DT-010 Exerts Cardioprotective Effects by Regulating the Crosstalk between the AMPK/PGC-1α Pathway and ERp57

The AMP-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) pathway performs a crucial role in energy metabolism and mitochondrial network. Our previous study found that DT-010, a novel danshensu (DSS) and tetramethylpyrazine (TMP) conjugate, had significant cardioprotective properties in vitro and in vivo. We also reported that ERp57 served as a major target of DSS using the chemical proteomics approach. In this article, we focus on exploring the interrelationship between the regulation of the AMPK/PGC-1α pathway and promoting ERp57 expression induced by DT-010 in tert-butylhydroperoxide- (t-BHP-) induced H9c2 cell injury. The results showed that DT-010 activated the AMPK/PGC-1α pathway and increased ERp57 protein expression. Importantly, the above phenomenon as well as the mitochondrial function can be partially reversed by siRNA-mediated ERp57 suppression. Meanwhile, silencing AMPK significantly inhibited the ERp57 expression induced by DT-010. In addition, molecular docking and kinase assay in vitro revealed that DT-010 had no direct regulation effects on AMPK activity. Taken together, DT-010 exerted cardioprotective effects by regulating the crosstalk of AMPK/PGC-1α pathway and ERp57, representing a potential therapeutic agent for ischemic heart disease.


Introduction
Ischemic heart disease (IHD) is the top leading cause of cardiovascular diseases (CVDs) health lost [1,2]. Variable treatment plans are available depending on the symptoms and types of CVDs. The health status of the general population has improved dramatically over the past few decades, benefiting from lifestyle changes, medications, surgery, cardiac rehabilitation, and active surveillance [3]. However, there remains no effective therapeutic agent for preventing myocardial ischemia-reperfusion injury (MIRI) in patients; hence, new strategies and methods are in urgent need. MIRI leads to mitochondrial damage by affecting energy metabolism, mitochondrial-related inflammation, mitochondrial dynamics, etc. [4] Multiple studies have also shown that improving mitochondrial function significantly reduced MIRI [5][6][7]. Mitochondrial-targeted clinical intervention trials to restore mitochondrial function have been underway for many years, although there are still no mitochondrialtargeted drugs (MTDs) for the treatment of CVDs specifically [8][9][10][11]. Novel MTDs for IHD are still needed.
Accumulating studies have demonstrated a positive interconnectedness between the AMP-activated protein kinase (AMPK) and the mitochondrial net [12]. Mitochondrial dysfunction and oxidative stress mediated by AMPK deficiency are essential processes in MIRI. Peroxisome proliferatoractivated receptor gamma coactivator-1 alpha (PGC-1α) is a key regulator of mitochondrial biogenesis [13], and it may contribute to enhanced mitochondrial DNA (mtDNA) copy number, an indicator of disease aggressiveness. Overexpression of PGC-1α improves I/R-induced cardiomyocyte apoptosis [14]. Activated PGC-1α/nuclear factor erythroid 2-related factor 2 (Nrf2) signaling exerted an antioxidant effect in oxygen-glucose deprivation/reperfusion-(OGD/R-) induced injury in H9c2 cells [15]. Emerging research has shown that the activation of the AMPK/PGC-1α pathway could decrease mitochondrial dysfunction and apoptosis [16]. Hence, activation of the AMPK/PGC-1α pathway may be one of the strategies for the treatment of IHD.
Our previous studies had shown that DT-010 exerted cardioprotective effects by activating the PGC-1α/Nrf2/heme oxygenase 1 (HO-1) pathway [17,18]. However, the upstream regulator in terms of DT-010 is unclear. Furthermore, a chemical proteomics approach was used, and we identified endoplasmic reticulum (ER) resident protein 57 (ERp57) as a potential target of DT-010 [18]. ERp57 is an ER resident thiol-disulfide oxidoreductase and acts as a redox-dependent chaperone. Prolonged ER stress causes cellular dysfunction that leads to CVDs. The study found that cell viability and clonogenic survival are reduced upon the depletion of ERp57 [19]. However, the function of ERp57 in cardioprotection has rarely been studied. It is also unclear whether DT-010 acts majorly on the crosstalk between the AMPK/PGC-1α pathway and ERp57. In the following, we explored the hypothesis of DT-010 in a model of tert-butylhydroperoxide-(t-BHP-) injured H9c2 cells and its associated signal pathways. This finding will provide new insight into CVD treatment.

Cell
Culture and Treatment. The H9c2 cardiomyoblast cell line was cultured in DMEM containing 10% fetal bovine serum at 37°C in a 5% CO 2 humidified incubator. After treatment, H9c2 cells were collected and processed for the MTT assay, cellular oxygen consumption measurement, and Western blot assays, respectively.

MTT Assay.
For the cytotoxicity test, H9c2 cells were treated with DT-010 (10,15,20,30, and 40 μM) diluted in serum-free DMEM for 24 h or DT-010 (30 and 100 μM) at different time points (1, 2, and 4 h). MTT (final concentration: 0.5 mg/mL) was then added and cultured for another 4 h. For the AMPK inhibitor test, H9c2 cells were preincubated with 1 mM compound C (an AMPK inhibitor) for 0.5 h. After discarding the medium, H9c2 cells were then treated with DT-010 (30 μM) for 1 h, the supernatant was aspirated, and a fresh medium containing 150 μM t-BHP was added for another 4 h. The microplate reader (BioTek Instruments, USA) was used to measure the absorbance at 570 nm. Cell viability (%) was calculated relative to the value of the group without any treatment. Cell viability (% of Ctrl) was measured by the MTT assay. * * P < 0:01, * * * P < 0:001, and * * * * P < 0:0001 versus the Ctrl group. Data are mean ± SD of three independent experiments. Data were analyzed using one-way ANOVA or two-way ANOVA and Dunnett's multiple comparison tests.      [20]. The reaction mixture (50 μL) was conducted at 30°C for 40 min. The kinase activity was measured by quantitating the amount of ATP remaining in the solution using the Kinase-Glo Plus luminescence kinase assay kit. The IC 50 values were calculated using GraphPad Prism software (nonlinear regression with normalized dose-response fit).
2.8. Data Analysis and Statistics. All data were processed and analyzed with GraphPad Prism software 9.0 (GraphPad, San Diego, CA, USA). Results were stated as mean ± standard deviation (mean ± SD). One-way ANOVA or two-way ANOVA and Dunnett's or Tukey's multiple comparison tests were assessed for comparison between groups. Differences with P < 0:05 were defined as significant.

Cardiovascular Therapeutics
were reduced, and DT-010 administration eliminated these differences (Figures 2(a)-2(d)). The above results demonstrated that the cardioprotective effect of DT-010 may be interconnected with the revision of ERp57 and the AMPK/ PGC-1α signaling pathway. Compound C, also named dorsomorphin, is a potent, reversible, selective AMPK inhibitor. As shown in Figure 2(e), the cell viability was significantly decreased when AMPK activity was blocked (P < 0:01).

Upregulation of ERp57 Expression by DT-010
Depends on AMPK Activation. Our results confirmed that DT-010 can promote AMPK phosphorylation and upregulate ERp57 protein expression; thus, the upstream and downstream regulatory relationship of DT-010 on AMPK and ERp57 was determined. As shown in Figure 4, compared with the DT-010+adenovirus vector group, the expression of ERp57 protein was significantly decreased after the knockdown of AMPK mediated by the adenovirus. The results indicated that DT-010 may regulate the expression of ERp57 by activating AMPK, thereby affecting mitochondrial function.

Upregulation of PGC-1α Expression by DT-010 Is
Dependent on ERp57 Activation. To further verify the upstream and downstream relationship of DT-010 referring to ERp57 and PGC-1α expression, we next evaluated the influence of DT-010 on PGC-1α protein expression by specific shRNA of ERp57. As revealed in Figure 5, DT-010 sig-nificantly increased the expression of ERp57 and PGC-1α when matched to the t-BHP+scrambled siRNA group (P < 0:01). Compared with the t-BHP+DT-010+scrambled siRNA group, the upregulation trend of PGC-1α was partially abolished by ERp57-specific shRNA. The result reflected that DT-010 may regulate PGC-1α expression by regulating ERp57 expression.

Discussion
IHD remains a major disease with an increasing overall global burden. Thrombolysis and percutaneous coronary intervention are effective and are extensively applied, which can restore myocardial perfusion timely and alleviate myocardial infarction or necrosis [21]. Myocardial ischemic reperfusion injury (MIRI) that comes along with restoring blood flow may further worsen the prognosis in these patients. According to the statistics of the World Health Organization, one out of every six deaths was attributed to MIRI [22]. Research is being carried on; however, there is still no effective therapeutic strategy to prevent MIRI [21]. Mitochondrial dysfunction is involved in the pathological process of myocardial infarction. The study found that impaired mitochondrial biosynthesis was the common cause of end-stage ischemic heart failure and cardiac hypertrophy [12]. Multiple clinical studies have found that mitochondrial targeting agents (MTAs) have limited benefit in the treatment of CVD, properly due to the poor uptake by ischemic tissue, the single regulatory target, the inefficient mitochondrial localization or tissue-specific delivery, etc. [23] Therefore, targeting mitochondria from multiple angles is still an important treatment strategy for CVDs, and efforts should be made to improve the clinical transformation rate [24,25]. In our previous study, we found that DT-010 exhibited brilliant antioxidant and antiapoptotic properties which were at least partially concerned with the activation of the PGC-1α/Nrf2/HO-1 pathway [17]. Continuing in-depth studies, we revealed that DT-010 displayed cardioprotective effects by regulating the axis of AMPK/PGC-1α pathway signaling and ERp57 crosstalk. Our results suggest that DT-010 maybe a promising MTA for myocardial ischemia therapy.
Overexpression of AMPK improved mitochondrial biosynthesis by maintaining mitochondrial redox homeostasis and mitochondrial respiration and inhibiting mitochondrial apoptosis [26]. The PGC-1α pathway was downregulated in rat and mouse models of heart failure [27]. Notably, PGC-1α transcription was blocked in heart failure caused by myocardial infarction [28]. Meanwhile, mitochondrial number and  Cardiovascular Therapeutics mtDNA copy number were reduced in rodent and human models of heart failure. The study found that AMPK could improve cardiac contractility after myocardial infarction by promoting PGC-1α expression and enhancing mitochondrial respiration [29]. AMPK/PGC-1α pathway activation prevented acute cardiotoxicity by maintaining mitochondrial homeostasis [30]. Therefore, targeting the AMPK/PGC-1α pathway might be one of the important strategies for the treatment of CVDs. Our results showed that DT-010 increased the expression of the AMPK/PGC-1α pathway. Importantly, the cardioprotective effects were reversed by AMPK inhibitor compound C in vitro. Interestingly, molecular docking and in vitro kinase experiments showed that DT-010 had no direct regulatory effect on AMPK. AMPK is widely expressed in vivo and regulates a variety of life activities, and AMPK heterodimeric complexes in different tissue subunit compositions vary widely, which may lead to reduced efficacy and increased drug toxicity. Although AMPK agonists have shown cardioprotective effects in ischemia-reperfusion mouse model. Proper regulation of AMPK should exert its protective potential while avoiding its potentially harmful effects. We speculated that DT-010 was superior to these AMPK activators as DT-010 not only had a significant cardioprotective effect by activating the AMPK/PGC-1α pathway but also had a high safety profile. Studies have found that ERp57 was involved in a range of disease processes, including neurological disease [31], cancer [32], and infection diseases [33]. However, it is unclear the association between ERp57 and cardioprotection. ERp57/PDIA3 overexpression inhibited the ER stress and apoptosis induced by the SOD1 mutant, suggesting ERp57's role in neuroprotection [34,35]. Wang et al. demonstrated that ERp57/PDIA3 deficiency reduced inflammation, oxidative pressure, and apoptosis in traumatic brain injury (TBI) mice [36], indicating that ERp57/PDIA3 had different roles in different cellular processes in different cellular compartments. ERp57/PDIA3 might also participate in the modulation of mitochondrial Ca 2+ indirectly through the interaction with calnexin [37]. The t-BHP-mediated intracellular Ca 2+ increase could induce ROS generation and mitochondrial dysfunction [38]. Our previous study has found that DT-010 could reduce t-BHP-induced intracellular free radical production, including hydroxyl radical (·OH), superoxide anion(·O 2− ), and peroxynitrite radical (ONOO − ). Moreover, DT-010 triggered Nrf2 nuclear translocation and upregulated the protein expression of Nrf2 as well as mitochondrial transcription factor A (Tfam) and HO-1 in H9c2 cells [17]. We speculated that DT-010 may affect mitochondrial calcium homeostasis by regulating ERp57 to control ROS generation and mitochondrial function. Our published literature revealed that ERp57 was a major target of biotinylated danshensu (DSS) using the chemical proteomics approach and showed that ADTM, a novel DSS and tetramethylpyrazine (TMP) conjugate, exhibited potent inhibition on the redox activity of ERp57/PDIA3 [18]. Based on the structural similarity between ADTM and DT-010, we reasoned that DT-010 might have a regulatory effect on ERp57. We next explored the relationship between the regulation of the AMPK/PGC-1α pathway and pro-moting ERp57 expression induced by DT-010 in H9c2 cell oxidative injury model. The results showed that DT-010 activated the AMPK/PGC-1α pathway and increased the ERp57 protein expression. Importantly, specific siRNAmediated ERp57 suppression partially reduced the PGC-1α protein expression and inhibited the mitochondrial functions of DT-010. Meanwhile, silencing AMPK significantly inhibited the expression of ERp57 of DT-010.

Conclusion
We speculated that DT-010 may have a different mode of action from that of ADTM. We found a feedback loop regulated by DT-010 among AMPK, PGC-1α, and ERp57, which are associated with oxidative stress and mitochondrial dysfunction. Our bountiful research work exhibited enormous promise for its involvement in CVDs.

Data Availability
The raw data supporting the conclusion of this article will be made available by the corresponding authors.