HBB contributes to individualized aconitine-induced cardiotoxicity in mice via interfering with ABHD5/AMPK/HDAC4 axis

Abstract

The root of Aconitum carmichaelii Debx. (Fuzi) is an herbal medicine used in China that exerts significant efficacy in rescuing patients from severe diseases. A key toxic compound in Fuzi, aconitine (AC), could trigger unpredictable cardiotoxicities with high-individualization, thus hinders safe application of Fuzi. In this study we investigated the individual differences of AC-induced cardiotoxicities, the biomarkers and underlying mechanisms. Diversity Outbred (DO) mice were used as a genetically heterogeneous model for mimicking individualization clinically. The mice were orally administered AC (0.3, 0.6, 0.9 mg· kg^-1 ·d^-1) for 7 d. We found that AC-triggered cardiotoxicities in DO mice shared similar characteristics to those observed in clinic patients. Most importantly, significant individual differences were found in DO mice (variation coefficients: 34.08%-53.17%). RNA-sequencing in AC-tolerant and AC-sensitive mice revealed that hemoglobin subunit beta (HBB), a toxic-responsive protein in blood with 89% homology to human, was specifically enriched in AC-sensitive mice. Moreover, we found that HBB overexpression could significantly exacerbate AC-induced cardiotoxicity while HBB knockdown markedly attenuated cell death of cardiomyocytes. We revealed that AC could trigger hemolysis, and specifically bind to HBB in cell-free hemoglobin (cf-Hb), which could excessively promote NO scavenge and decrease cardioprotective S-nitrosylation. Meanwhile, AC bound to HBB enhanced the binding of HBB to ABHD5 and AMPK, which correspondingly decreased HDAC-NT generation and led to cardiomyocytes death. This study not only demonstrates HBB achievement a novel target of AC in blood, but provides the first clue for HBB as a novel biomarker in determining the individual differences of Fuzi-triggered cardiotoxicity.

Keywords: ABHD5/AMPK/HDAC4 axis; aconitine; cardiotoxicity; hemoglobin subunit beta; individual differences; nitrogen monoxide.

Fig. 1. In DO mice, AC-triggered cardiotoxicity shared similar characteristics with clinical patients, and significant individual variabilities were also observed.

a The schematic diagram showed DO mice grouping and the dosages of AC administrations. ECGs (b) and the levels of CK-MB (c) in AC-sensitive and AC-tolerant mice were measured after treatments of AC (n = 5). Cardiotoxicity-related behavior scores (d) and the coefficient of dispersion (e) of DO mice were recorded on the d 1. f The proportion of sensitive and tolerant mice at three dosages of AC. (*P < 0.05, **P < 0.01).

Fig. 2. Differential gene background would be the key determinant for the individualized response of AC cardiotoxicity.

a The LD₅₀ of AC were measured in 8 progenitor mice of DO mice, either in female or in male mice, respectively (n = 10). b Survival ratio of 8 progenitor mice was measured (n = 10). c The behavior scores of progenitor mice were recorded within 0–360 min after exposed to AC at 0.405 mg/kg (1/3 of average LD₅₀) (n = 4). d Representative images of progenitor mice after AC exposure. NOD/ShiLtJ mice suffered from vomiting, tachyarrhythmia, desudation, diarrhea, and convulsion, while in 129S1/SvImJ, no significant physical changes were observed. (**P < 0.01).

Fig. 3. HBB is the only specifically expressed gene in AC-sensitive mice, overlayed in DO mice and progenitor mice.

a–c Show the results in DO mice(n = 9). d–f Show the results in progenitor mice (n = 5). a The Overlapping DEGs (Venn diagram) and DEGs (Heat map). b Nine signaling pathways were overlayed (Histogram) and among top 50 signaling pathways enriched (Venn diagram). c 96 of DEGs were overlayed (Venn diagram), and DEGs expression were validated (Heat maps). Then the interaction was analyzed (network diagram). d Overlapping DEGs (Venn diagram) and cluster analysis of DEGs (Heat maps). e The signaling pathways enriched (venn diagram) were overlayed and top of 5 overlayed pathways (histogram). f Overlapping DEGs (Venn diagram) and 14 overlayed DEGs expression were validated (Heat maps). KEGG and the interaction of 14 overlayed DEGs (network diagram) (fold change > 2).

Fig. 4. HBB overexpression enhanced the responsive sensitivity of AC-induced cardiotoxicity.

a, b HBB expressions in heart tissues were evaluated by Western blotting and immunohistochemistry (IHC) (n = 5). c Cell viability assay of cardiomyocytes after exposed AC (n = 3). d The ROS level in primary-isolated cardiomyocytes was quantify(n = 3). e HBB protein expression and purification. f HBB pure proteins were given to mice. (HBB and IgG, 125 mg/kg, i.v, n = 6) (*P < 0.05, **P < 0.01, ***P < 0.001; ^#P < 0.05, ^##P < 0.01).

Fig. 5. AC could directly bind to HBB with highest binding affinity.

a The solvent-induced protein precipitation (SIP) combined with proteomics were utilized to identify the key targets for AC-associated cardiotoxicity. b CO-IP assay was applied to verify the combination of AC with HBB, ADH1, ABHD5, and AMPK. AC at 1 µM and 10 µM were used as concentration gradient to further confirm the bind of HBB with these proteins. c–f Docking data indicated the interactions of AC with HBB, ADH1, ABHD5, and AMPK, respectively and precise amino acids involved.

Fig. 6. AC specifically binds to cell-free Hb to rapidly scavenge nitric oxide and induce cardiotoxicity.

a The hemolysis effects of AC were evaluated from 6.125 nM to 10 μM in vitro. b HE staining of DO mice heart tissues in 0.3 mg/kg, 0.6 mg/kg, and 0.9 mg/kg group; as well as Masson staining in 0.9 mg/kg. c The interaction between AC and Hb in red blood cells, plasma, and cardiomyocytes. d NO generations in heart tissues were examined in DO mice within 0–240 min after treated with AC (n = 3). Protein expression levels of eNOS, iNOS, nNOS (e), and S-nitrosylation (f) were detected (n = 3).

Fig. 7. HBB promotes AC-induced cardiotoxicity via interfering with ABHD5/AMPK/HDAC4 axis.

a The IP-mass assay was performed to determine HBB-binding proteins in HBB-overexpressed AC16 cells. b CO-IP assay was used to verify the binding of HBB with ADH1, ABHD5, and AMPK. Docking data (c–e) and the protein binding affinities (f) of HBB with ADH1, ABHD5, and AMPK before and after AC interfering. The protein levels of full length HDAC (HDAC-FL) (g), N-terminal polypeptide of HDAC4 (HDAC4-NT) (g), AMPK (h), p-AMPK (h), mTOR (h), and p-mTOR (h) were assessed (n = 3). i The protein levels of AMPK, ADH1, and ABHD5 were detected by IHC.