ZBED6 deficiency drives pathological cardiac hypertrophy through MEOX1–ERK1/2–dependent autophagy dysfunction

Figures

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  Fig 1. ZBED6 expression is downregulated in cardiac hypertrophy patients and cardiomyocytes subjected to hypertrophic stress. (A) Immunoblotting and quantitation of ZBED6 protein level of cardiac tissue in healthy individuals (n = 6) and cardiac hypertrophy patients (n = 5). (B) The mRNA level of ZBED6 were analyzed by RT-qPCR and normalized to GAPDH. (C and D) Representative immunofluorescence images of α-actinin and quantification of AC16 cell size treated with Ang II (1 μM) or vehicle for 24 hours (n = 50). Scale bars: 25 μm. (E) ZBED6 protein level in AC16 cells treated with vehicle or Ang II for 24h (n = 50). Data are presented as mean ± SEM; * P < 0.05, ** P < 0.01.

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  Fig 2. ZBED6 deficiency drives cardiac hypertrophy and diastolic dysfunction in pigs. (A) Representative images of longitudinal echocardiography (Echo) from 9-month-old ZBED6-deficient and WT pigs. (B and C) Echo parameters of panel A were calculated (n = 4). LVEF: left ventricular ejection fraction in (B), LVFS: left ventricular fraction shortening in (C). (D and E) Representative images of pulse-wave Doppler in (D) and the ratio between mitral E wave and A wave (E/A) (E). (F and G) Representative tissue Doppler tracings in (F) and quantitative analysis of the mitral E to e′ -wave ratio (E/e′) in (G). (H) The ratio of wet heart weight to body weight (HW/BW) (n = 3). (I) Representative photographs of general hearts, transverse cross sections and WGA-stained sections of heart in each group at 9 months (n = 3). Scale bars, 50 μm. (J) Frequency distribution of myofiber cross-sectional area (μm2) in the left ventricle for each group (n = 3). (K and L) Representative α-actinin immunofluorescence images and quantification of relative cross-sectional area in WT and ZBED6-deficient cells treated with vehicle or Ang II (1 μM) for 24 h (n = 50). Scale bars, 25 μm. Data are expressed as mean ±…

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  Fig 3. Loss of ZBED6 induces cardiac hypertrophy through autophagy impairment. (A) Representative electron microscopic images of autophagy in left ventricular myocardial from ZBED6-deficient and wild-type pigs. (White arrow indicated autolysosomes, black arrow autophagosome). Scale bars, 500 nm. (B and C) The protein levels of P62 and LC3B in ZBED6-deficient pigs (B) and ZBED6-depletion cardiomyocytes (C) compared with control (n = 3). (D) Representative immunofluorescence images of wild-type and ZBED6-deficient cardiomyocytes stained for LC3B (green), LysoTracker (red), and nuclei (blue). Scale bars, 50 μm. (E and F) Relative protein expression and quantification of LC3B and p62 in WT and ZBED6-dificient AC16 cells treated with BafA1 (50 nM) or DMSO for 24 hours (n = 3). (G and H) Representative immunofluorescence images of α-actinin staining and quantification of cell surface area in WT and ZBED6-deficient AC16 cells treatment with DMSO or Rapamycin (2 μM) for 24 hours (n = 50). Scale bars, 25 μm. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01.

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  Fig 4. ZBED6-MEOX1 axis regulates cardiomyocyte hypertrophy. (A) Integrated analysis of cardiac RNA-seq and ChIP-seq data from WT and ZBED6-deficient pigs, combined with human RNA-seq datasets, identifies six potential ZBED6 targets. (B) Relative mRNA levels of MEOX1, MCF2L2, BDNF, ARHGAP28, CX3CR1 and AGRN in WT and ZBED6 knockout pigs (n = 3). (C and D) Representative immunofluorescence images α-actinin and quantification of cell surface area in WT and ZBED6-deficient AC16 cells transfected with siNC or siMEOX1, stained for α-actinin (green) and nuclei (blue) (n = 50). Scale bar: 25 μm. (E) The relative mRNA levels of ANP, BNP and MYH7 in WT and ZBED6-deficient AC16 cells under the indicated groups (n = 3). (F and G) Immunoblot analysis and quantification of ANP protein level from samples (n = 3). (H and I) Western blot analysis and quantification of ANP in each group (n = 3). (J and K) Representative immunofluorescence images α-actinin and quantification of cell surface area in WT and ZBED6-OE cells transfected with pcDNA3.1 or MEOX1-overexpression vector, followed by Ang II treatment (n = 45). Scale bar: 25 μm. (L) The mRNA levels of ANP, BNP and MYH7 in WT and ZBED6-overexpression…

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  Fig 5. ZBED6 regulates cardiac hypertrophy through the MEOX1-MAPK signaling pathway. (A) GSEA identified enriched signaling pathways in ZBED6-deficient versus WT pig cardiac transcriptomes. (B) The protein levels of key MAPK pathway kinases in cardiac tissue of WT and ZBED6-dificient pigs (n = 3). (C). The protein levels of phosphorylated ERK1/2 (p-ERK1/2) and total ERK1/2 in cardiac tissue from healthy individuals (n = 6) and cardiac hypertrophy patients (n = 5). (D) Western blot analysis of key MAPK pathway kinases in WT or ZBED6 knockout (KO) AC16 cells transfected with siNC or siMEOX1 (n = 3). (E) Western blot analysis of key MAPK pathway kinases in WT and ZBED6-OE cells transfected with pcDNA3.1 or MEOX1-overexpression vector, followed by Ang II treatment (n = 3).

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  Fig 6. ZBED6 deficiency-induced cardiomyocyte hypertrophy is reversed by inhibition of MEOX1-induced pERK1/2 activation. (A) Representative immunofluorescence images of α-actinin in WT and ZBED6-deficient AC16 cells treated with vehicle or SCH772984 for 48 hours. Scale bar, 25 μm. (B) Quantification of cardiomyocyte cross-sectional area in A (n = 50). (C) Relative mRNA levels of ANP, BNP and MYH7 in WT and ZBED6-deficient AC16 cells treated with vehicle or SCH772984 (n = 3). (D and E) Immunoblot analysis of ANP, LC3B and p62 in wild type and ZBED6-deficient AC16 cells subsequently treated with vehicle or SCH772984 (n = 3). (F and G) Representative α-actinin immunofluorescence images and quantification of the cell size in ZBED6-deficient AC16 cells treated with either SCH772984 or BafA1 for 48 hours (n = 50). Scale bar, 25 μm. Data are expressed as mean ± SEM; * P < 0.05, ** P < 0.01, ns, not significant.