Abstract
Targeting the cardiomyocyte cell cycle is a promising strategy for heart repair following injury. Here, we identify a cardiac-regeneration-associated PIWI-interacting RNA (CRAPIR) as a regulator of cardiomyocyte proliferation. Genetic ablation or antagomir-mediated knockdown of CRAPIR in mice impairs cardiomyocyte proliferation and reduces heart regenerative potential. Conversely, overexpression of CRAPIR promotes cardiomyocyte proliferation, reduces infarct size and improves heart function after myocardial infarction. Mechanistically, CRAPIR promotes cardiomyocyte proliferation by competing with NF110 for binding to the RNA-binding protein PA2G4, thereby preventing the interaction of PA2G4 with the NF110–NF45 heterodimer and reducing NF110 degradation. The ability of CRAPIR to promote proliferation was confirmed in human embryonic stem cell-derived cardiomyocytes. Notably, CRAPIR serum levels are lower in individuals with ischemic heart disease and negatively correlate with levels of N-terminal pro-brain natriuretic peptide. These findings position CRAPIR both as a potential diagnostic marker for cardiac injury and as a therapeutic target for heart regeneration through the PA2G4–NF110–NF45 signaling axis.
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Data availability
The RNA sequencing raw data generated in this study have been deposited in Sequence Read Archive at the NCBI Center under accession number PRJNA1186848. Other data are available in the main article and Supplementary Information. Source data are provided with this paper.
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Acknowledgements
This work was supported by the National Natural Science Foundation of China (92168119 to B.C., 82100300 and 82470266 to W.M., 82272389 to Yu Liu, 82300302 to Yining Liu, 82330011 and U21A20339 to B.Y.), the HMU Marshal Initiative Funding (HMUMIF-21018 to B.C.) and the key project of Natural Science Foundation of Heilongjiang Province (ZD2023H001 to N.W.).
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B.C. and W.M. conceived the study concept. H.C., W.H., Yanan Tian, Z.R., Xin Wang, J.L., Q.O., Y.H., H.J., X.L., XiuXiu Wang and Yining Liu performed the experimental studies. H.C., Yanan Tian and W.H. carried out the data analysis. B.C., W.M., Yu Liu, Ye Tian, F.L., N.W. and B.Y. wrote the manuscript. B.C., W.M. and N.W. responded to the editors’ and reviewers’ comments. B.C., W.M., Yu Liu, Yining Liu and B.Y. provided the funding. All authors reviewed the manuscript.
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Extended Data
Extended Data Fig. 1 The conservation and abundance of piRNAs.
a, The distribution of piRNA length. b, Heatmap of differentially expressed piRNAs in heart tissues. c, The blast of piRNA sequences between the mouse and human using NCBI’s blast tool. d, The expression of small RNAs in heart and cardiomyocytes (n=3, 4 biological replicates). The high expression of these three piRNAs was confirmed by comparing their expression levels to those of the well-known highly expressed miR-1 and miR-208 in the heart, and the rarely expressed miR-122 and miR-124, which are specifically expressed in liver or brain tissue, respectively. e, The expression level of AB352916, DQ540981 and AB351100 in postnatal day 1 (P1) or day 7 (P7) mouse heart tissues (n=3, 4 mice). Data are presented as mean ± s.e.m. Statistical analyses were performed by Two-tailed Student’s t test with or without Welch’s correction (e). AR, apex resection.
Extended Data Fig. 2 The verification of piRNA AB352916 expression and features.
a, The expression of AB352916, DQ540981 and AB351100 in cardiomyocytes after transfecting with their antagomirs (n=4 independent experiment). b, The effects of DQ540981 and AB351100 antangomir on cardiomyocyte proliferation (n=3 independent experiment). c, The expression of AB352916 in cardiomyocytes after transfected with agomir (n=4 independent experiment). d, Detection of 2′‐O‐methylation at the 3′ end of AB352916 and miRNAs using RTL‐P approach. This experiment was repeated three times independently. e, The detection of the binding of PIWI proteins to CRAPIR using RNA pull down analysis and Western blot. This experiment was repeated three times independently. f, The expression of AB352916 in cardiomyocytes isolated from failing mouse hearts (n=5 biological replicates). g, The expression of AB352916 in different tissues (n=3 mice). h, The distribution of AB352916 between the nucleus and cytoplasm in cardiomyocytes (n=3 biological replicates). i, The distribution of AB352916 between the nucleus and cytoplasm analyzed using FISH. Scale bar: 5 μm. This experiment was repeated three times independently. Data are presented as mean ± s.e.m. Statistical analyses were performed by Two-tailed Student’s t test with (a, c) or without Welch’s correction (a, f, g), one-way ANOVA followed by Tukey’s Multiple Comparison tests (b). ago-NC, negative control for agomir; anta-NC, negative control for antagomir.
Extended Data Fig. 3 The effects of CRAPIR knockout on the proliferation of cardiomyocytes.
a, The schematic for the construction of CRAPIR KO mice. b and c, Representative P1 and P7 heart tissue sections stained with AuroraB, and the quantification of the percentage of AuroraB+ cardiomyocytes (n=3 mice). White arrows point the positive cardiomyocytes. Scale bar: 20 μm. Data are presented as mean ± s.e.m. Statistical analyses were performed by Two-tailed Student’s t test with Welch’s correction (c) or without Welch’s correction (b, c).
Extended Data Fig. 4 CRAPIR promotes cardiomyocyte proliferation and improves cardiac functions in P7 and adult mice with MI.
a, Masson staining of CKI mice subjected to MI. Scale bar: 500 μm. b, Cardiac sections from P7 mouse hearts stained with AuroraB at 3 days after MI (n=3 mice). Scale bar: 20 μm. c, The fibrosis of adult mice after MI for one month was studied using Masson staining (n=7 mice). Scale bar: 500 μm. d and e, Cardiac sections from adult mouse hearts stained with pH3 and Ki67 at 3 days after MI (n=3 mice). Scale bar: 50 μm. f, Cardiac sections from adult mouse hearts stained with AuroraB at 3 days after MI (n=3 mice). Scale bar: 20 μm. g, Echocardiography was used to observe cardiac function of adult mice at 28 days after MI (n=6 mice). Data are presented as mean ± s.e.m. Statistical analyses were performed by Two-tailed Student’s t test with (e, f) or without Welch’s correction (b-g). MI, myocardial infarction; ago, CRAPIR agomir; ago-NC, negative control for agomir.
Extended Data Fig. 5 The expression of PA2G4 and the proteins binding to PA2G4.
a and b, The mRNA and protein expression of PA2G4 in cardiomyocytes transfected with PA2G4 siRNA and plasmid (n=5, 6 biological replicates). c and d, The protein expression level of PA2G4 in heart tissues (n=4 mice for c, 5 mice for d). e, Venn diagram showing overlapping proteins bind to PA2G4. Proteins with PSM values exceeding 5, unique peptide values greater than 10, and decreased abundance in PA2G4-bound proteins post-CRAPIR overexpression, which falls below 0.2 times that of the control group, were identified and selected. f, The structure of ILF2 and ILF3 proteins. g, Secondary mass spectrum for ILF2 and ILF3. Data are presented as mean ± s.e.m. Statistical analyses were performed by Two-tailed Student’s t test with (a) or without Welch’s correction (a-d). AR, apex resection; NC siRNA, negative control for siRNA; OE, overexpression.
Extended Data Fig. 6 The effects of NF110 overexpression on cardiomyocyte proliferation and the influence among NF45, NF110, and PA2G4.
a, NF110 overexpression promotes cardiomyocyte proliferation (n=3 independent experiment). b, The effects of PA2G4 siRNA on the expression of NF90 and TCP80 (n=8 independent experiment). c, The effects of PA2G4 overexpression on the expression of NF110 in cardiomyocytes. This experiment was repeated three times independently. d, The quantification of Western blot results indicating the effects of PA2G4 on the expression of NF110 and NF45 in the nucleus and cytoplasm in cardiomyocytes (n≥4 independent experiment). e and f, The effects of NF110 and NF45 siRNAs on the protein expression level of PA2G4, NF110, and NF45 in cardiomyocytes (n≥5 independent experiment). g, The inhibitory effect of PA2G4 overexpression on the expression of NF110 was blocked by MG132 in cardiomyocytes. This experiment was repeated three times independently. h, Secondary mass spectrum for RNF13. Data are presented as mean ± s.e.m. Statistical analyses were performed by Two-tailed Student’s t test with (b, d, e) or without Welch’s correction (a, b, d-f). NC siRNA, negative control for siRNA; OE, overexpression.
Extended Data Fig. 7 The effects of CRAPIR on the expression of NF110 and NF45 in cardiomyocytes and knockdown of NF45 attenuates the effects of CRAPIR on cardiomyocyte proliferation.
a, The protein expression level of NF110 and NF45 in cardiomyocytes transfected with CRAPIR (n≥6 independent experiment). b, The effects of CRAPIR on the expression of NF110 and NF45 in the nucleus and cytoplasm analyzed by Western blot. This experiment was repeated three times independently. c, The proliferative ability of cardiomyocytes was analyzed by the detection of EdU, pH3 and Ki67 (n=3 independent experiment). d, Co-IP analysis revealing the ubiquitination of NF110 was induced by CRAPIR antagomir in cardiomyocytes. This experiment was repeated three times independently. Data are presented as mean ± s.e.m. Statistical analyses were performed by Two-tailed Student’s t test with or without Welch’s correction (a), one-way ANOVA followed by Tukey’s Multiple Comparison tests (c). NC siRNA, negative control for siRNA; ago, CRAPIR agomir; ago-NC, negative control for agomir; anta, CRAPIR antagomir; anta-NC, negative control for antagomir.
Extended Data Fig. 8 Silencing of NF110 inhibits the improvement of CRAPIR on heart function after MI.
a, The heart contractile function was analyzed using Echocardiographic analysis (n=6 mice). b, The quantification of the percentage of pH3+ and Ki67+ cardiomyocytes (n=3 mice). c, The expression of cell cycle genes (n=4 biological replicates). Data are presented as mean ± s.e.m. Statistical analyses were performed by one-way ANOVA followed by Tukey’s Multiple Comparison tests (a-c). NC siRNA, negative control for siRNA; ago-CRAPIR, CRAPIR agomir; ago-NC, negative control for agomir; MI, myocardial infarction.
Extended Data Fig. 9 The effects of PA2G4 mutants on cardiomyocyte proliferation.
The proliferative ability of cardiomyocytes was analyzed by the detection of pH3 and Ki67 (n=3 independent experiment). Data are presented as mean ± s.e.m. Statistical analyses were performed by one-way ANOVA followed by Tukey’s Multiple Comparison tests.
Extended Data Fig. 10 The schematic of CRAPIR function in cardiomyocyte proliferation.
CRAPIR directly binds to PA2G4, affecting the interaction of PA2G4 with NF110-NF45 heterodimer and recruiting the ubiquitin ligase RNF13, reducing the degradation of NF110, thus promoting cardiomyocyte proliferation.
Supplementary information
Supplementary Information
Supplementary Fig. 1.
Supplementary Tables 1–4
Supplementary Table 1 The proteomic data of CRAPIR pulldown. Supplementary Table 2 The quantification of western blot results indicating the binding of PA2G4 to NF110 and NF45. Supplementary Table 3 The quantification of western blot results indicating the binding of ILF3 to PA2G4 and NF45. Supplementary Table 4 The quantification of western blot results indicating the binding of NF45 to NF110 and PA2G4.
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Ma, W., Chen, H., Tian, Y. et al. The highly conserved PIWI-interacting RNA CRAPIR antagonizes PA2G4-mediated NF110–NF45 disassembly to promote heart regeneration in mice. Nat Cardiovasc Res 4, 102–118 (2025). https://doi.org/10.1038/s44161-024-00592-z
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DOI: https://doi.org/10.1038/s44161-024-00592-z
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