HL-1 cells inhibited the pyroptosis induced by AGEs (200 µg/mL). Data are represented as mean ± SEM. **P < 0.01 vs. NEG, ++P < 0.01 vs. AD-DICAR-over, n = 4

• 

  Fig. 4. Interaction between VCP and DICAR in the pyroptosis of cardiomyocytes induced by AGEs. a Representative images of VCP pulled down by DICAR probe and detected by CHIRP-MS. b Total VCP protein expression between WT and db/db mice. c RIP used to detect the combination between VCP and DICAR. d Model prediction of VCP combined with DICAR. e the KD of DICAR combined with VCP detected by SPR. f the effect of VCP-siRNA on anti-pyroptosis induced by AGEs. Data are represented as mean ± SEM. **P < 0.01, ***P < 0.001 vs. con; ++P < 0.01 vs. AGEs (200 µg/ml, 48 h); ##P < 0.01 vs. AGEs +mDICAR-JP, n = 4

• 

  Fig. 5. VCP mediated Med12 protein degradation via the Ub-protein system. a The FISH probe detected DICAR located in the endoplasmic reticulum. b VCP expression in ER, *P < 0.05 vs. WT, n = 3. c Co-IP was used to detect the ubiquitination level of VCP in the heart tissues of DICAR+/− mice. d LC-MS + PRM was used to detect the protein expression in the heart tissues of DICAR+/− mice. e Myom1, Med12, Myh6, Myl2, and Cavin2 protein expression in the heart tissues of DICAR+/− mice detected by WB. f Myom1, Med12, Myh6, Myl2 and Cavin2 protein expression in the heart tissues of db/db mice detected by WB. Data are represented as mean ± SEM. **P < 0.01 vs. WT, n = 8. g Med12 mRNA in the heart tissues of DICAR + /− and DICARTg detected by qPCR. h Representative images of WB detected pyroptosis of the heart in the DICARTg mouse treated by T2DM. Data are represented as mean ± SEM. ***P < 0.001 vs. WT, n = 8. i Representative images of DICAR-JP and VCP siRNA effect on the Med12 protein expression. Data are represented as mean ± SEM. **P < 0.01 vs. BSA, ++P < 0.01 vs. AGEs (200 µg/mL, 48 h), n = 3. j Representative images of Med12 siRNA-induced pyroptosis of HL-1. Data are represented as mean ± SEM.…