JP2 and JCN Crosstalk Abrogate MURF1-Mediated JCN Ubiquitination and Degradation in Cardiomyocytes

Figures

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  Figure 1. Effects of JP2 Expression on Lipid Overload-Induced Cardiac Injury (A and B) Neonatal mouse cardiomyocytes infected with Ad-JP2 or Ad-HA and exposed to palmitate or oleate for 24 hours. Caspase-3 activity and DNA fragmentation measurements are shown. (C and D) Neonatal mouse cardiomyocytes infected with Ad-shJP2 or Ad-HA for 24 hours. Caspase-3 activity and DNA fragmentation measurements are shown. Tg-JP2/tTA mice and wild-type (WT) littermates fed high-fat diet (HFD) or normal diet (ND) for 4 months. (E) Fractional shortening (FS)% assessed by echocardiography. (F) Caspase-3 activity in heart tissue lysates. (G) Cardiomyocyte cross-sectional area. (Left) Representative photomicrographs of Texas Red-WGA staining (red) with Hoechst 33,342 nuclear counterstain (blue); right panel: quantification. (H) Collagen deposition. (Left) Representative photomicrographs of sirius red staining (red); (right) quantification. Data are mean ± SD, n = 4-10. Two-way analysis of variance followed by Newman-Keuls post hoc test was performed for multiple pairwise comparisons. Student's t-test was used for comparison between 2 groups. ∗P < 0.05 vs oleate + Ad-HA, Ad-HA or ND + WT and †P < 0.05 vs…

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  Figure 2. Determination of JP2-JCN Interaction (A) Identification of JCN peptide sequences in JP2 immunoprecipitates and JP2 peptide sequences in JCN immunoprecipitates from mouse heart lysates by mass spectrometry. (B) Mouse heart lysates were immunoprecipitated with anti-JP2 or anti-JCN antibody followed by western blot analysis using the reciprocal antibodies. IgG served as a negative control. Representative bands from 3 different hearts are presented. (C to E) BiFC assay. (C) Structures of plasmids pVN155/JP2 and pVC155/JCN. (D) A representative band for JP2 and JCN expression in H9c2 cells transfected with indicated plasmid combinations. (E) Representative GFP fluorescence images of H9c2 cells cotransfected with indicated plasmid pairs. Experiments were repeated 3 times using independent cell cultures.

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  Figure 3. JP2 Regulation of JCN Stability A to D Neonatal mouse cardiomyocytes were incubated with palmitate or oleate, or infected with Ad-shJP2 or Ad-HA for 24 hours. JCN protein (A and C) and mRNA (B and D) levels were analyzed. (E) Neonatal mouse cardiomyocytes infected with Ad-JP2 or Ad-HA were exposed to palmitate or oleate for 24 hours. (Top) Representative Western blot band for JCN, (bottom) quantification of JCN protein levels relative to GAPDH. (F) Tg-JP2/tTA and WT mice were fed HFD or ND for 4 months. (Top) Representative Western blot band for JCN from 2 of 8 different hearts per group; (bottom) quantification. (G and H) JCN protein stability. Neonatal mouse cardiomyocytes infected with Ad-JP2 or Ad-HA were exposed to palmitate or oleate. Sixteen hours later, cardiomyocytes were incubated with cycloheximide for different periods and JCN protein levels were determined by Western blot. G Effect of palmitate on JCN protein half-life. H Effect of JP2 overexpression on JCN protein half-life in palmitate-incubated cardiomyocytes. (Top) Representative Western blots for JCN protein; (bottom) quantification. Data are mean ± SD, n = 4-8. Two-way analysis of variance followed by…

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  Figure 4. JCN Ubiquitination and Proteasomal Degradation (A) Neonatal mouse cardiomyocytes incubated with palmitate or oleate in the presence of MG132 or vehicle for 24 h. Upper panel: representative blot for JCN; bottom panel: quantification. (B) H9c2 cells transfected with a DDK-tagged JCN plasmid, infected with Ad-JP2 or Ad-HA, and incubated with palmitate or oleate for 24 hours. DDK-JCN protein was immunoprecipitated using anti-DDK antibody, and ubiquitination was measured using antiubiquitin antibody. Upper panel: representative blot for ubiquitinated DDK-JCN protein; bottom panel: quantification. (C) Neonatal cardiomyocytes infected with Ad-shJP2 or Ad-HA. JCN ubiquitination was assessed by immunoprecipitation (IP) with anti-JCN antibody followed by immunoblotting (IB) with antiubiquitin antibody. (Left) Representative blot for ubiquitinated JCN protein; (right) quantification. (D) Tg-JP2/tTA and WT mice were fed HFD or ND for 4 months. JCN ubiquitination was determined by IP. (Left) Representative blot for ubiquitinated JCN protein; (right) quantification. (E) Mass spectrometry identified JCN ubiquitination sites in palmitate-treated neonatal cardiomyocytes. (F and G) H9c2 cells…

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  Figure 5. Role of MURF1 in JCN Ubiquitination and Degradation A to D Neonatal mouse cardiomyocytes incubated with palmitate or oleate in the presence of MURF1 inhibitor EMBL or vehicle. (A) JCN ubiquitination was assessed by IP. (Top) Representative blot for ubiquitinated JCN protein; (bottom) quantification. (B) (Top) Representative blot for JCN; (bottom) quantification. Caspase-3 activity (C) and DNA fragmentation (D) were determined. (E) Neonatal mouse cardiomyocytes infected with Ad-JP2 or Ad-HA and exposed to palmitate or oleate for 24 hours. MURF1-JCN interaction was performed by immunoprecipitation with anti-MURF1 antibody followed by immunoblotting with anti-JCN antibody. (Top) Representative blot for JCN and MURF1 protein; (bottom) quantification. (F) Neonatal mouse cardiomyocytes were infected with Ad-shJP2 or Ad-HA. MURF1-JCN interaction was detected by IP. (Top) Representative blot for JCN and MURF1 protein; (bottom) quantification. (G) Tg-JP2/tTA and WT mice were fed HFD or ND for 4 months. MURF1-JCN interaction was assessed by IP. (Top) Representative blot for JCN and MURF1 protein; (bottom) quantification. (H to M) WT mice were fed HFD or ND and treated with MURF1 inhibitor…

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  Figure 6. Role of JCN in Caspase-3 Activity and DNA Fragmentation in Palmitate-Induced Cardiomyocytes (A and B) Neonatal mouse cardiomyocytes were infected with Ad-JCN or Ad-HA and exposed to palmitate or oleate for 24 hours. Caspase-3 activity (A) and DNA fragmentation were analyzed (B). (C and D) Neonatal mouse cardiomyocytes were infected with Ad-JCN or Ad-HA combined with Ad-shJP2 or Ad-HA. After 24 hours, caspase-3 activity (C) and DNA fragmentation (D) were analyzed. (E and F) Neonatal mouse cardiomyocytes were infected with Ad-JP2 or Ad-HA combined with Ad-AsJCN or Ad-HA and then exposed to palmitate or oleate for 24 hours. Caspase-3 activity (E) and DNA fragmentation (F) were determined. Data are mean ± SD, n = 4-6. Two-way analysis of variance followed by Newman-Keuls post hoc test was performed for multiple pairwise comparisons. ∗P < 0.05 vs oleate + Ad-HA, Ad-HA + Ad-HA, or oleate + Ad-HA + Ad-HA; †P < 0.05 vs palmitate + Ad-HA, Ad-shJP2 + Ad-HA, or palmitate + Ad-HA + Ad-HA. ∗ and †: P < 0.05; ∗∗ and ††: P < 0.01; ∗∗∗ and †††: P < 0.001. Abbreviations as in Figure 1.

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  Figure 7. Ca2+ Handling in Adult Mouse Cardiomyocytes (A to C) Adult mouse cardiomyocytes were infected with Ad-JP2 or Ad-HA and exposed to palmitate or oleate. RyR2 Ca2+ leak (F/F0), caffeine-induced peak (F/F0), and Tau-SERCA were measured. (D to F) Adult mouse cardiomyocytes were infected with Ad-JCN or Ad-HA and exposed to palmitate or oleate. RyR2 Ca2+ leak (F/F0), Caffeine-induced peak (F/F0), and Tau-SERCA were detected. (G to I) Adult mouse cardiomyocytes were infected with Ad-shJP2 or Ad-HA. RyR2 Ca2+ leak (F/F0), caffeine-induced peak (F/F0), and Tau-SERCA were detected. (J to L) Adult mouse cardiomyocytes were infected with Ad-AsJCN or Ad-HA. RyR2 Ca2+ leak (F/F0), caffeine-induced peak (F/F0), and Tau-SERCA were determined. Data are mean ± SD, n = 9-11 different adult mouse cardiomyocytes in each group. Two-way analysis of variance followed by Newman-Keuls post hoc test was performed for multiple pairwise comparisons. Student's t-test was used for comparison between 2 groups. ∗P < 0.05 vs Ad-HA or oleate + Ad-HA, and †P < 0.05 vs palmitate + Ad-HA. ∗ and †: P < 0.05; ∗∗ and ††: P < 0.01; ∗∗∗ and †††: P < 0.001. Abbreviations as in Figure 1.

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  Figure 8. Determination of JP2 and JCN Protein Levels, Cleaved Caspase-3, JCN-MURF1 Interaction, and JCN Ubiquitination in Heart Tissue Lysates Transverse aortic constriction (TAC)-induced heart failure model was created in Tg-JP2/tTA and WT mice. Heart tissue was analyzed as follows. (A) Western blot. (Left) Representative blots for JP2 and JCN protein. (Middle and Right) Quantification. (B) (Top) representative band of cleaved caspase-3; (bottom) quantification. (C) MURF1-JCN interaction was performed by immunoprecipitation with anti-MURF1 antibody followed by immunoblotting with anti-JCN antibody. (Top) representative bands for JCN and MURF1 protein; (bottom) quantification. (D) JCN ubiquitination was assessed by immunoprecipitation. (Top) representative band for ubiquitinated JCN protein; (bottom) quantification. Data are mean ± SD, n = 6. Two-way analysis of variance followed by Newman-Keuls post hoc test was performed for multiple pairwise comparisons. ∗P < 0.05 vs sham + WT, †P < 0.05 vs TAC + WT. ∗ and †: P < 0.05; ∗∗ and ††: P < 0.01; ∗∗∗ and †††: P < 0.001. Abbreviations as in Figure 1.

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  Figure 9. Schematic Mechanism In normal conditions, JP2 binds to JCN and blocks the MURF1-JCN interaction, thereby preventing MURF1-mediated ubiquitination and proteasome-dependent degradation of JCN. However, the protein level of JP2 is reduced under stress, enabling MURF1 to bind to JCN and promoting MURF1-mediated ubiquitination and degradation of JCN. JCN reduction triggers the excessive opening and Ca2+ release from RyR2, leading to cardiomyocyte injury. In contrast, overexpression of JP2 or JCN protects intracellular Ca2+ homeostasis and prevents cell death under stress.