Preservation of ALYREF Phase Separation Mitigates Doxorubicin-Induced Cardiomyocyte DNA Damage and Cardiotoxicity

Abstract

The clinical utility of the anticancer agent doxorubicin (DOX) is limited by its dose-dependent cardiotoxicity. ALYREF, a nuclear protein that preserves genomic stability through interactions with intranuclear components or as an m⁵C-binding regulator of mRNA maturation and export, has not been previously implicated in DOX-induced cardiotoxicity (DIC). Here, the role and underlying mechanisms of ALYREF in the pathogenesis of DIC are investigated. The findings demonstrate that ALYREF expression is markedly reduced in a murine model of DIC. Myocardial-specific overexpression of ALYREF attenuates DOX-induced DNA damage and cardiomyocyte apoptosis, whereas cardiac-specific knockout of ALYREF (ALYREF CKO) exacerbates DOX-induced cardiac dysfunction. Mechanistically, it is identified that nuclear DOX directly binds to the aspartate residue (D171) within the intrinsically disordered regions (IDRs) of ALYREF, disrupting its liquid-liquid phase separation (LLPS) and promoting its ubiquitin-mediated degradation. The condensate state of ALYREF is essential for maintaining the integrity of the NORAD-activated ribonucleoprotein complex 1 (NARC1). Consequently, disruption of ALYREF LLPS leads to dissociation of the NARC1 complex, resulting in DNA damage and apoptosis in CMs. Collectively, these findings reveal a previously unrecognized mechanism by which DIC via interference with ALYREF condensates, offering new insight into the molecular basis of DIC.

Keywords: ALYREF; DNA damage; cardiotoxicity; doxorubicin; liquid–liquid phase separation.

Figure 1

ALYREF regulates DNA damage and apoptosis in cardiomyocytes. a) Western blot analysis of ALYREF protein levels in DIC mouse hearts (n = 6). b,c) Western blot analysis of ALYREF protein levels in DOX‐treated CMs (n = 6) or hiPSC‐CMs (n = 6). d–k) CMs transfected with ALYREF siRNA or negative control (NC). (d) Representative images (left) and quantification (right) of immunostaining of the DNA damage marker γH2AX (green) in CMs (n = 5). CMs were counterstained with α‐actinin (red, a cardiac marker) and DAPI (blue). Scale bar = 50 µm. (e) Western blot analysis of γH2AX protein levels in CMs (n=6). (f) Representative images and quantification of comet assay in CMs (n = 6). Scale bar = 50 µm. (g) Representative images and quantification of immunostaining of the DAPI in CMs (n = 4). Scale bar=50 µm. (h) Representative images and quantification of TUNEL staining in CMs (n = 4). Scale bar = 50 µm. (i–k) Western blot analysis of Cleaved‐Caspase‐3 (n = 6), Bax (n = 6), and Bcl2 (n = 6) protein levels in CMs. l–s) CMs transfected with ALYREF plasmid and then treated with DOX. (l) Representative images and quantification of immunostaining of γH2AX (green), α‐actinin (red, a cardiac marker) and DAPI (blue) in CMs (n = 5). Scale bar = 50 µm. (m) Western blot analysis of γH2AX protein levels in CMs (n = 6). (n) Representative images and quantification of comet assay in CMs (n = 6). Scale bar=50 µm. (o) Representative images and quantification of immunostaining of the DAPI in CMs (n = 5). Scale bar = 50 µm. (p) Representative images and quantification of TUNEL staining in CMs (n = 6). Scale bar = 50 µm. (q–s) Western blot analysis of Cleaved‐Caspase‐3 (n=6), Bax (n=4), and Bcl2 (n = 6) protein levels in CMs. Data are presented as mean ± SEM. Statistical analysis was performed by (a–k) Student's t‐test, and (l–s) by one‐way ANOVA with Tukey's multiple comparisons test. ^** p < 0.01, and ^*** p < 0.001.

Figure 2

Cardiac‐specific ALYREF deficiency exacerbates doxorubicin‐induced cardiotoxicity in mice. a) Schematic diagram of the experimental procedure for constructing ALYREF CKO mice for DIC.Adult mice received continuous intraperitoneal injections of tamoxifen (30 mg/kg/day) for 5 days to induce ALYREF gene knockout in the heart at week 8. After 2 weeks, a DIC model was induced. b–f) Cardiac function was examined by echocardiography in ALYREF CKO or ALYREF^flox/flox mice treated with DOX or saline. Quantitative analysis of ejection fraction (EF), shortening fraction (FS), end‐diastolic left ventricular internal diameter (LVIDd), and end‐systolic left ventricular internal diameter (LVIDs) (n = 5). g,h) ELISA to measure creatine kinase‐MB (CK‐MB) and cardiac troponin T(cTnT) levels in the serum (n = 5). i) Representative hematoxylin and eosin (H&E), Masson, and Sirius red staining of heart (n = 5). j) Heart weight to tibia length (HW/TL) ratio (n = 5). k) Quantitative analysis of Masson staining (n = 5). l) Representative and quantitative analysis of WGA staining (n = 5). m) Representative images and quantification of immunostaining of γH2AX (green) in heart (n = 5). Cardiac tissue was counterstained with α‐actinin (red, a cardiac marker) and DAPI (blue). Scale bar = 50 µm. n) Representative images and quantification of TUNEL staining in heart (n = 5). Scale bar = 50 µm. o–t) Western blot analysis of γH2AX (n = 6), Cle‐Caspase‐3 (n = 6), Bax (n = 6), and Bcl2 (n = 6) protein levels in heart. Data are presented as mean ± SEM. (c–h,j–t) One‐way ANOVA with Tukey's multiple comparisons test was used. ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001.

Figure 3

Cardiac‐specific overexpression of ALYREF attenuates doxorubicin‐induced cardiotoxicity. a) Schematic representation of the cardiac‐specific overexpression of ALYREF mice and DOX administration. The time points in the picture are representative. b–d) Cardiac function was examined by echocardiography in AAV9‐ALYREF CKO or AAV9‐Vector mice treated with DOX or saline. Quantitative analysis of EF, and FS (n = 10). e,f) ELISA to measure CK‐MB and cTnT levels in the serum (n = 5). g) Representative hematoxylin and eosin (H&E), Masson, Sirius red, WGA, γH2AX, and TUNEL staining of heart (n = 5). Cardiac tissue was counterstained with α‐actinin (red, a cardiac marker) and DAPI (blue). Scale bar = 50 µm. h) Heart weight to tibia length (HW/TL) ratio (n = 10). i–l) Quantitative analysis of Masson, WGA, γH2AX, and TUNEL staining (n = 5). m–r) Western blot analysis of γH2AX (n = 6), Cleaved‐Caspase‐3 (n = 6), Bax (n = 6), and Bcl2 (n= 6) protein levels in heart. Data are presented as mean ± SEM. (c–f, h–r) Statistical analysis was performed by one‐way ANOVA with Tukey's multiple comparisons test. ^** p < 0.01 and ^*** p < 0.001.

Figure 4

ALYREF regulates doxorubicin‐induced DNA damage and apoptosis in cardiomyocytes independent of m⁵C reader function. CMs transfected with ALYREF or ALYREF △K163A plasmid and then treated with DOX. a) Representative images and quantification of immunostaining of γH2AX (green), α‐actinin (red, a cardiac marker) and DAPI (blue) in CMs (n = 6). Scale bar = 50 µm. b) Western blot analysis of γH2AX protein levels in CMs (n = 6). c) Representative images and quantification of comet assay in CMs (n = 4). Scale bar = 50 µm. d) Representative images and quantification of immunostaining of the DAPI in CMs (n = 4). Scale bar = 50 µm. e) Representative images and quantification of TUNEL staining in CMs (n = 4). Scale bar = 50 µm. f–h) Western blot analysis of Cleaved‐Caspase‐3 (n = 4), Bax (n = 3), and Bcl2 (n = 7) protein levels in CMs. Data are presented as mean ± SEM. (a–h) Statistical analysis was performed by one‐way ANOVA with Tukey's multiple comparisons test. **p < 0.01, ^*** p < 0.001.

Figure 5

Doxorubicin induces DNA damage by disrupting the ALYREF liquid–liquid phase separation condensate in cardiomyocytes. a) Representative images of ALYREF (green) immunostaining in CMs treated with DOX at different time points (n = 4). CMs was counterstained with α‐actinin (red, a cardiac marker) and DAPI (blue). Scale bar = 50 µm. b) Representative images of ALYREF (green) immunostaining in CMs isolated from DIC mice (n = 3). c) Western blot analysis of ALYREF protein levels in CMs treated with DOX at different time points (n = 3). d) PONDR Predicts Disordered Regions (IDRs) in the Mouse ALYREF Protein Sequence. e) Representative images and quantification of fluorescence intensity of the CMs overexpressing either EGFP or EGFP‐ALYREF and then EGFP‐ALYREF treated with 5% 1,6‐hexanediol (1,6‐HEX) or DOX. Scale bars = 10 µm. f) Live‐cell time‐lapse imaging shows two adjacent EGFP‐ALYREF aggregates fused (white arrows) and dividing (red arrows) in CMs. Scale bar = 2.5 µm. g) Live‐cell images of fluorescence recovery after photobleaching (FRAP) experiments in CMs overexpressing EGFP‐ALYREF. Left) Representative images of FRAP experiments. (1) The region of photobleaching and analysis. (2) Positive control, un‐photobleached area and analysis. (3) Negative control, the region of un‐photobleaching. Right) Quantification of fluorescence intensity during FRAP assay. Scale bars = 5 µm and 2.5 µm (zoomed‐in image). h) Formation of ALYREF droplets in 250 mmol L⁻¹ Tris‐Hcl and 10% PEG‐8000 solution, and then treated with 1,6‐HEX or DOX. Scale bars = 50 µm. i) The time‐lapse images display fusion of ALYREF droplets in vitro. Scale bars = 50 µm. j–l) CMs transfected with ALYREF and then treated with 1,6‐HEX (0.25%) or DOX. (j) Representative images and quantification of immunostaining of γH2AX (green), α‐actinin (red, a cardiac marker), and DAPI (blue) in CMs (n = 6). Scale bar = 50 µm. (k) Representative images and quantification of comet assay in CMs (n = 6). Scale bar = 50 µm. (l) Representative images and quantification of immunostaining of the DAPI in CMs (n = 6). Scale bar = 50 µm. m) Western blot analysis of ALYREF protein levels in CMs after treated with 1,6‐HEX (0.25%) (n = 6). Data are presented as mean ±SEM. Statistical analysis was performed by (c–m) Student's t‐test, and (j–l) by one‐way ANOVA with Tukey's multiple comparisons test. ^*** p < 0.001.

Figure 6

DOX binds directly to ALYREF proteins. a) Molecular docking analysis of ALYREF binding to DOX. b,c) Representative images and quantification curve of CESTA of ALYREF protein levels in CMs treated with DOX or DMSO (n = 5). d) DARTS analysis of DOX binding to ALYREF (n = 4). e) Live‐cell workstation analysis of EGFP‐ALYREF colocalized with DOX in CMs. Molecular dynamics simulation analysis. f) Root mean square deviation (RMSD) of the protein backbone. g) The root mean square fluctuation (RMSF). h) The radius of gyration (Rg). i) Hydrogen bonding. j,k) Gibbs energy landscape. l) Dynamic ALYREF‐DOX combined simulation diagrams.

Figure 7

Binding of doxorubicin to ALYREF D171^st amino acid induces impaired phase separation and causes DNA damage and apoptosis in cardiomyocytes. Molecular dynamics simulation analysis residue energy. b) Representative images and quantification curve of CESTA of ALYREF △D171 in CMs treated with DOX (n = 5). CMs were transfected by Flag‐tagged mutant plasmids with D171 deletion in ALYREF (ALYREF △D171). c) DARTS analysis of DOX binding to ALYREF△D171 (n = 4). d) Representative images of the CMs overexpressing EGFP, EGFP‐ALYREF, EGFP‐ALYREF deletion C‐terminal IDRs (EGFP‐ALYREF△IDR), or EGFP‐ALYREF △D171. Scale bars = 10 µm. e–l) CMs transfected with ALYREF△D171 plasmid and then treated with DOX. (e) Representative images and quantification of immunostaining of γH2AX (green), α‐actinin (red, a cardiac marker), and DAPI (blue) in CMs (n = 6). Scale bar = 50 µm. (f) Western blot analysis of γH2AX protein levels in CMs (n = 6). (g) Representative images and quantification of comet assay in CMs (n = 6). Scale bar = 50 µm. (h) Representative images and quantification of immunostaining of the DAPI in CMs (n = 6). Scale bar = 50 µm. (i) Representative images and quantification of TUNEL staining in CMs (n = 6). Scale bar = 50 µm. (j–l) Western blot analysis of Cleaved‐Caspase‐3 (n = 3), Bax (n = 6), and Bcl2 (n = 7) protein levels in CMs. Data are presented as mean ± SEM. (e–l) Statistical analysis by one‐way ANOVA with Tukey's multiple comparisons test. ^*** p < 0.001.

Figure 8

Doxorubicin affects genome stability by causing dissociation of the NARC1 complex. a,b) Immunoprecipitation analysis of ALYREF interaction with RBMX, CDCL5, TOP1, and PRPF19 in CMs treated with DOX or overexpressing Flag‐ALYREF D171 plasmid. c) Immunoprecipitation analysis of RBMX interaction with ALYREF, CDCL5, TOP1, and PRPF19 in CMs treated with DOX. d) Western blot analysis of RBMX protein levels in CMs treated with DOX at different time points (n = 3). e) Western blot analysis of RBMX protein levels in CMs after transfected with ALYREF siRNA (n = 3). f) Immunoprecipitation analysis of RBMX interaction with CDCL5, TOP1, PRPF19 in CMs after transfected with ALYREF siRNA. g–n) CMs after cotransfection with RBMX and ALYREF plasmid and then treated with DOX. (g) Representative images and quantification of immunostaining of γH2AX (green), α‐actinin (red, a cardiac marker), and DAPI (blue) in CMs (n = 6). Scale bar = 50 µm. (h) Western blot analysis of γH2AX protein levels in CMs (n = 6). (i) Representative images and quantification of comet assay in CMs (n = 5). Scale bar = 50 µm. (j) Representative images and quantification of immunostaining of the DAPI in CMs (n = 3). Scale bar = 50 µm. (k) Representative images and quantification of TUNEL staining in CMs (n = 3). Scale bar = 50 µm. (l–n) Western blot analysis of Bax (n = 5), Bcl2 (n = 5), and Cle‐Caspase‐3 (n = 5) protein levels in CMs. Data are presented as mean ± SEM. Statistical analysis was performed by (d,e) Student's t‐test, and (g–n) by one‐way ANOVA with Tukey's multiple comparisons test. ^* p < 0.05, ^** p < 0.01, and ^*** p < 0.001.