Fig 2: RBM22 is required for cardiomyocyte proliferation and neonatal heart regeneration.a Mouse experimental strategy. Tamoxifen was injected from P0 to P3 to induce Rbm22 deletion in cardiomyocytes. AR was performed on the control mice and Rbm22fl/flαMHC-MerCreMer+ mice (Rbm22-iCKO) at P3. Heart tissues were collected at 7 and 21 dpr. EdU incorporation was quantified at 7 dpr to assess cardiomyocyte proliferation. Histology and echocardiography analyses were performed at 21 dpr to evaluate cardiac morphology and function. b–f Representative left ventricular M-mode echocardiography images (b) and echocardiography analyses (c–f) in neonatal control mice or Rbm22-iCKO mice at 21 dpr (n = 8 mice per group). g, h Masson trichrome staining (g) and quantification (h) of fibrotic scars of the control and Rbm22-iCKO mouse hearts at 21 dpr (n = 5 mice per group). i Statistical analysis of the regeneration number of mice between heart regeneration and non-regeneration (n = 10 mice per group). j, k WGA staining (j) and quantification of the cardiomyocyte size (k) in heart tissue sections from control and Rbm22-iCKO mice at 21 dpr (n = 5 mice per group). WGA (green), DAPI (blue). l, Anlysis of total cardiomyocyte counts per heart across different experimental groups (n = 5 mice per group). m–r Representative images of EdU incorporation (m), phospho-histone H3 (pH3)- (o), and Aurora B- (q) positive cardiomyocytes, with the corresponding quantification presented in panels (n, p, and r), in the control and Rbm22-iCKO hearts at 7 dpr (n = 5 mice per group). α-actinin (green), EdU, pH3, or Aurora B (red, indicated by yellow arrows), DAPI (blue). Data are presented as mean ± SD. Statistical analyses were performed by one-way ANOVA with Tukey’s multiple comparisons test. ns, not significant. Source data are provided as a Source Data file.

Fig 3: Rbm22 knockout impairs cardiac repair by inhibiting cardiomyocyte proliferation in adult mice post-MI.a Scheme of mouse experimental design. Eight-week-old control and Rbm22-iCKO mice were administered tamoxifen and then subjected to MI. Cardiomyocyte proliferation detection by immunofluorescence staining was performed at 14 dpi. Adult cardiomyocytes were isolated at 28 dpi, while echocardiography and histological analyses were conducted at 28 dpi. b, c Representative M-mode tracing images (b) and echocardiographic analysis of cardiac function (c) in the hearts of control and Rbm22-iCKO mice at 28 dpi (n = 5 mice per group). d, e Masson trichrome staining (d) and quantification (e) of fibrotic scar in sequential heart sections from control and Rbm22-iCKO mice at 28 dpi (n = 5 mice per group). f, g Representative images (f) and quantification of the number of total rod-shaped cardiomyocytes per heart (g) dissociated at 28 dpi (n = 5 mice per group). h–j Representative images (h) and quantification of cardiomyocyte nucleation (i) and diploidy of isolated cardiomyocytes (j) from Rbm22-iCKO mice heart at 28 dpi (n = 3 mice per group). k, l WGA staining (k) and quantification of the size of cardiomyocytes (l) in heart sections from control and Rbm22-iCKO mice at 28 dpi (n = 5 mice per group). m–r Representative images and quantification of cTnT-positive cardiomyocytes co-labeled with EdU (m, n), pH3 (o, p), and Aurora B (q, r) in control and Rbm22-iCKO hearts at border zones at 14 dpi (n = 5 mice per group). cTnT (green), EdU, pH3, or Aurora B (red, indicated by yellow arrows), DAPI (blue). Data are presented as mean ± SD. Statistical analyses were performed by one-way ANOVA with Tukey’s multiple comparisons test (c, e, g, j, l, n, p, and r) and two-way ANOVA with Sidak’s multiple comparisons test (i). Source data are provided as a Source Data file.

Fig 4: Loss of Rbm22 reprograms the transcription networks that govern cardiomyocyte cell cycle activation.a–c Representative images (left) and quantification (right) of neonatal mouse cardiomyocyte cultures stained with α-actinin and Ki67 (a), pH3 (b), and Aurora B (c). Yellow arrows indicate Ki67-, pH3-, and Aurora B-positive cardiomyocytes, respectively (n = 5 per group). d Flow cytometry analysis of cell cycle distribution in cardiomyocytes transfected with si-Ctrl #1 and si-Rbm22 #1 (n = 5 per group). e GSEA plot based on the whole transcriptome. f GSEA analysis of genes involved in DNA replication pathways. g Heatmap of genes enriched in the DNA replication pathway identified by GSEA as in (f). h Heatmap of DEGs involved in cell cycle and cell division. A color scale indicating the relative signal intensity plotted on each heatmap is shown in the middle. i Gene interaction network of the DEGs. j, k,Validation of the key genes encoding cell cycle regulator proteins from RNA-seq by qRT-PCR (j) and immunoblot assays (k) in cardiomyocytes silenced with si-Ctrl #1 and si-Rbm22 #1 (n = 6 per group in j, n = 3 per group in k). Data are presented as mean ± SD. Statistical analyses were performed by one-way ANOVA with Tukey’s multiple comparisons test (a–c right), two-way ANOVA with Sidak’s multiple comparisons test (d), two-tailed unpaired t test (j and k), and multiple hypothesis test (f). Source data are provided as a Source Data file.

Fig 5: RBM22 overexpression induces mitotic proliferation in human iPSC-derived cardiomyocytes and promotes cardiac repair through cell cycle re-entry post-injury.a Representative immunoblot analysis (left) and relative quantification (right) of RBM22 expression in hiPSC-CMs treated with AAV9-Ctrl or AAV9-Rbm22 (n = 3 per group). b qRT-PCR analysis of RBM22 mRNA levels in hiPSC-CMs (n = 3 per group). c, d Representative images and quantification of EdU incorporation (c) and Aurora B– (d) positive hiPSC-CMs treated with AAV9-Ctrl or AAV9-Rbm22 (n = 5 per group). e qRT-PCR analysis of function, maturation genes, and cell cycle regulatory genes in hiPSC-CMs (n = 5 per group). f Flow cytometry analysis of the proliferative ability of CTV-labeled hiPSC-CMs in sorted populations (n = 5 per group). g Schematic summary showing that RBM22 promotes cardiomyocyte proliferation after myocardial injury by enhancing chromatin accessibility and transcriptionally activating cell cycle-related gene expression. Under physiological conditions, RBM22 expression gradually decreases with age, coinciding with the exit of the cardiac regenerative window. Upon myocardial injury, RBM22 recruits SMARCA4 to the promoters of cell cycle-related genes for chromatin accessibility, and facilitates RNA Pol II-mediated transcription of these genes, ultimately promoting cardiomyocyte proliferation and cardiac regeneration. AAV9-mediated gene therapy enhances RBM22 expression in cardiomyocytes, thereby promoting cardiomyocyte proliferation and facilitating cardiac regeneration after myocardial injury. Created in BioRender. Xia, Q. (2026) https://BioRender.com/v9wqipa. Data are presented as mean ± SD. Statistical analyses were performed by two-tailed unpaired t test (a–f). Source data are provided as a Source Data file.