DOX targets GSTP1 and inhibits its activity to protect against Doxorubicin-Induced Cardiomyopathy

///'2.1 DOX targets to GSTP1 and inhibits its activity.//nTo explore the potential target of DOX in the pathogenesis of DIC, we conducted DOX-pulldown to investigate the proteins which specifically bind with DOX in the murine heart. Seven candidate proteins were revealed by SDS-PAGE and LC-MS/MS (Figure 1A). Given the close correlation with myocardial damage(32, 33), GSTP1 was selected to further study. BIAcore assay was next used to detect the binding constant of them, which showed specifical binding with GSTP1 in different concentration of DOX in vitro (Figure 1B, C). Then the mRNA and protein expression level of GSTP1were detected under different concentration of DOX treatments. The mRNA and protein expression level of GSTP1 exhibited no significant changes under DOX treatment (Figure 1D, E). Although DOX could not affect the mRNA and protein expression level of GSTP1, we speculated that whether it affects the activity of GSTP1. To determine the speculation, we purified recombinational GSTP1 protein and validated its purity and characteristics by SDS-PAGE and western blot, respectively (Figure S1A, B). Surprisedly, we found that DOX can inhibit the activity of GSTP1 significantly in vitro experiment (Figure 1F). Meanwhile, we detected the activity of GSTP1 in H9c2 cells under DOX treatment, which was consistent with the vitro results (Figure 1G).//nIn general, the initial //u200bfindings revealed that DOX could target to GSTP1 and inhibit its activity intracellularly or extracellularly, which is not reported before.//n//n2.2 GSTP1 protects against DOX-Induced Cardiomyopathy.//nSince DOX could target to GSTP1 specifically and inhibit its activity, we speculated that GSTP1 may be an important mediator or regulator in DIC. To identify the role of GSTP1 in vivo, AAV9 was applied via tail vein injection to manipulate the expression profile of GSTP1.//nThe DIC model was established, cardiac function and the extent of myocardial injury were analyzed. DOX treatment led to a conceivable decline of heart rate (BPM), ejection fraction (EF), fractional shortening (FS), and stroke volume compared to the control (AAV-NC), as indicated by echocardiography assessment. Interestingly, these aberrant alterations were alleviated by cardiac-specific GSTP1 overexpression (Figure 2A-E). Moreover, several types of heart failure biomarkers, including Myh7 (myosin-7), Anp (atrial natriuretic peptide), and Bnp (brain natriuretic peptide), were detected to further evaluate cardiac injuries. As shown in our results of q-PCR, GSTP1 overexpression substantially downregulated DOX-induced increase expression of Myh7, Anp, and Bnp (Figure 2F-H). In addition, the fibrotic state of murine hearts was investigated by Masson’s Trichrome staining, which revealed that DOX administration resulted in cardiac fibrosis, which was mitigated by GSTP1 overexpression (Figure 2I, 2J).//nWe further validated these findings in zebrafish and cardiomyocytes (H9c2, a cell line of rat). In detail, DOX-induced cardiotoxicity was determined in Tg (cmlc2: GFP) zebrafish embryos, an animal model in which the cardiomyocytes’ nuclei and plasma membranes are labeled with GFP(34). We found that overexpression of GSTP1 relieved the DOX induced abnormal alterations including decreased heart rate, elongated shape of heart with a compact ventricle and thin atrial wall (Figure S2A). For cardiomyocytes, lentivirus was used to get H9c2 cells with overexpressed GSTP1 (Figure S2B) and cells with Lenti-Flag-GSTP1 bear lower cytotoxicity compared to the control (Lenti-NC), as exhibited by microscopic examination (Figure S2C). Besides, different concentrations of DOX treatment induced cytotoxicity and inhibited intracellular activity of GSTP1. These aberrant alterations were alleviated by GSTP1 overexpression and accelerated by GSTP1 gene disruption (Figure S2D-F). Taken together, these findings indicated that GSTP1 overexpression alleviated the progression of DIC.//n//n2.3 GSTP1 ameliorates DOX-induced ferroptosis in vitro.//nSeveral previous studies indicated that ferroptosis plays an important role in the progression of DIC. Besides, selective inhibition of ferroptosis improved cardiac function significantly after DOX administration(5, 35). In this study, we first tried to determine whether or not GSTP1 was participated in the modulation of ferroptosis in DOX-treated cardiomyocytes. Our initial analysis revealed that RSL3, a ferroptosis activator, led to a distinct decline in cardiomyocyte survival. Interestingly, this ferroptotic cell death was alleviated by GSTP1 overexpression and aggregated by GSTP1 interference, suggesting that GSTP1 was involved in the process of ferroptosis (Figure S3A, S4A). Next, DCFH-DA staining demonstrated that DOX treatment led to an overwhelming generation of ROS, a key marker of ferroptosis. Of note, the increased lipid ROS was attenuated by GSTP1 overexpression and accentuated by GSTP1 knockdown (Figure S3B-D, S4B-D). However, apoptosis and necrosis induced by DOX were no affected by GSTP1 overexpression, indicating a ferroptosis-specific role of GSTP1 (Figure S3F). Ferroptosis is also characterized by mitochondrial shrunken and iron overload. And RSL3-induced mitochondrial injury and iron overload alleviated by GSTP1 overexpression (Figure S3F-G, S4F-G).//nTo further verify the dominant role of GSTP1 in DOX-induced cardiac ferroptosis, ferroptosis-related parameters were detected in H9c2 cells. Specifically, DOX-induced ferroptotic cell death, higher ROS and lipid-ROS levels were alleviated by GSTP1 overexpression (Figure 3A-D). Next, DOX-induced mitochondrial injury was detected by immunofluorescence analysis and transmission electron microscope (TEM) in vitro. It was observed that DOX treatment led to significant mitochondrial damage, which was attenuated by GSTP1 overexpression (Figure 3E-F). In regard to the detection of intracellular iron, our results showed that DOX treatment increased the level of iron (Fe2+ and total iron), and this aberrant alteration was alleviated by GSTP1 introduction (Figure 3G).//nOverall, these findings suggested that GSTP1 inhibited ferroptosis to attenuate DOX-induced cardiomyocyte injury both in vitro. Meanwhile, GSTP1 may be another key mediator of ferroptosis like GPX4 (Figure S3).///