Post-infarction KLHL40-mediated regulation of cardiac sarcomeric integrity and function

Figures

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  Figure 1. Sarcomeric molecular dynamic changes after MI. (A–E) GEO dataset analysis showed the mRNA expression levels of Myot at sham, 10 min, 1 h, 6 h,1 day, 3 days, 7 days, 28 days, 8 weeks after MI. (F) PXD020193 dataset analysis showed that the protein expression levels of MYOT at sham, 10 min, 1 h, 6 h, 1 day, and 3 days after MI. (G–I) GEO datasets analysis showed that the mRNA expression levels of Capza1 and Capza2 at sham, 10 min, 1 h, 6 h, 1 day, 3 days, 7 days, 28 days, and 8 weeks after MI. (J) PXD020193 dataset analysis showed that the protein expression levels of CAPZA2 at sham, 10 min, 1 h, 6 h, 1 day, and 3 days after MI. (K–O) GEO dataset analysis showed that the mRNA expression levels of Tcap at sham, 10 min, 1 h, 6 h, 1 day, 3 days, 7 days, 28 days, 8 weeks after MI. (P) PXD020193 dataset analysis showed that the protein expression levels of TCAP at sham, 10 min, 1 h, 6 h, 1 day, and 3 days after MI. ns indicates no significant difference . *P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 2. Dynamic expression of KLHL40 after MI. (A–C) GEO dataset analysis showed that the mRNA expression levels of KLHL40 at sham, 10 min, 1 h, 6 h, 1 day, 3 days, and 7 days after MI. (D) PXD020193 dataset analysis showed that the protein expression levels of KLHL40 at sham, 10 min, 1 h, 6 h, 1 day, and 3 days after MI. (E and F) qRT-PCR and Western blotting semiquantitative analysis of KLHL40 expression in the early stage of human MI. (G and H) qRT-PCR and Western blotting semiquantitative analysis of KLHL40 expression in the late stage of human MI. (I) IHC staining of KLHL40 expression and Masson’s trichrome staining. (J and K) qRT-PCR and Western blotting semiquantitative analyses for KLHL40 in H9C2 cells after 1 h, 3 h, 6 h, 12 h, 18 h, and 24 h hypoxia. ns indicates no significant difference. *P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 3. qRT-PCR and Western blotting verified the knockdown and overexpression of KLHL40. (A and B) The efficiency of KLHL40 knockdown was evaluated by qRT-PCR and Western blotting. (C and D) The efficiency of KLHL40 overexpression was evaluated by qRT-PCR and Western blotting. ns indicates no significant difference. *P < 0.05, **P < 0.01, ***P < 0. 001.

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  Figure 4. Selective regulation of Z- disc proteins by KLHL40. (A, C, E) Western blotting of MYOT, CAPZA1/2, TCAP expression in sh-KLHL40 cells. (B, D, F) Western blotting of MYOT, CAPZA1/2, TCAP expression in oe -KLHL40 cells. (G and I) Western blotting of ACTN2 and FLNC expression in sh-KLHL40 cells. (H and J) Western blotting of ACTN2 and FLNC expression in oe -KLHL40 cells. ns indicates no significant difference. *P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 5. KLHL40 regulates selective sarcomeric protein turnover through intracellular calcium and calpain signaling. (A and B) Western blotting for CAST expression in sh-KLHL40 cells and oe-KLHL40 cells. (C and D) Western blotting for CAPN1 and CAPN2 expression in sh-KLHL40 cells and oe-KLHL40 cells. (E and F) Western blotting for ATP2A2 expression in sh-KLHL40 cells and oe-KLHL40 cells. (G) Representative calcium fluorescence images and related quantification of sh-KLHL40 cells and oe-KLHL40 cells by Fluo-4 Assay. (H and I) Western blotting for DES in sh-KLHL40 cells and oe-KLHL40 cells. ns indicates no significant difference . *P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 6. KLHL40 attenuates inflammasome activation and apoptosis in cardiomyocytes. (A and B) Western blotting for NLRP3 expression in sh-KLHL40 cells and oe-KLHL40 cells. (C and D) Western blotting for Cleaved Caspase-1 expression in sh-KLHL40 cells and oe-KLHL40 cells. (E and F) Western blotting for BAX and BCL2 expression in sh-KLHL40 cells and oe-KLHL40 cells. ns indicates no significant difference . *P < 0.05, **P < 0.01, ***P < 0.001.

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  Figure 7. Mechanisms of KLHL40 degradation and stability. (A and B) MTT assay was used to determine the optimal MG132 treatment conditions. (C) Representive light microscopy images of cells treated with MG132 (0 μM, 5 μM, 10 μM). (D and E) qRT-PCR and Western blotting for KLHL40 expression of the cells treated with MG132 (0 μM, 5 μM, 10 μM). (F and G) MTT assay was used to determine the optimal chloroquine treatment conditions. (H) Representative light microscopy images of cells treated with chloroquine (0 μM, 50 μM, 100 μM) for 6 h. (I and J) qRT-PCR and Western blotting for KLHL40 expression of the cells treated with chloroquine (0 μM, 50 μM, 100 μM) for 6 h. ns indicates no significant difference . *P < 0.05, **P <0.01, ***P < 0.001.