ROS‐ATM‐CBP axis‐mediated PARP1 lactylation aggravates doxorubicin‐induced cardiotoxicity

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  FIGURE 1. CBP is the lactyltransferase for PARP1 lactylation. (A, B) Lactylation of endogenous PARP1 was determined by Co‐IP using anti‐PARP1 or anti‐PanKla antibodies in HL‐1 cells. (C) CBP can specifically catalyze lactylation of PARP1 in vitro. (D, E) Co‐IP of endogenous proteins from HL‐1 cell lysates using anti‐CBP or Anti‐PARP1 antibody, followed by immunoblot of CBP, PARP1 and Tubulin. (F, G) HL‐1 cells treated with or without CBP activator or CBP inhibitors were lysed and immunoprecipitated using anti‐PARP1 antibody, followed by detection of Pan‐KLA. (H) Full‐length Myc‐PARP1 or six truncated Myc‐PARP1 plasmids were transfected, respectively, and total lysates were subjected to immunoprecipitation with anti‐Myc antibody, followed by detection of Pan‐KLA. (I) The mass spectrometry analysis results of the lactylation site of PARP1. (J) HL‐1 cells transfected with Myc‐PARP1‐WT, Myc‐PARP1‐mut 5KR and Flag‐CBP were lysed and immunoprecipitated using anti‐Myc antibody, followed by detection of Pan‐KLA. (K) HL‐1 cells transfected with Myc‐PARP1‐WT and Myc‐PARP1‐mut 5KR, with or without the treatment of CBP inhibitor, were lysed and immunoprecipitated using anti‐Myc antibody, followed by…

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  FIGURE 2. SIRT1 is the delactylase for PARP1. (A) SIRT1 can specifically erase lactylation of PARP1 in vitro. (B, C) Co‐IP of endogenous proteins from HL‐1 cell lysates using anti‐SIRT1 or Anti‐PARP1 antibody, followed by immunoblot of SIRT1, PARP1 and Tubulin. (D–F) HL‐1 cells were transfected with SIRT1 siRNAs or treated with SIRT1 inhibitor or SIRT1 activator. IP of endogenous proteins from HL‐1 cell lysates was performed using anti‐PARP1 antibodies, followed by detection of Pan‐KLA. (G) HL‐1 cells transfected with Myc‐PARP1‐WT, Myc‐PARP1‐mut 5KR and SIRT1 siRNA were lysed and immunoprecipitated using anti‐Myc antibody, followed by detection of Pan‐KLA (n = 3 per group). (H) HL‐1 cells transfected with Myc‐PARP1‐WT and Myc‐PARP1‐mut 5KR and Flag‐SIRT1 were lysed and immunoprecipitated using anti‐Myc antibody, followed by detection of Pan‐KLA. All immunoblots had three biological repetitions, with similar results.

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  FIGURE 3. LDHA regulates lactate production and PARP1 lactylation. (A–C) Relative intracellular lactate content of HL‐1 cells after transfection with Myc‐LDHA or LDHA siRNA or LDHA inhibitor treatment (n = 10 per group). (D) Relative intracellular lactate content in myocardial tissues of LDHA‐cKO and LDHA‐cWT mice (n = 10 per group). (E–G) HL‐1 cells were transfected with Myc‐LDHA or LDHA siRNA or LDHA inhibitor treatment, and endogenous proteins in HL‐1 cell lysates were immunoprecipitated using anti‐PARP1 antibody, followed by detection of Pan‐KLA. (H) Cardiomyocytes from LDHA‐cWT or LDHA‐cKO mice were lysed and immunoprecipitated with anti‐PARP1 antibody, followed by detection of Pan‐KLA. (I) HL‐1 cells transfected with Myc‐PARP1‐WT and Myc‐PARP1‐mut 5KR and Myc‐LDHA were lysed and immunoprecipitated using anti‐Myc antibody, followed by detection of Pan‐KLA. (J) HL‐1 cells transfected with Myc‐PARP1‐WT and Myc‐PARP1‐mut 5KR and LDHA siRNA were lysed and immunoprecipitated using anti‐Myc antibody, followed by detection of Pan‐KLA. Based on the central limit theorem, the data were considered to be normally distributed. Relative protein levels were calculated as fold changes vs. the first…

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  FIGURE 4. ROS‐ATM‐CBP signalling pathway activates PARP1 lactylation. (A, B) Intracellular ROS and lactate levels in HL‐1 cells treated with 1 µM doxorubicin for 0, 2 and 4 h. (C) Representative immunoprecipitation, western blots and quantification for detecting PARP1 lactylation. HL‐1 cells were stimulated by doxorubicin (1 µM for 0, 2 and 4 h), and the cells were lysed and immunoprecipitated using an anti‐PARP1 antibody, followed by detection of Pan‐KLA. (D) Schematic of the effect of SIRT1 activation on the lactylation level of PARP1 in mice. DOX (5 mg/kg) or the same volume of saline was administered intraperitoneally once weekly. One week before the DOX treatment, mice were pre‐treated with resveratrol (SIRT1 activator, 10 mg/kg/day) via daily intraperitoneal injections. (E) The SIRT1 activity was detected. (F–H) Representative immunoprecipitation, western blots and quantification for detecting PARP1 lactylation and protein expression of SIRT1. (I) Schematic of the effect of CBP inhibition on the lactylation level of PARP1 in mice. DOX (5 mg/kg) or the same volume of saline was administered intraperitoneally once weekly. One week before the DOX treatment, mice were pre‐treated with…

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  FIGURE 5. CBP exacerbates DOX‑induced myocardial injury primarily through the PARP1 lactylation–PARylation axis. (A, B) Representative flow cytometry analysis and quantitative analysis of apoptosis in rat primary cardiomyocytes induced by DOX (1 µM for 24 h). (C) The CCK‐8 assay was used to assess the viability of rat primary cardiomyocytes. (D, E) Representative PARylation staining and quantitative analysis induced by DOX (1 µM for 24 h) in rat primary cardiomyocytes. (F–L) Representative western blots and quantification of PARP1 lactylation/PARylation, as well as apoptosis marker (cleaved‐caspase 3, Bcl‐2 and BAX) in rat primary cardiomyocytes under DOX (1 µM for 24 h) treatment. Based on the central limit theorem, the data were considered to be normally distributed. Relative protein, apoptosis, viability and PARylation levels of rat primary cardiomyocytes were calculated as fold changes vs. the first group. Data are presented as mean ± SD. (A, C, E, G–L) Statistical significance was assessed by one‐way ANOVA with Tukey multiple comparisons test (p‐values adjusted for 6 comparisons). All immunoblots had three biological repetitions, with similar results. ns p > .05, *p < .05, **p < .01,…

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  FIGURE 6. Myocardium‐specific CBP knockout mice decrease PARP1 lactylation and prevent doxorubicin‐induced cardiac dysfunction. (A) Schematic of DOX‐induced cardiac dysfunction in CBP‐cKO mice and CBP‐cWT mice. DOX was intraperitoneally injected once a week with a dose of 5 mg/kg or the same volume of saline for 4 weeks (20 mg/kg cumulative dose of doxorubicin). (B–D) Representative echocardiography and quantitative analysis of EF% and FS% (n = 10 per group). (E–G) Quantitative analysis of Serum cTnT, CK‐MB and LDH levels (n = 10 per group). (H) Representative TUNEL staining, WGA staining, DHE staining and immunohistochemistry staining (n = 10 per group). (I) Quantification of apoptotic cardiomyocytes by TUNEL staining (n = 10 per group). (J) Quantification of relative cardiomyocyte cross‐sectional areas by WGA staining (n = 10 per group). (K) The ratio of heart weight to tibia length (HW/TL, mg/mm) (n = 10 per group). (L) Quantification of DHE staining intensity (n = 10 per group). (M) Quantification of total PARylation by immunohistochemistry staining (n = 10 per group). (N–R) Representative immunoprecipitation, western blots and quantification of PARP1 lactylation/ PARylation as well as…

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  FIGURE 7. Model showing the molecular signalling cascade of CBP‐PARP1 lactylation–PARylation axis. The schematic depicts how oxidative stress‐induced ROS activates the ATM‐CBP‐PARP1 signalling pathway via a lactylation–PARylation cascade to promote cardiomyocyte apoptosis.