Cardiomyocyte-Specific RNF128 Attenuates Pathological Cardiac Hypertrophy Progression by Stabilizing SERCA2a through Lys63-Linked Polyubiquitination

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  Figure 1. RNF128 is downregulated in cardiomyocytes during cardiac hypertrophy. A, Heatmap of differentially expressed RNF family members in GSE151254 datasets. B, single-cell RNA sequencing (scRNA-seq) showing 8 clusters of cells from mouse hearts. C to E, RNF128 is highly enriched in cardiomyocytes. F, scRNA-seq revealed a reduced proportion of total positive expression abundance of Rnf128 among total cardiac cells after TAC surgery. G, Representative images of in situ RNAscope and immunofluorescence staining of RNF128 in sham and hypertrophic heart sections (scale bar = 50 μm). H, The percentage of RNF128 positive area in sham and hypertrophic heart (n = 6 mice, vs sham, one-way ANOVA). I, Quantification of RNF128 RNA dots in sham and hypertrophic heart tissue (n = 6 mice per group, vs sham, one-way ANOVA). J, Representative immunoblotting images of RNF128 protein levels in sham, Ang II and TAC tissue. K, Representative western blot images showing RNF128 level in dilated cardiomyopathy patients (DCM, n = 5), hypertrophic patients (n = 5) and non-cardiac disease control donors (NCC, n = 4). L to M, Quantification of RNF128 level in mouse tissues (n ≥ 5 mice, vs sham, one-way ANOVA). N,…

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  Figure 2. RNF128 deficiency aggravates TAC (transverse aortic constriction) induced cardiac hypertrophy. A, Representative M-mode, pulse-wave doppler (PW) and tissue doppler (TD) images from each group in mice. B, Ejection fraction (EF), fractional shortening (FS) and early diastolic mitral inflow velocity to early diastolic mitral annular velocity ratio (E/e’). C, Representative image of whole heart (scale bar = 2 mm). D, Heart weight to tibial length ratio (HW/TL). E, Heart weight to body weight ratio (HW/BW). F, Representative image of hematoxylin-eosin staining (H&E). G, Gross heart sectional area is elevated in TAC treated RNF128cKO group. H, Wheat germ agglutinin (WGA) staining showing cardiomyocyte cross-sectional area. I, Quantification of cardiomyocyte cross-sectional area measured by WGA (n = 60 cells from 6 mice of each group, Kruskal–Wallis H test, and post-hoc pairwise comparisons were conducted with the Mann–Whitney U test). J, Masson and Sirius Red staining in heart sections. K, Statistical analysis of left ventricular fibrosis by Sirius Red staining. L to M, Immunohistochemical Staining of BNP and MYH7 in heart sections. N, The serum ANP and BNP concentrations in TAC models…

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  Figure 3. RNF128 knockout promotes angiotensin II (Ang II)-induced myocardial hypertrophy. A, Representative M-mode image from each group. B, Cardiac function is significantly impaired in RNF128cKO mice. C, Representative gross-heart images (scale bar = 2 mm). D, RNF128cKO mice show exacerbated cardiac hypertrophy, indicated by elevated HW/BW and HW/TL ratios. E, representative H&E-stained sections. F, quantification of cardiac cross-sectional area. G to H, WGA staining shows significantly enlarged cross-sectional area of single cardiomyocytes in RNF128cKO mice. (scale bar = 50 μm, n = 60 cells from 6 mice, Kruskal–Wallis H test, and post-hoc pairwise comparisons were conducted with the Mann–Whitney U test). I to K, Masson and Sirius Red staining revealed higher left ventricular fibrosis in RNF128cKO mice. L, Immunohistochemical staining of BNP in heart sections. M, Immunohistochemical staining of MYH7 in heart sections. N, The serum ANP and BNP concentration. (n = 6 mice per group, Student’s t-test). O, Real-time qPCR analysis of Nppa, Nppb, MYH7, Tgfb1 and Col1a1 in heart tissues. (B, D, F, H, K, L, M, O: n = 6 mice per group. All histogram data are displayed as mean ± standard…

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  Figure 4. RNF128 ameliorates cardiomyocyte hypertrophy and maintains calcium metabolism in cardiomyocytes. A to B, neonatal mouse cardiomyocytes (NMCM) treated with PBS or Ang II stained by TRITC Phalloidin and cell area quantification (scale bar = 50 μm, n = 9 cells). C to D, Representative images of NMCM immunoblotting and relative protein expression of BNP and MYH7. E, the UMAP of 10 cell subtypes among TAC treated RNF128fl/fl and RNF128cKO mice. F, Gene Ontology (GO) pathway of upregulated genes in RNF128cKO cardiomyocytes. G, Gene Set Enrichment Analysis (GSEA) of cardiac muscle contraction and action potential pathway. H, Workflow diagram of protein profiling and multi-omics sequencing. I, five common genes of altered proteins and RNF128 interacting proteins (Gene names: names of common proteins; Intensity: intensity of interacting protein mass spectrometry; Score: score of Co-IP mass spectrometry; Fold Change: expression fold change in protein profiling of heart tissue; p-value: heart tissue protein profiling). J, Coimmunoprecipitation of RNF128 and SERCA2a in NMCMs. K, Representative immunoblotting image of SERCA2a and RNF128 in NMCMs (n = 5 biological replicates, one-way ANOVA).…

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  Figure 5. RNF128 directly catalyzes SERCA2a’s K63-linked ubiquitination and prevents its degradation. A, Schematic illustration of 6 mutants of RNF128 plasmids. B, Coimmunoprecipitation of Flag-tagged RNF128 mutants and Myc-tagged SERCA2a in HEK293T (293T) cells. C, RNF128 catalyzes K63, not K48 polyubiquitination of SERCA2a. D, the regulatory effect of RNF128 on SERCA2a ubiquitination is completely abolished when ubiquitin is mutated at lysine 63 (K63R). E, the catalytic function of RNF128 requires RING domain. F to G, RNF128 increased SERCA2a’s stability in a dose-dependent way in 293T cells (n = 3 biological replicates, vs 0 μg, one-way ANOVA). H, RNF128 catalyzed SERCA2a’s ubiquitination in vivo. I, RNF128 catalyzed SERCA2a’s ubiquitination in NMCMs. J to K, RNF128 increased SERCA2a’s stability in a concentration-dependent way in NMCMs cells (n = 4 biological replicates, vs 0 MOI, one-way-ANOVA). L, Common modification sites of SERCA2a in 293T cells and heart tissues (sites with localization probability > 0.75 were defined as confident identifications. Intensity indicates peak intensity of the peptide fragment containing the modification site). M, Lysine 476 to alanine mutation (K476A)…

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  Figure 6. RNF128 maintains calcium homeostasis in cardiomyocytes by suppressing autophagy-lysosome degradation of SERCA2a. A, Representative western blotting for SERCA2a in NMCMs treated with MG132 (Carbobenzoxy-L-leucyl-L-leucyl-L-leucinal), CQ (Chloroquine), NH4Cl (Ammonium chloride), and 3-MA (3-Methyladenine) for 6 hours. B, Representative western blotting for SERCA2a in NMCMs treated with BafA1 (bafilomycin A1), VPS34-IN1 and Epoxomicin for 6 hours. C to D, Quantification of SERCA2a (n = 4 biological replicates, vs Si-NC of each inhibitor, two-way ANOVA). E to F, Silencing ATG5 or ATG7 inhibits SERCA2a’s degradation in RNF128 knockdown NMCMs (F, n = 5 biological replicates, vs Si-RNF128+Si-NC, one-way ANOVA). G, Immunofluorescence staining of SERCA2a and lysosome-associated membrane protein 2 (LAMP2, Scale bar = 2μm). H, Pearson correlation coefficient of SERCA2a and LAMP2 in RNF128fl/fl and RNF128cKO cardiomyocytes (n = 5 biological replicates, Student’s t-test). I, Representative western blotting of coimmunoprecipitation of myc-SERCA2a with Flag-tagged autophagy cargo receptors in 293T cells. J, Representative western blotting of coimmunoprecipitation of SERCA2a with Flag-tagged…

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  Figure 7. RNF128 rescues cardiac dysfunction and pathological remodeling through SERCA2a in vivo. A, Representative western blotting of cardiac tissues from RNF128fl/fl or RNF128cKO mice injected with AAV9-Vector/ AAV9-RNF128/ AAV9-SERCA2a. B to E, cardiac dysfunction was attenuated by RNF128 or SERCA2a restoration. M-mode images of each group (B), EF (C), FS (D) and E/e’ (E) quantification. F, Whole heart images (scale bar = 2 mm). G, Cardiac hypertrophy indicated by HW/TL was reduced after restoration of RNF128 or SERCA2a. H, Representative Hematoxylin–eosin (H&E) images. I, Quantification of heart sectional area. J to K, WGA staining and quantification of cardiomyocyte cross-sectional area (K, scale bar = 50 μm, n = 60 cells from 6 mice per group, Kruskal–Wallis H test with Mann–Whitney U test post-hoc pairwise comparisons). L to M, Masson and Sirius Red staining of heart sections and quantification of left ventricular fibrosis. (A, C, D, E, G, I, M, Bar graphs represent mean ± SD, n = 6 mice per group, vs RNF128cKO + AAV9-Vector, one-way ANOVA with Dunnett’s post-hoc test was used for intergroup comparisons, with adjusted p-values displayed for multiple testing. ns, p > 0.05, * p <…

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  Figure 8. RNF128 overexpression relieves Ang II-induced hypertrophic remodeling. A, Flowchart for cTnT promote-driven Adeno-Associated Virus Serotype 9 (AAV9cTnT) injection followed by Ang II-infusion. B, Representative M-mode images. C, EF, FS and E/e’. D, RNF128 upregulation mitigates Ang II-induced cardiac enlargement (scale bar = 2 mm). E, RNF128 upregulation reverses the increased HW/BW and HW/TL ratios triggered by cardiac hypertrophy. F to G, H&E staining showing gross heart sectional area. H and I, WGA staining showed decreased cardiomyocyte cross-sectional area after RNF128 overexpression (n = 60 cells from 6 mice per group, Kruskal–Wallis H test, and post-hoc pairwise comparisons were conducted with the Mann–Whitney U test). J to K, Masson and Sirius Red in sections of hearts suggesting reduced left ventricular fibrosis in RNF128oe mice. L to M, Immunohistochemical (IHC) of BNP and MYH7 in heart sections. N, The serum ANP and BNP concentration. (n = 6 mice for each group, Welch’s t-test). O, Real-time qPCR analysis of Nppa, Nppb, Myh7, Col1a1 and Tgfb1 in heart tissues. (C, E, G, K, M, O, Bar graphs represent mean ± SD, n = 6 mice per group, Šídák’s multiple comparisons test was…