ESI-09

Epac1 Signaling Pathway Mediates the Damage and Apoptosis of Inner Ear Hair Cells after Noise Exposure in a Rat Model
Fanfan Sun, a,b Junge Zhang, a Li Chen, a Yuhao Yuan, b Xiaotao Guo, a Liuyi Dong b* and Jiaqiang Sun a*

Abstract—To investigate the role of the exchange protein directly activated by cAMP (Epac) signaling pathway in inner ear hair cell damage and apoptosis after noise exposure, we analyzed the expression level of Epac1 in a rat model of noise-induced hearing loss (NIHL), based on rat exposure to a 4-kHz and 106-dB sound pressure level (SPL) for 8 h. Loss of outer hair cells (OHCs), mitochondrial lesions, and hearing loss were examined after treat- ment with the Epac agonist, 8-CPT, or the Epac inhibitor, ESI-09. The effects of 8-CPT and ESI-09 on cell prolifer- ation and apoptosis were examined by CCK-8 assays, holographic microscopy imaging, and Annexin-V FITC/PI staining in HEI-OC1 cells. The effects of 8-CPT and ESI-09 on Ca2+ entry were evaluated by confocal Ca2+ fluo- rescence measurement. We found that the expression level of Epac1 was significantly increased in the cochlear tissue after noise exposure. In NIHL rats, 8-CPT increased the loss of OHCs, mitochondrial lesions, and hearing loss compared to control rats, while ESI-09 produced the opposite effects. Oligomycin was used to induce HEI- OC1 cell damage in vitro. In HEI-OC1 cells treated with oligomycin, 8-CPT and ESI-09 increased and reduced cell apoptosis, respectively. Moreover, 8-CPT promoted Ca2+ uptake in HEI-OC1 cells, while ESI-09 inhibited this pro- cess. In conclusion, our data provide strong evidence that the Epac1 signaling pathway mediates early patholog- ical damage in NIHL, and that Epac1 inhibition protects from NIHL, identifying Epac1 as a new potential therapeutic target for NIHL.

Key words: Epac, CaMKII, noise-induced hearing loss (NIHL), apoptosis.

INTRODUCTION
Noise-induced hearing loss (NIHL) is a major type of sensorineural hearing loss, accounting for approximately 16% of adult hearing loss cases worldwide (Li et al., 2017). Its incidence tends to increase annually (Liu et al., 2019). To date, no Food and Drug Administration (FDA)-approved drugs protecting against noise-induced hearing loss are available (Sha and Schacht, 2016). The molecular mechanism associated with NIHL is similar to that underlying hearing loss caused by age and drugs (Kujawa and Liberman, 2009; Kurabi et al., 2017). This mechanism involves the loss of hair cells and synaptic connections, increased levels of reactive oxygen species (ROS) (Seidman et al., 2004), changes in mitochondrial

*Corresponding authors.
E-mail addresses: [email protected] (L. Dong), sunjq0605@126. com (J. Sun).
Abbreviations: ABR, auditory brainstem response; CaMKII, calmodulin-dependent protein kinase II; FDA, Food and Drug Administration; HCs, hair cells; OHCs, outer hair cells; PMSF, phenylmethylsulfonyl fluoride; PTS, permanent threshold shift; RIPA, radioimmunoprecipitation assay; ROS, reactive oxygen species; SPL, sound pressure level; TTS, temporary threshold shift.

DNA (Oh et al., 2020), apoptosis, and cell necrosis (Hu et al., 2008). However, recent studies have shown that multiple and complex mechanisms may be responsible for hearing loss, involving different cell death signaling and homeostasis pathways (Breitzler et al., 2020). It has been reported that after noise exposure calcium accumu- lates in sensory hair cells, and calcium channel blockers protect from NIHL; however, the exact molecular events underlying NIHL pathogenesis remain unknown (Wang et al., 2019). Noise exposure increases the expression of the calcium-dependent phosphatase, calcineurin, and the subsequent elevation of calcium level may lead to cell death (Chen et al., 2012).
Exchange protein directly activated by cAMP (Epac) is implicated in calcium intake (de Rooij et al., 1998; Schmidt et al., 2013). cAMP acts as an intracellular second messenger and regulates a variety of patholog- ical processes such as cell regeneration and repair (Murray and Shewan, 2008). Epac serves as an exchange factor for the small GTPase, Rap, a member of the Ras protein family. In particular, Epac promotes the conversion of inactive GDP-bound Rap1 and Rap2 into their active GTP-bound forms (Yang et al., 2012).

Acting through these small GTPases; Epac links cAMP signaling to calcium mobilization, kinase activation, gene transcription, and cytoskeleton dynamics, regulat- ing multiple cellular functions (Robichaux and Cheng, 2018). In mammals, there are two forms of Epac, Epac1 and Epac2, encoded by Rapgef3 and Rapgef4, respectively (Grandoch et al., 2010). Early studies have shown that high Epac expression results in the activa- tion of downstream Rap1 pathways, ultimately increas- ing calcium uptake (Pereira et al., 2007). Moreover, it has been reported that Epac regulates calcium release and uptake from the endoplasmic reticulum via calmodulin-dependent protein kinase II (CaMKII), thereby affecting the concentration of cytoplasmic cal- cium (Lezcano et al., 2018).
Calcium overload is associated with the apoptosis of hair cells (HCs). The involvement of the caspase- mediated cell death pathway in HC loss is widely recognized (Hua et al., 2002; Nicotera et al., 2003). A previous study suggested that hearing loss induced by temporary threshold shift (TTS) is associated with the upregulation of anti-apoptotic Bcl-2 in HCs, while hearing loss induced by permanent threshold shift (PTS) is associated with the upregulation of pro- apoptotic Bax (Yamashita et al., 2008). Moreover, cal- cium overload promotes the activation of the apoptotic protein, Bax, and activates caspase-3 and caspase-9 within damaged outer hair cells (OHCs), leading to hair cell death (Chen et al., 2020). Therefore, we explored the involvement of Epac in noise-induced damage of inner ear hair cells.
We hypothesized that Epac1 activation would exert neurotoxic effects through Ca2+/CaMKII, and that the pharmacologic inhibition of Epac would reduce apoptosis and attenuate NIHL. To establish whether the Epac1 signaling pathway was involved in NIHL, we examined the loss of OHCs, hearing loss, and apoptosis after rat exposure to noise. To explore the role of CaMKII in the effects of Epac1 on OHC apoptosis, we measured CaMKII expression, as well as the intracellular Ca2+ concentration, in HEI-OC1 cells after oligomycin exposure.

EXPERIMENTAL PROCEDURES
Animal preparation
Specific pathogen-free (SPF) male Sprague-Dawley rats (weighing 250 ± 20 g) were obtained from the Experimental Animal Center of Anhui Medical University (license: SCXK (Wan) 2017-001, Hefei, China). All rats were kept in a stable environment (22–23 °C, 60–75% humidity, 12:12 light/dark cycle), with food and water available ad libitum for 1 week before the experiments to allow for animal acclimatization to the experimental conditions. All animal experiments conformed to the guidelines of the National Institutes of Health (NIH) on the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee of Anhui Medical University (license: SYXK (Wan) 2017-006).

Intraperitoneal drug administration to rats
8-CPT (C3912, Sigma) was dissolved in 0.9% oxygenated saline (10 mg/mL, 8-CPT) as a stock solution, aliquoted, and stored at 20 °C. ESI-09 (SML0814, Sigma) was dissolved in DMSO (20 mg/mL, ESI-09) as a stock solution, aliquoted, and stored at 20 °C. The 8-CPT stock solution was diluted to 5 mg/ kg with 0.9% oxygenated saline, and the ESI-09 stock solution was diluted to 1 mg/kg with 0.9% oxygenated saline before being injected into animals. 8-CPT and ESI-09 were intraperitoneally injected 7 d before noise exposure, and the control group was injected with the same volume of saline. The rats were randomly divided into four groups (n = 10): control, noise exposure (NE), pretreatment with 8-CPT after noise exposure (NE + 8- CPT, 5 mg/kg), and pretreatment with ESI-09 after noise exposure (NE + ESI-09, 1 mg/kg).

Noise exposure
All noise-exposed rats in standard housing cages received 4 kHz octave band noise at a sound pressure level (SPL) of 106 dB for 8 h to obtain acute noise- induced hearing loss (Fu et al., 2016). The rats of the con- trol group in standard housing cages were exposed to ambient noise levels in the animal facility (55–65 dB SPL) and kept for 8 h without noise exposure. The noise was generated digitally and routed to a soundcard on a personal computer. The noise was sent to a power ampli- fier and presented with a loudspeaker. Sound levels within each test chamber were measured by a sound meter to ensure uniformity and stability.

Auditory brainstem response (ABR)
The rats in each group were tested for ABR before and after 24 h of noise exposure. The rats were anesthetized by intraperitoneal injection of 3% pentobarbital sodium solution (30 mg/kg) and placed on a heating pad in a soundproof box, maintaining the temperature at 37 °C. A subcutaneous needle electrode was inserted into the apex (active) and below the left ear (reference) and right ear (ground). The wide-band click sound (10 ms, 22/s) and tone burst sound (8, 12, 16, 24, 32 kHz, 0.5 ms rise/fall period, no plateau period, alternating phase) were used as the stimulus, and Tucker Davis Technology (TDT) System III hardware and Biosig software (Tucker-Davis Technologies, 26 Alachua, USA) were used to record the ABR thresholds. Upton 521 responses were averaged for each stimulus level. The thresholds were determined for each frequency by reducing the intensity by 10 dB increments until no organized responses were detected. ABR wave I was used to determine the ABR thresholds for each frequency. An expert who was blinded to the treatment conditions assigned the ABR scores.

Surface preparation
The rats were sacrificed after the last ABR recording, and the temporal bone was removed, perfused with 4% paraformaldehyde, and kept in this fixative overnight at 4 °C. After three PBS washes, the cochlea was soaked in 10% EDTA decalcification solution for 5 days. The side wall, and Reimes, Sossner membrane, and cover film were removed under a dissecting microscope, the basilar membrane was stained with iFluorTM 488-labeled phalloidin (diluted 1:1000 in PBS) (Yeasen Biotech Co., Ltd, China) for 30 min, rinsed with PBS, and placed on a glass slide. OHC morphology was evaluated using a fluorescent microscope.

Transmission electron microscope
The basilar membrane tissue was fixed in 2.5% glutaraldehyde solution overnight. The tissue was washed with PBS, fixed with 1% osmotic acid at 4 °C for 1 h, and then subjected to gradient dehydration, followed by epoxy resin penetration and embedding. Finally, the cochlear tissue was sliced into 50 nm sections. The sections were stained with a 50% alcohol- saturated solution of uranyl acetate and lead citrate for 5–10 min, and then washed and dried. The ultrastructure of the mitochondria was examined and imaged by transmission electron microscopy (TEM, Hitachi, Japan).

Cell culture
HEI-OC1, an inner ear cell line, was provided by the Department of Otolaryngology Head and Neck Surgery, the First Affiliated Hospital of USTC, Division of Life. The cells were cultured in DMEM/high glucose (HyClone Laboratories, USA) supplemented with 10% fetal bovine serum (FBS, CellMax, Australia) and 1% penicillin/ streptomycin in an incubator (ThermoFisher Scientific, USA) at 37 °C with 5% CO2, and 0.25% trypsin was used for subculturing at 80% cell confluence.
Oligomycin (J911096, Aladdin) was used to damage HEI-OC1 cells at a concentration of 1 mM in DMEM/high glucose without FBS in an incubator with 10% CO2 at 37 °C. HEI-OC1 cells were randomly divided into four groups: control, oligomycin (OA), pretreatment with 8- CPT after oligomycin-treated (8-CPT + OA), and pretreatment with ESI-09 after oligomycin treatment (ESI-09 + OA).

Cell viability assay
HEI-OC1 cells were incubated for 12 h in 96-well plates, and oligomycin was used at different concentrations (0.125, 0.25, 0.5, 1, and 2 mM) in triplicate wells for 12 h. The cell viability was tested using the CCK-8 Cell Counting Kit (Dojindo Laboratories, Japan). The live cell imaging system was used for live cell observation. HEI- OC1 cells were treatment with oligomycin, and holographic microscopy images were acquired using a HolomonitorTM M4 (Phase Holographic Imaging AB, PHIAB, Lund, Sweden) equipped with a 0.8 mW HeNe laser (633 nm).

Immunofluorescence staining
The rats were euthanized 24 h after the last ABR recording. The cochlea was removed and kept in 4% paraformaldehyde overnight at 4 °C. The cochleae were then rinsed in PBS prior to decalcification with 10% EDTA. After 5 days of decalcification, the basilar membrane was removed from the cochlea. The basilar membrane was incubated in 3% TritonX-100 solution for
30 min, washed three times with PBS (10 min each), and blocked with goat serum for 30 min at 25 °C. The cochlea basilar membrane was probed at 4 °C for 72 h with primary anti-Epac1 antibodies (1:100, bs-8682R, Bioss, Beijing, China). After three washes with PBS, the tissue was incubated with a TRITC-conjugated secondary antibody (ZB-0316, ZSGB-BIO, China) at a concentration of 1:50 at 4 °C overnight in the dark. The tissue was then washed three times with PBS and incubated with iFluorTM 488 phalloidin at a concentration of 1:100 for 30 min in the dark at 25 °C, and imaged by fluorescence microscopy or confocal microscopy (ZEISS LSM880 with Airyscan, Carl Zeiss AG, Germany).
HEI-OC1 cells were washed three times with PBS and kept in 4% paraformaldehyde for 15 min. The samples were permeabilized in 3% Triton X-100 solution and 5% BSA for 30 min, and then incubated with primary anti- Epac1 antibodies (1:100) for 72 h at 4 °C. After three washes with PBS, the samples were incubated with TRITC-conjugated secondary antibody (1:50) at 4 °C overnight in the dark. The samples were then washed three times with PBS and incubated with DAPI for 15 min. The samples were imaged using an ImageXpress Micro 4 imaging system (Molecular Devices, USA).

Western blotting
Protein expression was detected by western blotting. The rats were sacrificed after the last ABR recording. Rat cochlear samples were homogenized in ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer and phenylmethylsulfonyl fluoride (PMSF) using an automatic sample rapid grinding machine (Shanghai Jinxing Industrial Development Co., Ltd, Shanghai, China) at 70 Hz for 60 s. HEI-OC1 cells were lysed with cold RIPA lysis buffer plus PMSF. After 30 min on ice, the samples were centrifuged at 12,000 rpm for 15 min at 4 °C and the supernatants were stored as total protein. Protein concentrations were tested using a BCA Protein Assay kit (Beyotime Institute of Biotechnology, Beijing, China). The proteins were separated by 12% SDS-PAGE at 120 V for 1 h. The proteins were transferred to PVDF membranes and blocked with 5% skim milk at 25 °C for 1 h. The membranes were incubated with antibodies against Epac1, Rap1, CaMKII, Bax, Bcl-2, and cleaved caspase-3 (Abcam, Affinity Biosciences, USA) at 4 °C overnight, washed three times with TBST, and then incubated with goat anti- rabbit or goat anti-mouse antibodies (ZB-2306, ZSGB- BIO, China). b-actin (TA-09, ZSGB-BIO, China) was used as a sample loading control. The protein bands were imaged using a ChemiQ4600mini chemiluminescence imaging system (Bioshine, China), and the relative band density was quantified by Image J.

Apoptosis detection
Annexin V-FITC (Vazyme Biotech Co., Ltd, China) was used to analyze apoptosis, and propidium iodide (PI) was used to distinguish living from non-living cells. HEI- OC1 cells were washed three times with PBS and kept in binding buffer containing a saturating concentration of Annexin V-FITC for 30 min at 25 °C. Then, the cells were incubated for 15 min with 5 ml of PI and imaged with an ImageXpress Micro 4 imaging system (Molecular Devices, USA).

Measurement of intracellular Ca2+ concentration
Ca2+ levels were assessed using fura-2 AM fluorescence and a confocal laser microscope. The treated HEI-OC1 cells were loaded with fura-2 AM by incubation with 2 mM fura-2 AM (Dojindo, Japan) and 0.02% F127 for
30 min, and then transferred to a physiological salt solution (PSS: 145 mM NaCl, 3 mM KCl, 4 mM MgCl2,
10 mM glucose, and 10 mM Hepes, pH 7.4). Thapsigargin (2 mmol/L) was added to the bath solution to empty the intracellular calcium stores. Extracellular Ca2+ was added back to the cells to reach a final concentration of 4 mM. Fluorescence signals from bound Ca2+ and unbound fura-2 were measured using an Olympus Fluorescence Imaging System and excitation wavelengths of 340 and 380 nm.

Statistical processing
The statistical analysis was performed using GraphPad Prism 6 software. One-way analysis of variance (ANOVA) and t-tests were used to analyze all results. P values <0.05 were considered indicative of statistically significant differences. All data were expressed as mean ± SD. RESULTS Epac1 stimulation increases the ABR threshold after noise exposure ABR measurements were performed before and 24 h after noise exposure (Fig. 1A). The results showed that the rat model of TTS was successfully established (Fig. 1B). Noise exposure significantly increased the ABR threshold from 31.00 ± 1.871 dB SPL to 73.00 ± 2 .550 dB SPL at 12 kHz. Treatment with the Epac agonist, 8-CPT, further elevated the ABR threshold, while Epac inhibition with ESI-09 decreased the ABR threshold at click, 8 kHz, and 12 kHz (Fig. 1C–F). The hearing loss was more severe at high frequencies than at low frequencies. Epac1 stimulation after noise exposure promotes apoptosis-mediated hair cell death The loss of OHCs was examined by phalloidin staining after noise exposure. The results showed that control OHCs were arranged neatly, and intact at all frequency Fig. 1. Effects of Epac regulators on ABR threshold after noise exposure. Rats received an intraperitoneal (i.p.) injection of 8-CPT and ESI-09 for 7 consecutive days before noise exposure. (A) The protocol of the noise exposure (NE) experiments is illustrated (n = 10 per group). (B) ABR waveforms for the four group of rats at 12 kHz tone burst. (C–F) Analysis of ABR thresholds at click and tone burst at 8 kHz, 12 kHz, 24 kHz in the four groups of rats after noise exposure (n = 10 per group). Data represent the mean ± SEM; *P < 0.05 vs. Control, #P < 0.05 vs. NE. Fig. 2. Effects of Epac regulators on the morphology and apoptosis of cochlear inner hair cells. (A) Representative fluorescent microscopic images of OHC loss from cochlear surface preparations stained with phalloidin processed 24 h after noise exposure (n = 3 per group); scale bar = 50 mm. (B) Representative transmission electron microscopy (TEM) images of cochlear mitochondria (n = 3 per group); scale bar = 20 mm. (C) Western blot analysis of cleaved caspase-3, Bax, and Bcl-2 expression in the cochlea after noise exposure (n = 3 per group). b-actin was used as the loading control. The data represent the mean ± SEM; *P < 0.05 vs. Control group, #P < 0.05 vs. NE group. Noise trauma activates Epac1 in OHCs The level of Epac1 expression in OHCs was evaluated by immunolabeling of cochlear surface preparations, and found to be increased 24 h after noise exposure (Fig. 3A, B). Additionally, western blot analysis showed that the level of Epac1 was significantly increased 24 h after noise exposure. Furthermore, the activity of the direct downstream effector of Epac, Rap1, was markedly increased 24 h after noise exposure by western blotting (Fig. 3C, D). To further clarify the role of Epac1 signaling pathway in NIHL, the Epac agonist, 8-CPT, and inhibitor, ESI-09, were employed. Both Epac1 and Rap1 levels were increased in OHCs and cochlear tissue after treatment with 8-CPT, while treatment with ESI-09 yielded the opposite effects, as determined by both immunolabeling and western blotting. Oligomycin exposure increases Epac1 expression in HEI-OC1 cells The toxicity of oligomycin toward the cochlear cell line, HEI-OC1, was tested by CCK-8 assay. HEI-OC1 cells were incubated with different concentrations of oligomycin (0.125, 0.25, 0.5, 1, and 2 mM) for 12 h. The CCK8 assay showed that the number of surviving cells was reduced by treatment with 0.5 mM oligomycin, and further decreased with increasing drug concentrations (Fig. 4C). Thus, in view of these and previous results (Fan et al., 2020), the oligomycin concentration of 1 mM was chosen to induce HEI-OC1 cell damage. Fig. 3. Expression of Epac1 in cochlear tissue after noise exposure. (A) Representative fluorescent microscopic images of Epac1 in OHCs 24 h after noise exposure; green: phalloidin-stained OHCs; scale bar = 10 mm. (B) Bar graph representing the intensity of Epac1-specific signal in OHCs (n = 3 per group). (C) Western blot analysis of Epac1 and Rap1 expression in the cochlea after noise exposure. b-actin was used as loading control. The data represent the mean ± SEM (n = 3 per group); *P < 0.05 vs. Control group, #P < 0.05 vs. NE. Epac1 activation increases apoptosis in HEI-OC1 cells following oligomycin-induced damage To explore the role of Epac in oligomycin-induced apoptosis, we used PI and Annexin V-FITC to label dead and apoptotic cells, respectively. The proportions of apoptotic and dead cells were significantly increased after treatment with oligomycin compared to controls. Notably, these proportions were further increased after pretreatment with the Epac agonist, 8-CPT, and reduced by the Epac inhibitor, ESI-09, compared to cells treated with oligomycin alone (Fig. 5A, B). Furthermore, the expression of the proapoptotic proteins, cleaved caspase-3, cleaved caspase-9, and Bax was analyzed by western blotting in HEI-OC1 cells treated with oligomycin (1 mM) for 12 h, with or without a 2 h pretreatment with 8-CPT or ESI-09. Cleaved caspase-3, cleaved caspase-9, and Bax were found upregulated after oligomycin treatment. Cell pretreatment with 8-CPT further increased the expression of cleaved caspase-3, cleaved caspase-9, and Bax, while pretreatment with ESI-09 attenuated the upregulation of these proteins (Fig. 5C, D). Fig. 4. Effects of Epac regulators on HEI-OC1 cell survival after oligomycin exposure. (A) HEI-OC1 cells were pre-incubated with the Epac agonist, 8-CPT, and the Epac inhibitor, ESI-09, for 2 h, and then incubated with 1 mM oligomycin for 24 h. Cell images were captured by holographic microscopy, and show the morphological changes over time after treatment with oligomycin; scale bar = 50 mm, n = 3. (B) CCK-8 assay of HEI- OC1 cells treated with different oligomycin concentrations for 12 h, n = 3. (C, D) HEI-OC1 cell confluence (%) was calculated by holographic microscopy, n = 3; *P < 0.05 vs. Control group. upregulated in oligomycin-treated compared to control cells (Fig. 6C, D). To further clarify the role of the Epac signaling pathway in HEI-OC1 cells, the effects of the Epac stimulation or inhibition on Epac1 and Rap1 expression were investigated by both immunofluorescence and western blotting. Cell pretreatment with 8-CPT increased Epac1 immunostaining, as well as Epac1 and Rap1 protein expression, as determined by western blotting, while the opposite results were obtained after HEI-OC1 cell pretreatment with ESI-09. Fig. 5. Effect of Epac1 on oligomycin-induced HEI-OC1 cell apoptosis. (A) HEI-OC1 cells stained with Annexin V-FITC (green) and propidium iodide (PI, red); scale bar = 20 mm, n = 3. (B) Proportions of early apoptotic and death cells in oligomycin-treated HEI-OC1 cells. (C, D) Western blot analysis of cleaved caspase-3 and Bax expression in oligomycin-treated HEI-OC1 cells. b-actin was used as the loading control. The data represent the mean ± SEM, n = 3; *P < 0.05 vs. Control group, #P < 0.05 vs. OA. abolished by Epac inhibition but further increased by Epac stimulation (Fig. 7A–C). Moreover, analysis of CaMKII expression in the cochlear tissue revealed that CaMKII was upregulated after rat exposure to noise, in line with the in vitro results obtained with cultured cells (Fig. 7E). DISCUSSION In this study, we demonstrated that the expression level of Epac1 was increased in a rat model of NIHL. Moreover, we found that the Epac1 signaling pathway played a key role in oligomycin-induced, CaMKII-mediated death of HEI-OC1 cells. We also showed that pharmacologic blockade of Epac protected OHCs from NIHL-related injury. Damage to inner ear hair cells may cause irreversible hearing loss. However, no effective drugs or surgical treatments are still available (Youm and Li, 2018), and interventions capable of reducing hair cell loss and preserving the hearing function are urgently needed (Campbell et al., 2007). Our study suggests that Epac1 is a novel therapeutic target for NIHL and deserves further investigation. Fig. 6. Expression of Epac1 in HEI-OC1 cells after oligomycin exposure. (A) Representative fluorescent microscopic images showing Epac1 expression in HEI-OC1 cells 12 h after oligomycin exposure; blue: DAPI; scale bar = 20 mm, n = 3. (B) Bar graph representing the intensity of Epac1 signal in HEI-OC1 cells. (C) Western blot analysis of Epac1 and Rap1 expression in HEI-OC1 cells 12 h after oligomycin exposure. b-actin was used as the loading control. The data represent the mean ± SEM, n = 3; *P < 0.05 vs. Control group, #P < 0.05 vs. OA. Fig. 7. Effects of Epac regulators on intracellular Ca2+ in HEI-OC1 cells. Epac1 activation induces OHC death through CaMKII. (A) Representative images of Ca2+ in HEI-OC1 cells 12 h after oligomycin exposure; green: fura-2 AM; scale bar = 100 mm, n = 3. (B) The intracellular Ca2+ concentration was determined by confocal Ca2+ measurements; time courses of changes in calcium fluorescence are shown; n = 3. (C) A summary of data showing [Ca2+]i alterations induced by Epac regulators. (D) Western blot analysis of CaMKII expression in cochlear tissue after noise exposure; n = 3. (E) Western blot analysis of CaMKII expression in oligomycin-treated HEI-OC1 cells; b-actin was used as the loading control. The data represent the mean ± SEM, n = 3; *P < 0.05 vs. Control group, #P < 0.05 vs. OA.(Wehbe et al., 2020), blood vessels, and kidney, where it regulates a wide range of cellular functions (Le Prell et al., 2007) such as cell growth, adhesion, differentiation, divi- sion, cytokinesis, and inflammation. A recent study showed that a variety of kinases are involved in the patho- physiological events induced by Epac activation (de Rooij et al., 2000), including CaMKII, JNK (Kayyali et al., 2018), p38 MAPK, PKC (Chen et al., 2015), and AKT (Kou et al., 2013), which are all important in NIHL pathogenesis. Jamesdaniel et al. (2011) showed that noise activates p38 MAPK in cochlear hair cells, leading to hair cell death and hearing loss. Wang et al. (2019) showed that noise stimulation increases the expression of MCU, a unidirec- tional calcium transporter, in cochlear hair cells, downreg- ulates the expression of the sodium-calcium transporter, and causes cellular calcium overload, ultimately leading to hair cell damage and hearing loss. Chen et al. (2012) showed that noise stimulation activates Rho GTPases and NADPH oxidase, and increases ROS formation, lead- ing to hair cell death. The present study showed that the expression levels of Epac1 and Rap1 were significantly upregulated after noise exposure, suggesting that the Epac1 signaling pathway was involved in the progression of NIHL. Moreover, noise exposure induced mitochondrial damage in hair cells, causing hair cell loss and impaired hearing in rats. In order to verify the role of Epac1 in NIHL, we used the Epac1 agonist, 8-CPT, and the inhibitor, ESI- 09. HEI-OC1 cell pretreatment with 8-CPT increased Epac1 expression but did not exacerbate noise-induced cell damage, possibly because noise exposure induced a maximal effect on Epac1 expression. ESI-09 inhibited noise-induced Epac1 and Rap1 overexpression, and reduced hearing loss and mitochondrial damage in rats. This suggests that Epac1 downregulation could be a strategy to reduce hearing loss due to noise exposure, and identifies the Epac1 signaling pathway as a potential effective target for hearing loss treatment. Several studies have shown that CaMKII is a downstream effector of the Epac1 signaling pathway and participates in the regulation of cell death. Oestreich et al. (2009) showed that Epac1 increases the intracellular Ca2+ concentration through PLC-e inositol 1, 4, 5- triphosphate receptor signaling. Elevation of intracellular Ca2+ leads to the activation of CaMKII via autophospho- rylation on threonine 286, resulting in enhanced and pro- longed kinase activity. Rap1, an immediate effector of Epac, has been shown to contribute to Epac-mediated cell death. Liu and Schneider (2013) showed that the Epac/CaMKII pathway regulates Ca2+ homeostasis, stimulates transcription coupling and gene expression, and may contribute to cardiac hypertrophy and arrhyth- mia. Zhang et al. (2020) showed that Epac/CaMKII is implicated in neuronal damage, and emphasized the potential value of Epac/CaMKII inhibition in the treatment of various retinopathies. In the current study, we used in vivo and in vitro approaches to examine the expression of CaMKII, which reflected the level of Ca2+ in the cochlear tissue and HEI-OC1 cells. The expression level of CaMKII increased after noise exposure as well as treat- ment with oligomycin, and the Epac1 inhibitor, ESI-09, partially abolished CaMKII upregulation induced by noise and oligomycin. These results indicated that CaMKII- mediated Ca2+ regulation was involved in Epac1-related hearing loss. Overall, our study demonstrated that Epac1 was rapidly activated after noise exposure, functioned downstream of Rap1, and promoted calcium overload by activating CaMKII, resulting in increased cochlear damage. Cochlear hair cell apoptosis is a phenomenon related to noise-induced irreversible injury. Therefore, the modulation of this process could be an effective strategy for preventing NIHL. Mitochondrial calcium overload has been associated with apoptosis (He et al., 2017), and the increase in free Ca2+ in outer hair cells has been shown to lead to Bax activation, which in turn alters the permeability of the outer mitochondrial membrane and promotes the release of cytochrome C, followed by cas- pase activation and apoptosis (Kim et al., 2019). On the other hand, Bcl-2 antagonizes Bax expression and regu- lates apoptosis (Kale et al., 2017). Epac1 has been reported to induce apoptosis in cardiomyocytes and smooth muscle through the CaMKII pathway (Lewis et al., 2016; Szanda et al., 2018). Our results showed that the pharmacological inhibition of Epac1 significantly decreased the level of cleaved caspase-3 and signifi- cantly increased the Bcl-2/Bax ratio. Notably, Epac1 inhi- bition reduced cochlear hair cell loss and attenuated mitochondrial damage in hair cells. Moreover, ESI-09 reduced CaMKII expression and inhibited calcium- mediated activation of apoptosis. These results clearly indicated a role of Epac1 in NIHL.
In this study, we found that Epac1 expression was increased in experimental rat models of NIHL.

Moreover, noise exposure and oligomycin treatment caused Epac1 activation, which in turn induced CaMKII- dependent apoptosis in HCs and HEI-OC1 cells.
To our knowledge, this study is the first to link the Epac/CaMKII pathway to NIHL, and suggests that the inhibition of Epac1 expression is a novel potential therapeutic strategy for this condition.

DECLARATION OF COMPETING INTEREST
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

ACKNOWLEDGMENTS
The authors thank all members of the Center for Scientific Research of Anhui Medical University for their valuable help in our experiment. And we would like to thank AJE (American Journal Experts) for English language editing. This work was supported by The Natural Science Foundation of Anhui Province (1808085MH250).

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