Abstract

   Cardiovascular complications are a well-documented limitation of
   conventional cancer chemotherapy. As a notable side effect of
   cisplatin, cardiotoxicity represents a major obstacle to the treatment
   of cancer. Recently, it has been reported that cyclic GMP-AMP synthase
   (cGAS) stimulator of interferon genes (STING) signaling pathway was
   associated with the occurrence and development of cardiovascular
   diseases. However, the effect of STING on cardiac damage caused by
   cisplatin remains unclear. In this study, cisplatin was shown to
   activate the cGAS-STING signaling pathway, and deficiency of STING
   attenuated cisplatin-induced cardiotoxicity in vivo and in vitro.
   Mechanistically, the STING-TNF-α-AP-1 axis contributed to
   cisplatin-induced cardiotoxicity by triggering cardiomyocyte apoptosis.
   In conclusion, our results indicated that STING might be a critical
   regulator of cisplatin-induced cardiotoxicity and be considered as a
   potential therapeutic target for preventing the progression of
   chemotherapy-associated cardiovascular complications.

   Keywords: stimulator of interferon genes, cisplatin, tumor necrosis
   factor-α, activator protein 1, cardiotoxicity

Highlights

     * ➢ STING-TNF-α-AP-1 axis contributed to cisplatin-induced
       cardiotoxicity by triggering cardiomyocyte apoptosis.
     * ➢ Deficiency of STING attenuated cisplatin-challenged cardiac
       dysfunction.
     * ➢ STING may be a potential therapeutic target for preventing the
       progression of chemotherapy-associated cardiovascular
       complications.

Introduction

   Cardiovascular complications associated with conventional cancer
   chemotherapy are a global problem ([40]El-Awady et al., 2011;
   [41]McGowan et al., 2017). Cisplatin (cis-Diaminodichloroplatinum,
   CDDP), one of the most potent chemotherapeutic agents, has been proven
   effective against various solid tumors, including sarcoma, soft tissue
   cancer, bone cancer, muscle cancer, and vascular cancer. However,
   cisplatin-associated toxicity is a well-documented limitation of
   conventional cancer chemotherapy ([42]de Jongh et al., 2003; [43]Topal
   et al., 2018; [44]Ma et al., 2020). Cisplatin-induced cardiotoxicity
   has been reported to mainly include arrhythmia, acute myocardial
   infarction, supraventricular tachycardia, and atrial fibrillation
   ([45]Guglin et al., 2009; [46]Ma et al., 2010; [47]El-Awady et al.,
   2011). Eventually, cardiac toxicity led to congestive heart failure and
   sudden cardiac death. Therefore, it is urgent to find an effective
   strategy to prevent cardiovascular complications associated with
   conventional cancer chemotherapy.

   DNA is an important genetic medium, but it can also induce an innate
   immune response. DNA exposed to the cytoplasm is recognized by cyclic
   guanosine monophosphate-adenosine monophosphate (cGAMP) synthase
   (cGAS), triggering signaling cascades that lead to the production of
   pro-inflammatory factors and interferons (IFNs) ([48]Burdette et al.,
   2011; [49]Ablasser et al., 2013; [50]Bai and Liu, 2019). As an
   important intracellular inflammatory signal regulatory protein,
   stimulator of interferon genes (STING) can recognize not only the
   second messenger produced by bacteria or viruses, such as cyclic
   guanosine monophosphate (cGMP) and cyclic adenosine monophosphate
   (cAMP) but also cyclic dinucleotide produced by the interaction of
   intracellular DNA and cGAS ([51]Burdette et al., 2011; [52]Ahn et al.,
   2012; [53]Ablasser et al., 2013; [54]Hu and Shu, 2020); subsequently,
   it activates TANK binding kinase 1 (TBK1)-interferon regulatory factor
   3 (IRF3) signaling pathway and nuclear factor-κB (NF-κB), which
   regulate the production of IFNs and inflammatory factors ([55]Ishikawa
   and Barber, 2008; [56]Fang et al., 2017; [57]Ablasser and Chen, 2019;
   [58]Zhang et al., 2019). Therefore, STING plays a key role in the
   inflammatory response. Accumulating evidence has found that STING was
   associated with acute and chronic diseases ([59]Mao et al., 2017;
   [60]Luo et al., 2018; [61]Chung et al., 2019; [62]Li et al., 2019;
   [63]Maekawa et al., 2019; [64]Hu et al., 2020; [65]Zhang et al., 2020).
   Mao et al. found that Sting gene knockout could inhibit endothelial
   inflammation and improve insulin resistance induced by a high-fat diet
   in mice ([66]Mao et al., 2017). In addition, STING-mediated
   inflammation induced by mitochondrial stress has been reported to
   promote myocardial damage ([67]Sliter et al., 2018). Not only that, the
   activation of STING is caused by cisplatin-induced tubular inflammation
   and the progression of acute kidney injury ([68]Maekawa et al., 2019).
   However, the effect of STING on cisplatin-induced cardiotoxicity is
   unknown.

   In the present study, we investigated the effect of STING in
   cisplatin-induced cardiotoxicity. Our results showed that cisplatin
   activated the cGAS-STING signaling pathway, and of note, deficiency of
   STING attenuated cisplatin-induced cardiotoxicity in vivo and in vitro.
   Mechanistically, the STING-tumor necrosis factor-α (TNF-α)- activator
   protein 1 (AP-1) axis contributed to cisplatin-induced cardiotoxicity
   by triggering cardiomyocyte apoptosis. Altogether, our results
   indicated that STING might be a critical regulator of cisplatin-induced
   cardiotoxicity and be considered as a potential therapeutic target for
   preventing the progression of chemotherapy-associated cardiovascular
   complications.

Materials and Methods

Materials

   Cisplatin and AP-1 inhibitor T5224 were purchased from MedChemExpress
   (Monmouth Junction, NJ, United States). Cisplatin was dissolved in DMF
   for in vitro experiments and saline for in vivo experiments. T5224 was
   dissolved in DMSO for in vitro experiments. Antibodies for p-STING
   (1:1,000, #72971), STING (1:1,000, #13647), p-TBK1 (1:1,000, #5483),
   TBK1 (1:1,000, #3504), BAX (1:1,000, #2772), p-c-Jun (1:1,000, #3270),
   c-Jun (1:1,000, #9165), p-c-Fos (1:1,000, #5348), c-Fos (1:1,000,
   #2250), and TNF-α (1:1,000, 11,948) were purchased from Cell Signaling
   Technology (Danvers, MA, United States). Antibody for BCL2 (1:5,000,
   BF9103) was purchased from Affinity Biosciences LTD. (Changzhou,
   Jiangsu, China). Antibody for α-Tubulin (1:1,000, 11,224) was purchased
   from Proteintech Group (Chicago, Illinois, United States).

Cell Culture

   HL-1 cells were purchased from the Type Culture Collection of the
   Chinese Academy of Sciences (Shanghai, China) and incubated in DMEM
   media with 10% fetal bovine serum, 100 U/ml of penicillin, and 100U/ml
   of streptomycin at 37°C in a humidified 5%CO[2] incubator.

Generation of Sting-Knockout HL-1 Cells Using CRISPR-Cas9

   Lentiviral vectors that express guide RNAs (gRNAs:
   5′-TAC​TTG​CGG​TTG​ATC​TTA​CC-3′) that target STING were produced by
   co-transfecting HEK293T cells with a cocktail of gRNA/Cas9-expressing
   lentiCRISPRv2 and pPAX2 plasmids. Lentiviruses were collected at 48 h
   after transfection, supplemented with 10% FBS, and stored at −80°C.
   HL-1 cells were transduced with lentiviral vectors for 24 h in
   combination with 8 μg/ml polybrene. Then, the lentivirus-containing
   medium was replaced with fresh DMEM with 10%FBS and cultured for
   another 24 h. Cells were subjected to puromycin (1 μg/ml) selection to
   isolate Sting-knockout (STING KO) HL-1 cells. LentiCRISPRv2 without
   gRNA was used as an empty vector for mock transduction.

Cell Counting Kit-8 Assays

   HL-1 cells were seeded into 96-well plates at the density of 5,000
   cells per well. CCK8 (10 μL) was added to each well for 2 h at 37°C
   after incubator with different concentrations of cisplatin overnight.
   Optical density was measured at 450 nm using a microplate
   spectrophotometer.

Animal Experiments

   Male WT or STING^−/- mice (8 weeks old) were used for experiments. WT
   C57BL/6J mice were obtained from Beijing Vital River Laboratory Animal
   Technology Co. Ltd. (Beijing, China). STING^−/- mice with C57BL/6J
   background were purchased from Jackson Lab (Stock#025805). All animal
   care and experimental procedures were performed following the
   directives outlined in the Guide for the Care and Use of Laboratory
   Animals (US National Institutes of Health). Animal care and
   experimental protocols were approved by the China Pharmaceutical
   University Animal Policy and Welfare Committee (Nanjing, China;
   approval no. 2020–11–007).

   According to previous studies, the dose of cisplatin covered by the
   clinical dosage range 10–100 mg/m2 was chosen ([69]Zhao et al., 2018).
   Mice were randomly divided into four groups: saline group (Saline, n =
   6), cisplatin group (CDDP, n = 6), STING^−/- group (STING^−/-, n = 6),
   STING^−/- + cisplatin group (STING^−/- + CDDP, n = 6). Cisplatin
   (6 mg/kg) in 0.9% normal saline was administered by intraperitoneal
   injection on the first day, eighth day and 15th day as previously
   reported ([70]Zhao et al., 2018). On the 22nd day, transthoracic
   echocardiography was conducted on the mice to examine the cardiac
   function, and then all the animals were sacrificed under sodium
   pentobarbital anesthesia. The blood samples and heart tissues were
   collected.

Cardiac Function

   Transthoracic echocardiography (VisualSonics, Toronto, Canada) was
   performed to measure cardiac function in a non-invasive manner. The
   left ventricular internal dimension in diastole (LVIDd), left
   ventricular end-systolic volume (LVESV), left ventricular internal
   dimension in systole (LVIDs), left ventricular end-diastolic volume
   (LVEDV), left ventricular posterior wall thickness at end-diastolic
   (LVPWd), left ventricular posterior wall thickness at end-systolic
   (LVPWs), interventricular septal thickness at end-diastolic (IVSd), and
   interventricular septal thickness at end-systolic (IVSs) were assessed
   from M-mode images. The equations of fractional shortening (FS) =
   [(LVIDd - LVIDs)/LVIDd] × 100% and ejection fraction (EF) =
   [(LVEDV-LVESV)/LVEDV]×100% were respectively used to calculate FS and
   EF.

Pathological Staining

   The cardiac tissues were collected and fixed in 4% paraformaldehyde and
   embedded in paraffin. The cardiac tissue sections (5 μm) were stained
   with hematoxylin and eosin for light microscopy examination (Leica,
   Germany).

Terminal Deoxynucleotidyl Transferase Mediated dUTP Nick End Labeling
Staining

   Cardiac tissue sections or HL-1 cells were stained with TUNEL following
   the manufacturer’s operating procedures (Yeasen, Shanghai, China).
   Images were taken with the fluorescence microscope (Leica, Germany).

Determination of Serum Creatine Kinase Isoenzyme

   The serum was collected, and the content of serum creatine kinase
   isoenzyme (CK-MB) was detected by the corresponding kit purchased from
   Nanjing Jiancheng Co., Ltd (Nanjing, Jiangsu, China).

RNA Sequencing

   Total RNA of cardiac tissues was prepared with trizol reagent
   (Invitrogen), and RNA-seq was performed by Major Biotechnology Inc.
   (Shanghai, China). For data analysis, differentially expressed genes
   were then identified by using P-adjust 0.05 as a cutoff. Heap map
   analysis and KEGG enrichment analysis were performed with online
   software ([71]http://geneontology.org/).

Western Blot Analysis

   The cardiac tissue (20–40 mg) or HL-1 cells were lysed, the protein
   concentration was measured, and each protein’s loading amount was
   balanced. The protein was separated by SDS gel and then transferred to
   the PVDF membrane. After 90 min, the membranes were preincubated with
   5% non-fat milk and incubated with specific primary antibodies
   overnight. The second antibody conjugated with horseradish peroxidase
   was incubated for 1 h and visualized using chemiluminescence reagents.

2.12 Real-Time Quantitative PCR

   Protocol of RT-qPCR was described in a previous paper ([72]Wang et al.,
   2016). The primer sequences of interleukin 6 (Il6), chemokine (C-X-C
   motif) ligand 2 (Cxcl2), FBJ osteosarcoma oncogene (Fos), activating
   transcription factor 4 (Atf4), JunB proto-oncogene (Junb), tumor
   necrosis factor-alpha-induced protein 3 (Tnfaip3), chemokine (C-X-C
   motif) ligand 1 (Cxcl1), and β-actin listed in [73]Table 1 were
   synthesized with Sangon Biotech Co., Ltd (Shanghai, China). The
   quantitative analysis of genes was normalized to β-actin.

TABLE 1.

   Primers used for real-time qPCR assay.
   Gene Species Primers(FW) Primers(RW)
   Il6 Mouse CCA​AGA​GGT​GAG​TGC​TTC​CC CTG​TTG​TTC​AGA​CTC​TCT​CCC​T
   Cxcl-2 Mouse CCA​ACC​ACC​AGG​CTA​CAG​G GCG​TCA​CAC​TCA​AGC​TCT​G
   Fos Mouse CGG​GTT​TCA​ACG​CCG​ACT​A TTG​GCA​CTA​GAG​ACG​GAC​AGA
   Atf4 Mouse ATG​GCG​CTC​TTC​ACG​AAA​TC ACT​GGT​CGA​AGG​GGT​CAT​CAA
   Junb Mouse TCA​CGA​CGA​CTC​TTA​CGC​AG CCT​TGA​GAC​CCC​GAT​AGG​GA
   Tnfaip3 Mouse ACA​GTG​GAC​CTG​GTA​AGA​AAC​A
   CCT​CCG​TGA​CTG​ATG​ACA​AGA​T
   Cxcl1 Mouse CTG​GGA​TTC​ACC​TCA​AGA​ACA​TC CAG​GGT​CAA​GGC​AAG​CCT​C
   β-actin Mouse CCG​TGA​AAA​GAT​GAC​CCA​GA TAC​GAC​CAG​AGG​CAT​ACA​G
   [74]Open in a new tab

Enzyme-Linked Immunosorbent Assay

   The contents of TNF-α and IL-6 in cardiac tissues or TNF-α in HL-1
   cells were detected by the corresponding mouse ELISA kits for TNF-α or
   IL-6, following the manufacturer’s instructions. First, coat the ELISA
   plate with 100 μL/well of capture antibody in Coating Buffer overnight
   at 4°C. The cells were washed 3 times with 250 μL/well Wash Buffer and
   incubated with 200 μL of ELISA Diluent (1×) at room temperature for
   1 h. Next, add 100 μL/well of samples and standard to the appropriate
   wells and incubate at room temperature for 2 h. Aspirate, wash and then
   incubate with 100 μL/well diluted Detection Antibody to all wells for
   1 h. Afterward, aspirate, wash and then incubate with 100 μL/well of
   diluted Streptavidin-HRP for 30 min. Finally, the cells were washed and
   incubated with 100 μL/well of 1× TMB Solution for 15 min. Add
   100 μL/well of Stop Solution, and read the plate at 450 nm.

Statistical Analysis

   All measurement data were shown as mean ± SEM. Statistical analyses
   were performed using GraphPad Pro Prism 6.0 (GraphPad, San Diego, CA).
   Parametric data were used for the statistical analysis. We used one-way
   ANOVA followed by multiple comparisons test with Bonferroni correction
   when comparing data from more than two groups and a Student’s t-test
   when comparing data from two groups. It is considered to be significant
   when p < 0.05.

Results

Activation of the Cyclic GMP-AMP Synthase-Stimulator of Interferon Genes
Pathway Was Detected in Cisplatin-Induced HL-1 Cells

   First, we evaluated the effects of CDDP in HL-1 cells. The IC[50]
   (half-inhibitory concentration) value of cisplatin is different in
   different tumor cell lines, ranging from 10 to 200 μM ([75]Sato et al.,
   2018; [76]Cesna et al., 2018; [77]Morovati et al., 2019; [78]Kou et
   al., 2020). In addition, it has been reported that a certain
   concentration of cisplatin has toxic effects on H9c2 cell line (5 μM)
   and primary rat cardiomyocytes (200 μM) ([79]Zhang et al., 2017;
   [80]Qian et al., 2018; [81]Zhao, 2019). Here, the doses of 10, 20, 30,
   and 40 μM of CDDP were chosen for in vitro experiments. Incubation with
   CDDP (10, 20, 30, 40 μM) for 24 h reduced the viability of HL-1 cells
   in a dose-dependent manner ([82]Figure 1A). In addition, incubation
   with CDDP (10, 20, 30, 40 μM) for 24 h resulted in an increased
   expression of pro-apoptotic BAX and a decreased expression of
   anti-apoptotic BCL2 ([83]Figure 1B). Furthermore, CDDP induced
   apoptosis in HL-1 cells ([84]Figures 1C,D). It has been reported that
   the cGAS-STING pathway participated in myocardial ischemia-reperfusion
   injury and cardiac hypertrophy ([85]Cao et al., 2018; [86]Hu et al.,
   2020; [87]Zhang et al., 2020). Notably, the activation of STING and
   TBK1 was observed in CDDP-challenged HL-1 cells ([88]Figure 1E).

FIGURE 1.

   [89]FIGURE 1
   [90]Open in a new tab

   Activation of the cGAS-STING pathway was detected in cisplatin-induced
   HL-1 cells (A) HL-1 cells were incubated with CDDP (10, 20, 30, 40 μM)
   for 24 h and then the cell viability of HL-1 cells was examined by CCK8
   analysis (n = 5 independent experiments; ^### p < 0.001, vs. the 0
   group). (B) HL-1 cells were incubated with CDDP (10, 20, 30, 40 μM) for
   24 h, and then total protein was collected. The protein level of BAX
   and BCL2 in HL-1 cells was detected by Western blot. (C–D) HL-1 cells
   were incubated with CDDP (10, 20, 30, 40 μM) for 24 h, and then cell
   apoptosis induced by CDDP was detected by TUNEL staining. (C)
   Representative images of TUNEL staining in HL-1 cells (Green: TUNEL
   positive cell, DAPI: nucleus; Scale Bar: 25 μm, ×400 magnification).
   (D) Quantification of TUNEL-positive cells (E) HL-1 cells were
   incubated with CDDP (10, 20, 30, 40 μM) for 1 h, and total protein was
   collected. The protein level of p-STING, STING, p-TBK1, TBK1 in HL-1
   cells was detected by Western blot (n = 3 independent experiments; ^# p
   < 0.05, ^## p < 0.01, ^### p < 0.001, vs. Vehicle group).

Suppression of Stimulator of Interferon Genes Ameliorated Cisplatin-Induced
Injury in HL-1 Cells

   To confirm the effect of STING on the progression of apoptosis in
   cisplatin-induced HL-1 cells, we generated the Sting-knockout HL-1
   cells using CRISPR-Cas9. As shown in [91]Figures 2A,B, CDDP failed to
   induce the phosphorylation of TBK1 in Sting-knockout HL-1 cells. Here,
   the increased expression of BAX and the reduced expression of BCL2
   induced by CDDP were reversed by Sting knockout ([92]Figure 2C).
   Additionally, cardiomyocyte apoptosis and decreased cell viability
   induced by CDDP were attenuated by Sting knockout in HL-1 cells
   ([93]Figures 2D,E). Altogether, these results indicated that blockage
   of STING exerted protective effects against CDDP-induced cell injury.

FIGURE 2.

   [94]FIGURE 2
   [95]Open in a new tab

   Deficiency of STING ameliorated cisplatin-induced injury in HL-1 cells
   (A) STING was knocked out by CRISP-Cas9. HL-1 cells were subjected to
   puromycin (1 μg/ml) selection to isolate STING KO cells, and then total
   protein was collected. The protein level of STING was detected by
   Western blot. (B) HL-1 cells were incubated with CDDP (40 μM) for 1 h,
   and total protein was collected. The protein levels of p-TBK1, TBK1,
   STING were detected by Western blot. (C) HL-1 cells were incubated with
   CDDP (40 μM) for 24 h, and the protein levels of BAX and BCL2 in HL-1
   cells were detected by Western blot. (D) Representative images and
   quantification of TUNEL staining in HL-1 cells. HL-1 cells were
   incubated with CDDP (40 μM) for 24 h, and cell apoptosis was detected
   by TUNEL staining (Green: TUNEL positive cell, DAPI: nucleus; Scale
   Bar: 25 μm, ×400 magnification). (E) HL-1 cells were incubated with
   CDDP (40 μM) for 24 h, and then the cell viability of HL-1 cells was
   examined by CCK8 analysis (n = 3 independent experiments; ^# p < 0.05,
   ^## p < 0.01, ^### p < 0.001, vs. Vehicle group; ^* p < 0.05, **p <
   0.01, ***p < 0.001, vs. CDDP group).

Deficiency of Stimulator of Interferon Genes Blocked Cisplatin-Induced
Cardiac Dysfunction and Injury

   Next, we investigated the role of STING in CDDP-induced cardiotoxicity
   in vivo. [96]Figure 3A showed that the phosphorylation of STING and
   TBK1 was elevated in cardiac tissues of CDDP-treated mice, but that
   induced by CDDP was inhibited in STING^−/- mice. Furthermore, compared
   with the saline group, echocardiographic results showed that EF and FS
   in CDDP-treated mice were notably lowered ([97]Figure 3B and [98]Table
   2). Serum CK-MB level was significantly increased by intraperitoneal
   injection of CDDP. Moreover, the histopathological analysis showed the
   disruption of cardiac muscle fibers and the loss of striations and
   intercalated disc induced by CDDP in cardiac tissues. TUNEL staining
   showed a significant increase in cell death in cardiac tissues of
   CDDP-challenged mice ([99]Figures 3E,F), and immunoblotting analysis
   showed an increased expression of BAX and a reduced expression of BCL2
   in cardiac tissues of CDDP-treated mice ([100]Figure 3G). However,
   these changes were reversed by Sting knockout in mice ([101]Figure 3;
   [102]Table 2). These results indicated that deficiency of STING blocked
   CDDP-induced cardiac dysfunction and injury.

FIGURE 3.

   [103]FIGURE 3
   [104]Open in a new tab

   Deficiency of STING blocked cisplatin-induced cardiac dysfunction and
   injury (A) The protein levels of p-STING, STING, p-TBK1, TBK1 in
   cardiac tissues were detected by Western blot. (B) Representative
   images of M-mode of left ventricular (C) The levels of serum CK-MB were
   detected by the corresponding kit. (D) Representative images of H and E
   staining using paraffin section of heart tissues (Scale Bar: 25 μm,
   ×400 magnification). (E–F) Representative images (E) and quantification
   (F) of TUNEL staining using paraffin section of heart tissues. (Green:
   TUNEL positive cell, DAPI: nucleus; Scale Bar: 25 μm, ×400
   magnification) (G) The protein levels of BAX and BCL2 in cardiac
   tissues were detected by Western blot (n = 6 in each group; ^### p <
   0.001, vs. Saline group; ^* p < 0.05, **p < 0.01, ***p < 0.001, vs.
   CDDP group).

TABLE 2.

   Echocardiographic measurements.
   Parameter Saline CDDP STING^−/- CDDP + STING^−/-
   N 6 6 6 6
   EF (%) 62.36 ± 2.36 47.37 ± 2.00[105]^a 64.12 ± 2.93 62.90 ±
   4.17[106]^b
   FS (%) 33.07 ± 1.64 22.87 ± 1.32[107]^a 34.47 ± 2.18 32.85 ±
   2.80[108]^b
   LVID d(mm) 3.61 ± 0.09 3.86 ± 0.06 3.53 ± 0.09 3.48 ± 0.16
   LVID s(mm) 2.41 ± 0.09 2.6 ± 0.23 2.36 ± 0.13 2.38 ± 0.15
   LVPW d(mm) 0.76 ± 0.03 0.67 ± 0.04 0.85 ± 0.03 0.73 ± 0.07
   LVPW s(mm) 1.25 ± 0.07 0.96 ± 0.06[109]^a 1.36 ± 0.08 1.16 ± 0.09
   IVS d(mm) 0.77 ± 0.02 0.92 ± 0.06[110]^a 0.73 ± 0.05 0.83 ± 0.14
   IVS s(mm) 1.16 ± 0.03 1.22 ± 0.10 1.19 ± 0.01 1.13 ± 0.14
   [111]Open in a new tab
   ^a

   p < 0.05 vs Saline group.
   ^b

   p < 0.05 vs CDDP group.

Tumor Necrosis Factor-α Signal Pathway Is Involved in Cisplatin-Induced
Cardiac Injury

   We performed RNA sequencing (RNA-seq) on cardiac tissues from wild-type
   (WT) and STING^−/- mice to better characterize the cardioprotective
   mechanism by deficiency of STING in CDDP-induced mice. Venn analysis
   showed 318 genes were present in the genome Saline vs. CDDP and the
   genome CDDP vs. STING KO + CDDP ([112]Figure 4A). Heat map analysis
   with KEGG enrichment analysis confirmed a strong downregulation of TNF
   signaling pathway-related genes in cardiac tissues of STING^−/- mice
   ([113]Figures 4B–D).

FIGURE 4.

   [114]FIGURE 4
   [115]Open in a new tab

   TNF signaling pathway was the most significantly enriched pathway
   identified by RNA-seq (A) Venn diagram of identified differentially
   expressed genes in the genome Saline vs. CDDP and the genome CDDP vs.
   STING KO + CDDP. (B) Differential gene-expression heat maps of
   identified genes were present in the genome Saline vs. CDDP and the
   genome CDDP vs. STING KO + CDDP (C) KEGG pathway enrichment analysis of
   the identified differentially expressed genes present in the genome
   Saline vs. CDDP and the genome CDDP vs. STING KO + CDDP. The 10 most
   significantly enriched pathways were shown (D) Differential
   gene-expression heat maps of identified genes were present in the TNF
   signaling pathway.

   The mRNA levels of Il6, Cxcl2, Fos, Atf4, Junb, Tnfaip3, and Cxcl1 and
   the protein levels of TNF-α and IL-6 in cardiac tissues were determined
   to identify the potential drivers of the hyperactive TNF signal
   pathway. Consistent with the results of RNA-seq, CDDP increased the
   mRNA levels of a variety of TNF signaling pathway-related genes,
   including Il6, Cxcl2, Fos, Atf4, Junb, Tnfaip3, and Cxcl1 in cardiac
   tissues, but these changes were dramatically reversed by Sting knockout
   ([116]Figure 5A). Congruously, the increased protein levels of TNF-α
   and IL-6 induced by CDDP in cardiac tissues were reduced by Sting
   knockout ([117]Figures 5B–D). The downstream of TNF signal pathway,
   AP-1, a heterodimer composed of c-Fos and c-Jun, was activated in
   cardiac tissues of CDDP-challenged mice but inactivated in cardiac
   tissues of CDDP-challenged STING^−/- mice ([118]Figures 5E,F). These
   results indicated that the TNF signal pathway was involved in
   cisplatin-induced cardiac injury.

FIGURE 5.

   [119]FIGURE 5
   [120]Open in a new tab

   TNF signal pathway was involved in cisplatin-induced cardiac injury.
   (A) RT-qPCR was used to detect the expression of TNF signal
   pathway-related genes including Il6, Cxcl2, Fos, Atf4, Junb, Tnfaip3,
   and Cxcl1 in cardiac tissues (B–C) The expression of TNF-ɑ and IL-6 in
   cardiac tissues were detected by ELISA. (D–F) The protein expression of
   TNF-ɑ (D), p-c-Fos (E), c-Fos (E), p-c-Jun (F), c-Jun (F) in cardiac
   tissues were detected by Western blot (n = 6 in each group; ^# p <
   0.05, ^## p < 0.01, ^### p < 0.001 vs Saline group; ^* p < 0.05, **p <
   0.01, ***p < 0.001 vs. CDDP group).

Stimulator of Interferon Genes-Tumor Necrosis Factor-α-AP-1 Mediated
Cisplatin-Induced Cardiomyocyte Injury

   Next, we investigated the role of the STING-TNF-α-AP-1 axis in
   CDDP-induced cardiomyocytes injury. Exposed to CDDP, c-Jun and c-Fos
   were time-dependently phosphorylated in HL-1 cells ([121]Figure 6A).
   Analogously, the protein level of TNF-α was time-dependently increased
   in HL-1 cells ([122]Figure 6B). To further validate the regulatory role
   of STING in CDDP-induced activation of AP-1, we examined whether
   deficiency of STING inhibited the phosphorylation of AP-1 in
   CDDP-induced HL-1 cells. Expectedly, the protein and secretion level of
   TNF-α was suppressed ([123]Figures 6C,D), and the phosphorylation of
   c-Jun and c-Fos was blocked by Sting knockout ([124]Figure 6C). Of
   note, activation of STING by cGAMP promoted the phosphorylation and
   expression of c-Jun and c-Fos ([125]Figure 6E). In addition,
   cardiomyocyte apoptosis induced by CDDP was attenuated by AP-1
   inhibitor T5224 in HL-1 cells ([126]Figures 6F,G). Moreover, the
   decrease in cell viability induced by CDDP was improved ([127]Figure
   6H), and the increased expression of BAX induced by CDDP was reversed
   by T5224 in HL-1 cells ([128]Figure 6I). These results suggested that
   STING-TNF-α-AP-1 mediated CDDP-induced cardiomyocyte apoptosis.

FIGURE 6.

   [129]FIGURE 6
   [130]Open in a new tab

   STING-TNF-α-AP-1 mediated cisplatin-induced cardiomyocyte injury. (A–B)
   HL-1 cells were incubated with CDDP (40 μM) for a time course, and then
   the total protein was collected. The protein levels of p-c-Fos, c-Fos,
   p-c-Jun, c-Jun, and TNF-ɑ were detected by Western blot. (C) HL-1 cells
   were incubated with CDDP (40 μM) for 24 h, and then the total protein
   was collected. The protein levels of STING, TNF-ɑ, p-c-Fos, c-Fos,
   p-c-Jun, c-Jun were detected by Western blot. (D) HL-1 cells were
   incubated with CDDP (40 μM) for 24 h, and the medium supernatant and
   the total protein were collected. The contents of TNF-ɑ and IL-6 in the
   medium supernatant were detected by ELISA and calibrated with protein
   concentration. (E) HL-1 cells were incubated with cGAMP (1 μg/ml) for a
   time course, and then the total protein was collected. The protein
   levels of p-c-Fos, c-Fos, p-c-Jun, c-Jun were detected by Western blot.
   (F–G) Representative images and quantification of TUNEL staining in
   HL-1 cells. HL-1 cells were pretreated with T5224 (10 μM) for 30 min
   and incubated with CDDP (40 μM) for 24 h. Cell apoptosis induced by
   CDDP was detected by TUNEL staining (Green: TUNEL positive cell, DAPI:
   nucleus; Scale Bar: 25 μm, ×400 magnification). (H) HL-1 cells were
   pretreated with T5224 (10 μM) for 30 min and incubated with CDDP
   (40 μM) for 24 h, and the cell viability of HL-1 cells was examined by
   CCK8 analysis. (I) HL-1 cells were pretreated with T5224 (1, 5, 10 μM)
   for 30 min and incubated with CDDP (40 μM) for 24 h, and the total
   protein was collected. The protein levels of BAX were detected by
   Western blot (n = 3 independent experiments; ^# p < 0.05, ^## p < 0.01,
   ^### p < 0.001 vs. Vehicle or DMSO group; ^* p < 0.05, **p < 0.01, ***p
   < 0.001, vs. CDDP group).

Discussion

   This study demonstrated that STING played a crucial regulatory role in
   cisplatin-induced cardiotoxicity and demonstrated that cisplatin
   activated STING, which promoted the activation of the TNF-α-mediated
   AP-1 signaling pathway. However, deficiency of STING attenuated
   cisplatin-induced cardiac dysfunction and apoptosis in vivo and
   abolished cisplatin-induced cardiomyocyte apoptosis in vitro.

   Apoptosis plays a key role in the development of
   chemotherapy-associated cardiopathy. Recently, accumulated studies have
   suggested that the mtDNA-cGAS -STING pathway proved critical to the
   progression of cardiovascular diseases ([131]Mao et al., 2017; [132]Cao
   et al., 2018; [133]Sliter et al., 2018; [134]Li et al., 2019; [135]Hu
   et al., 2020; [136]Huang et al., 2020; [137]Zhang et al., 2020). In the
   present study, our results showed that STING was an essential regulator
   for cisplatin-induced cardiotoxicity. In our in vivo experiments, STING
   and TBK1 in cardiac tissues were phosphorylated by intraperitoneal
   injection with cisplatin. The deficiency of STING reduced the
   phosphorylation of TBK1 in cardiac tissue.

   Moreover, deficiency of STING attenuated not only cisplatin-induced
   cardiac dysfunction and the increase in serum CK-MB but also improved
   cisplatin-induced myocardial fiber disorder and cardiomyocyte
   apoptosis. In vitro experiments, deficiency of STING abolished
   cisplatin-induced apoptosis of HL-1 cells. These results indicated that
   suppression of STING protected against cardiotoxicity induced by
   cisplatin.

   Recent studies suggested that targeting the TNF signal pathway might
   comprise a viable therapeutic strategy to reduce disease burden in
   ischaemic vascular disease progression, particularly endothelial
   dysfunction, chronic inflammation, and atherosclerotic plaque
   development ([138]Hu et al., 2015; [139]Kojima et al., 2016; [140]Nash
   et al., 2019). Heat map analysis with KEGG enrichment analysis in
   RNA-seq from cardiac tissues confirmed a strong correlation between
   cisplatin-induced cardiotoxicity and TNF signaling pathway, and
   deficiency of STING alleviated cisplatin-induced cardiac dysfunction,
   which is significantly correlated with the inactivation of the TNF
   signal pathway. Consistently, the expression of TNF-α and the
   phosphorylation of c-Jun and c-Fos were increased, but these were
   reversed by deficiency of STING. cGAMP bind to the endoplasmic
   reticulum (ER)-localized STING, which promotes STING dimerization and
   translocation from ER to the endoplasmic reticulum-Golgi intermediate
   compartment (ERGIC) ([141]Sliter et al., 2018; [142]Ablasser and Chen,
   2019; [143]Gui et al., 2019; [144]Shang et al., 2019; [145]Zhang et
   al., 2019). Subsequently, STING recruits and activates TBK1 to initiate
   an IRF3-dependent innate immune response, resulting in the production
   of IFNs and a NF-κB-dependent inflammatory response, which leads to the
   production of pro-inflammatory factors, including TNF-α, IL6, IL1β,
   etc. ([146]Burdette et al., 2011; [147]Bai and Liu, 2019; [148]Zhang et
   al., 2019; [149]Hu and Shu, 2020). Our results showed that the
   activation of AP-1 was dependent on an increased expression of TNF-α.
   Intriguingly, the expression of c-Jun and c-Fos was increased when
   exposed to both cisplatin and cGAMP, indicating that AP-1 might be
   mediated by the STING-NF-κB signaling pathway.

Conclusion

   Our study found that the STING-TNF-α-AP-1 axis contributed to
   cisplatin-induced cardiotoxicity by triggering cardiomyocyte apoptosis
   and deficiency of STING attenuated cisplatin-induced cardiac
   dysfunction. Moreover, STING may be a potential therapeutic target for
   preventing the progression of chemotherapy-associated cardiovascular
   complications.

Acknowledgments