Breviscapine remodels myocardial glucose and lipid metabolism by regulating serotonin to alleviate doxorubicin-induced cardiotoxicity

Reagents

The following reagents were used: DMEM, FBS, penicillin/streptomycin (Meilunbio, Dalian, China), cTnI Elisa kits (Sangon Biotech, Shanghai, China), TNF-α, IL-1β, and IL-6 Elisa kits (MyBioSource, San Diego, United States), MDA kits, SOD kits, and NADH kits (Solarbio, Beijing, China), CCK-8 kits (Beyotime Biotechnology, Shanghai, China), ROS fluorimetric kits, JC-1 fluorescent dye, protein extraction kits, and BCA kits (Meilunbio, Dalian, China). IL-1 (1:100), PINK1(1:1,000), Parkin (1:1,000), AMPK(1:1,000), Akt (1:1,000), P13K(1:1,000), Tom20 (1:1,000), β-tubulin (1:1,000), mTOR (1:1,000), and an HRP-conjugated GAPDH (1:1,000) antibody were obtained from Abclonal Biotechnology (Wuhan, China). The RIPA lysis buffer and loading buffer were purchased from Meilunbio (Dalian, China). PVDF membranes were obtained from Merck (New Jersey, United States). DMSO was purchased from Macklin (Shanghai, China), and doxorubicin, breviscapine, and dexrazoxane were purchased from Widely (Wuhan, China). The Mdivi-1 inhibitor was purchased from Selleck Chemicals (Houston, United States).

Cell culture conditions and cell lines

Culture medium and supplements for cell culture were purchased from Gibco-Invitrogen (Carlsbad, CA, United States), and plasticware was purchased from Corning (Corning, NY, United States). H9c2 embryonic rat heart-derived cardiomyoblast cells (ATCC, CRL-1446) were cultured in DMEM containing 10% fetal bovine serum, 100 units/ml penicillin G sodium, and 100 μg/ml streptomycin sulfate (37°C, 5% CO₂). MCF-7 human metastatic breast cancer cells (iCell, iCell-h129) were cultured in 1,640 media supplemented with 10% fetal bovine serum, 100 units/ml penicillin G sodium, and 100 μg/ml streptomycin sulfate. 4T1 mouse breast cancer cells (ATCC, FS-0158) were cultured in DMEM medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin G sodium, and 100 μg/ml streptomycin sulfate.

The cells were passaged regularly when they reached 80–90% confluence and seeded in 96-well plates (1 × 10⁴ cells/well). H9c2 cells were exposed to control, 5 μM Dox (Guo et al., 2013), 5 μM Dox+20 μM Dexra (Sangweni et al., 2020), or 5 μM Dox+200 μM breviscapine (Li et al., 2022) for 24 h, respectively. Cells treated with DMEM only were used as blank controls, and Dexra was used as a positive control. Finally, we also injected Mdivi-1 (5μM, in DMSO), an inhibitor of mitochondrial division (Nhu et al., 2021).

Assessment of oxidative stress

Intracellular ROS levels were measured using the fluorescence dye DCFH-DA according to the manufacturer’s instructions. Briefly, DCFH-DA solution (1 μM) was prepared in PBS and added to H9c2 cells cultured in a 6-well plate before incubation at 37°C for 30 min. After incubation, the cells were washed with PBS. Fluorescent photomicrographs were taken at 20× magnification using a fluorescence microscope from Leica Microsystems, Ltd. ROS levels were quantified with a Beckman flow cytometer (Beckman, California, United States).

Determination of enzymatic indices

The activity of SOD and NADH was measured using an activity assay kit according to the manufacturers’ instructions. The absorbance and luminescence were measured using a microplate reader (Perkin Elmer, Massachusetts, United States).

Electron microscopy

Cells were fixed with 0.5% glutaraldehyde fixative at 4 °C for 15–30 min and were collected by centrifugation at 10,000–13,000 rpm for 5 min. The cells were further fixed with 3% glutaraldehyde at 4°C overnight and then treated with 1% osmium tetroxide at room temperature for 2 h. Thereafter, the samples were dehydrated in an acetone gradient, embedded in Epon 812, subjected to optical positioning, and cut into ultrathin sections. The sections were double-stained with uranyl acetate and lead citrate. The mitochondrial ultrastructure was examined using an H-7650 transmission electron microscope (Hitachi, Toyko, Japan).

Determination of mitochondrial respiration

Approximately 10⁶ cells were used to measure mitochondrial respiration with an O2K respirometer (Oroboros Instruments, Austria). The oxygen concentration was determined and analyzed by using Oroboros DatLab 7.4 software. In brief, a leak respiratory state was recorded in cells alone, electron transfer was coupled to phosphorylation by the addition of 5 mM ADP, and state 3 respiration supported by complex I was recorded. Maximal state 3 respiration with parallel electron input from complex I and complex II was recorded through the addition of 10 mM succinate, and complex II-supported respiration was measured in the presence of 6.25 µM rotenone. Maximal electron transfer capacity was recorded in the presence of 5 µM carbonyl cyanide p-(trifluoro-methoxy) phenyl-hydrazone (FCCP). Finally, antimycin (Ama), which completes complex Ⅲ and blocks all electron transport, was administered, and the oxygen consumption rate was measured as nonmitochondrial oxygen consumption.

Determination of changes in the mitochondrial membrane potential (ΔΨm)

5.5′,6.6′-Tetrachloro-1.1′,3.3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining was used to assess the mitochondrial membrane potential in H9c2 cells. Briefly, according to the predetermined experimental conditions, H9c2 cells were washed with warm Dulbecco’s phosphate-buffered saline (DPBS) before being stained with 100 μL of 2 μM JC-1 solution (mixed in DMEM without phenol red) and incubated under standard cell culture conditions for 30 min in the dark. After incubation, the cells were washed with warm DPBS, and fluorescence photomicrographs were taken at 20× magnification using a Leica Microsystems CMS GmbH inverted fluorescence microscope (Leica Camera AG, Barnack, German).

Immunofluorescence

To assess mitochondrial localization, cells grown on coverslips were washed with cold PBS and fixed with 4% paraformaldehyde for 15 min. Then, the cells were permeabilized with 0.5% Triton X-100 for 20 min and blocked with 5% goat serum for 30 min. The cells were incubated with primary antibodies overnight at 4°C and with secondary antibodies for 1 h. After washing three times, the cells were stained with 4′-6-diamidino-2-phenylindole (DAPI) and imaged with a confocal laser scanning microscope.

ATP measurement

Additionally, the intracellular ATP concentration was measured by using an ATP assay kit following the manufacturer’s protocol. The cells were lysed in the lysis buffer by repeated pipetting and centrifuged at 4 °C and 13,000 g for 10 min. The supernatants were used for the analysis of ATP levels, and the protein concentrations were determined by the BCA assay. A volume of fifty microliters of supernatant was added to 100 μl of ATP detection solution, incubated at room temperature for 5min, and mixed immediately, and the luminescence was determined using a luminometer (Flex Station 3). The concentration of ATP was calculated according to a standard curve and converted into nmol/μg protein.

Western blotting

Protein expression was evaluated by Western blotting and quantified with densitometry. The Dox and Dox + Brev-treated H9c2 cells in 10 cm cell culture dishes were lysed with the RIPA lysis buffer. Taking 5 mg of tissue and adding 10 ml of lysis buffer. The Bradford method was used to measure the protein concentration, utilizing BSA as a standard. Equivalent amounts of proteins were mixed with loading buffer (5X) and boiled at 95°C for 5 min. The proteins were then resolved by electrophoresis on 10%–12% SDS-polyacrylamide gels (SDS–PAGE) and transferred onto PVDF membranes. After blocking with 5% milk in TBST for 2 h at room temperature, the membranes were incubated overnight at 4°C with specific antibodies, such as rabbit polyclonal GAPDH. WB analysis of protein expression and phosphorylation was performed using antibodies against AMPK, Parkin, PINK1, Akt, PI3K, Tom20, and GAPDH was used as an internal control. Specific signals were visualized using the Bio-Rad gel doc system (Bio-Rad Laboratories, California, United States). The data are presented as the mean ± SD (n = 3) and quantified by ImageJ analysis.

Animal husbandry

Eight-week-old female C57BL/6 mice (SiPeiFu, Beijing, China) were used in the experiments. The animals were housed in cages and on a 12-h light/12-h dark cycle at 50% humidity and 25°C ± 2°C. The animals were given free access to a standard pellet diet and water. Plasma was obtained by centrifugation at 200 g for 10 min at 4°C and kept at −80°C prior to extraction.

The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1985).

Animal experimental protocols

The mice were randomly assigned to six treatment groups, including a control group, model group (Dox group), three dose Dox + Brev groups, and Dox + Dexra group.

To mimic human therapeutic regimens, a cumulative dose of 12 mg/kg of Dox was administered via three weekly i.p. injections (4 mg/kg on days 0, 7, and 14) except for the control group.

To investigate the effect of Brev in vivo, mice were treated with Brev, followed by daily i.p. injection of 4.8, and 16 mg/kg of Brev for 3 weeks, and mice in the Dox + Dexra treatment group received 12 mg/kg Dexra by intraperitoneal injection for 3 weeks in addition to Dox treatment. (Alexandre et al., 2020). Brev and Dexra were treated after Dox injections. Survival was monitored daily and heart function was assessed by echocardiography, 3 weeks after the first injection of Dox (Li et al., 2018).

The control group was injected with the same volume of normal saline. The mice were sacrificed 1 week after Dox administration.

Tumor studies

Mice were subcutaneously injected with 1×10⁵ 4T1 breast tumor cells. One week after cell injection, when the mean diameter of the tumors was greater than 2 mm, the mice were treated with Dox or breviscapine as described previously. Tumor size was measured twice a week for up to 4 weeks, and tumor volumes were calculated with the following equation: V = 4π/3×(L/2)2× (W/2), where V, L, and W represent the volume, length, and width of the tumor, respectively.

¹⁸F-fluorodeoxyglucose PET/CT imaging

Dox and breviscapine group mice (fasted overnight) were anesthetized with 2% isoflurane and injected with ∼11MBq/0.2 ml of fluorodeoxyglucose (FDG) PET via a tail vein, then returned to their cages. Forty minutes later, mice were anesthetized again with 2% isoflurane and placed on a stereotactic bed in a microPET Focus 220 (Siemens Company, Berlin, Germany). A 20 min static PET scan was then initiated. Afterwards, mice were imaged in a microCAT II (Siemens Company, Berlin, Germany) at an X-ray beam intensity of 180mAs and an X-ray tube voltage of 80 kVp for anatomical co-registration with the PET images.

Patients

Following more than 4 h of fasting and ensuring that the blood glucose level was less than 120 mg/dl, all patients received an intravenous administration of ¹⁸F-FDG (5.5 MBq/kg). PET/CT scans were started 60 min after injection using a combined PET/CT biograph (Siemens Company, Berlin, Germany). All scans were performed in a three-dimensional model. A low-dose CT scan was obtained first for attenuation correction and anatomical correlation. The ethical clearance conforms to the Guide for the Approval Document of Ethics Committee published by the Affiliated Hospital of Qingdao University (QDU-HEC-2022057).

Analysis of cardiac function and histopathology

Mouse heart tissue from each group was stored in 4% paraformaldehyde, embedded in paraffin wax, and cut serially into 4 mm sections. Tissue sections were deparaffinized via immersion in xylene (3 times, for 5 min each) and rehydrated using a descending series of alcohols (100%, 90%, 85%, and 75% alcohol, 5 min each). The sections were stained with hematoxylin and eosin for histological analysis. Biopsy samples were stained using Masson’s trichrome stain to investigate any morphological and fibrotic changes in the heart. Blue staining represented collagen accumulation. Immunohistochemistry was performed using the Histone Simple stain kits (Nichirei, Tokyo, Japan) according to the manufacturer’s instructions. The sections were treated for 15 min with 3% H2O2 in methanol to inactivate endogenous peroxidases and were then incubated at room temperature for 1 h with the primary antibodies IL-1, 1:100. The ultrastructure was then observed at 20× magnification using a Leica Microsystems’ CMS GmbH inverted fluorescence microscope (Leica Camera AG, Barnack, Germany).

Measurement of glucose levels

Mice were maintained on a normal chow diet. On the day of experimentation, the mice were fasted for 6 h (beginning at 8 a.m.), and blood was collected via the tail vein for the measurement of glucose levels.

H9c2 rat cardiomyoblast cells were washed with phosphate-buffered saline (PBS) and centrifuged and ultrasonically crushed, and incubated under standard cell culture conditions for 10 min. After incubation, the absorbance was measured using a microplate reader (Perkin Elmer, Massachusetts, United States).

Enzyme-linked immunosorbent assay

Mouse tissues were lysed with the lysis buffer. The samples were sonicated with a Qsonica homogenizer using 30 Hz pulses for 20s and then centrifuged at 12,000 g for 10 min. The supernatant was collected, aliquoted into 200 µl vials, and stored at −80°C. The protein concentrations of the samples were quantified by the BCA assay. The samples were analyzed using enzyme-linked immunosorbent assay kits for TNF-α, IL-1β, and IL-6 according to the manufacturer’s protocol. The optical density was measured at 450 nm by using a VICTOR Nivo™ microplate reader (Perkin Elmer, Massachusetts, United States).

Determination of changes in the mouse metabolome

Blood samples were resuspended, incubated on ice, centrifuged, diluted to the final concentration, and centrifuged. Finally, the supernatant was injected into the LC–MS/MS system for analysis. UHPLC–MS/MS analyses were performed using a Vanquish UHPLC system (Thermo Fisher, Massachusetts, United States) coupled with an Orbitrap Q Exactive^TM HF mass spectrometer (Thermo Fisher, Massachusetts, United States). The identified metabolites were annotated using the KEGG database, HMDB database, and LIPID Maps database. Principal component analysis (PCA) and partial least square discriminant analysis (PLS-DA) were performed using metaX. We applied univariate analysis (t-test) to calculate the statistical significance (p-value). Metabolites with a VIP>1, p value < 0.05, and fold change ≥2 or ≤0.5 were considered to be differentially abundant metabolites.

Anthracyclines increase myocardial oxygen consumption and decrease the ATP production rate

Interestingly, this metabolic compensation increased myocardial ROS levels, leading to higher oxidative stress (Figure 2A). O2K energy metabolism analysis showed that Dox increased myocardial oxygen consumption (Figure 2B) and led to hyperpolarization of the mitochondrial membrane (Figure 2C) (this is also a sign of elevated levels of mitochondrial oxidative stress and is often seen in models of cardiac ischemia–reperfusion). JC-1 staining also showed that Dox exposure caused an increase in the membrane potential (enhanced red fluorescence intensity) in a few surviving cells (Figure 2D). Although compensatory processes such as increased oxygen consumption and lipid oxidation occurred in the heart, the ATP production rate in the myocardium was unfortunately drastically reduced in response to the reduction in glucose metabolism (Figure 2E). Comparison of the oxygen consumption rate and ATP production rate showed that Dox exposure led to the consumption of a large amount of oxygen by the myocardium but the production of only a small amount of ATP (Figure 2F), greatly increasing the burden on the heart. This deficit in energy output caused a significant increase in the level of the energy receptor AMPK in the Dox group (Figure 2G). Therefore, we hypothesize that the heart exhibits a high oxygen consumption rate and a low ATP production rate in response to impairment of glucose metabolism caused by anthracyclines and that this phenomenon does not fully meet the energy requirements of the myocardium but leads to excessive oxidative stress. It is therefore speculated that in response to the energy gap, lipid oxidation in the heart is increased and more ROS accumulates, and this poor feedback pathway may be an important driver of DIC.

The new cardioprotective agent breviscapine promotes cardiac glucose metabolism and inhibits Dox-induced cardiomyopathy

Inspired by the aforementioned results, we investigated the protective effect of the flavonoid breviscapine, which is used to treat cardiovascular diseases, against the progression of DIC from a metabolic perspective. The experimental protocol is shown in Figure 3A, and ¹⁸F-FDG PET/CT imaging was performed using mice 1 week after the first Doxorubicin injection. As shown in Figure 3B, cardiac uptake was significantly higher in the breviscapine-treated group than in the Dox-treated group alone, suggesting that breviscapine intervention could reverse the Dox-induced decrease in cardiac glucose metabolism and remodel cardiac energy metabolism. Cardiac function and pathological changes after treatment with breviscapine were next examined, and the clinical drug dexrazoxane (Dexra) was used as a positive control. The echocardiography in Figure 3C showed that LVEF and LVFS in the Dox group were significantly lower than those in the control group, and LVESV and LVD were significantly higher than those in the control group. These results suggest that the left ventricular systolic function of the mice decreased after Dox treatment, and the Dox induced myocardial injury in this study was similar to dilated cardiomyopathy. Dox reduced systolic function, which is similar to what has been reported previously (Venturini et al., 1996; Sun et al., 2022). All functions were restored in the hearts of animals treated with Brev and Dox compared with those of animals treated with doxorubicin only. The cTnI is a biomarker of myocardial injury (Solaro and Solaro, 2020). The concentration of cTnI was significantly increased in mice treated with doxorubicin alone compared with those in the control group, and different concentrations of Brev showed better effects (Figure 3D). Moreover, the staining of pathological cardiac tissues after different treatments showed that the cardiac tissue structure in mice in the control group was normal. Unlike those in the control group, mice in the Dox group exhibited a large number of cardiac lesions, including necrosis, intracellular edema, myofibrillary disorder and rupture, and wavy degeneration of cardiac fibers. Interestingly, preconditioning with breviscapine prevented this type of injury, alleviated excessive accumulation of cardiac lipids, and restored myocardial homeostasis (Figure 3F). Masson staining of the myocardium showed that interstitial fibrosis was significantly increased after Dox treatment and that Brev intervention reduced the degree of cardiac fibrosis (Figure 3E). Cardiac ultrasound data showed that the LVESV and LVIDs were significantly higher in the Dox group than in the control group, suggesting ventricular cavity diameter enlargement and that breviscapine treatment could significantly alleviate these changes. These results suggest that breviscapine can reshape cardiac metabolism, increase cardiac glucose uptake, reduce cardiac steatosis, and alleviate doxorubicin-induced cardiac dysfunction and changes in myocardial morphology.

Breviscapine remodels cardiac glucose and lipid metabolism by regulating peripheral serum levels and myocardial AKT expression

Next, we studied how breviscapine regulates the balance of sugar and fat metabolism. As shown in Figure 4E, the level of serotonin in the peripheral serum in breviscapine-treated mice was dramatically increased compared with that in Dox-treated mice, and the degree of the decrease showed a linear relationship with the dose of breviscapine, with a decrease of more than 95% being observed in the high-dose group. Serotonin (5-HT) is a monoamine that has a variety of functions in neuronal and nonneuronal systems (Okaty et al., 2019). In the central nervous system, 5-HT acts as a neurotransmitter that regulates mood and feeding behavior (Berger et al., 2009). Recent studies have shown that peripheral 5-HT plays an important role in the metabolic regulation of peripheral tissues by inhibiting adaptive thermogenesis in brown adipose tissue. Inhibition of 5-HT synthesis reduces weight gain and improves metabolic dysfunction in a diet-induced obesity mouse model (Zemdegs et al., 2016; Xia et al., 2021). Genome-wide association studies have also revealed a genetic link between the serotonergic system and obesity. Serotonin has two different effects on glucose metabolism: it directly induces insulin secretion in pancreatic B cells, lowering blood glucose levels; and it inhibits the uptake of glucose from the blood by tissues other than the liver and skeletal muscle, raising blood sugar levels (Watanabe et al., 2011; Carmean et al., 2019; Yabut et al., 2019). Studies have shown that serotonin treatment can lead to a sudden and large increase in blood glucose levels, but how serotonin inhibits tissue uptake of glucose from the blood remains unclear. Our experimental results showed that after breviscapine treatment, peripheral serotonin levels decreased sharply, corresponding to an increase in cardiac glucose intake (Figure 4E), while liver glucose intake did not change significantly (Supplementary Figure S2) and fasting glucose (FBG) levels were decreased (Figure 4A). In the breviscapine group, additional supplementation with 5-HT increased the level of serotonin in mice and decreased cardiac glucose uptake (Figure 4B). To further analyze how serotonin regulates cardiomyocyte metabolism, we added additional 5-HT to increase serotonin levels in Dox-induced myocardial injury model mice and then collected the hearts of the mice for transcriptome analysis. The results showed that oxidation of fatty acids in the group treated with Dox and 5-HT was significantly higher than that in the Dox group alone (Supplementary Figure S3). We then analyzed the changes in the PI3K-protein kinase B (Akt/PKB) pathway, an important regulator of myocardial glucose metabolism that plays an important role in regulating glucose uptake; promoting cell division, proliferation, and survival; and inhibiting apoptosis. The results showed that the addition of 5-HT directly inhibited the gene expression of Akt but did not significantly change the PI3K catalytic subunit or regulatory subunit level (Figure 4C). Loss of AKT reduces the ability of cardiomyocytes to metabolize glucose. These results suggest that breviscapine relieves the inhibitory effect of serotonin on the tissue glucose uptake by significantly reducing the level of serotonin and ensures the absorption of glucose from the blood under pathological conditions, revealing the possibility that the body may participate in the regulation of cardiac metabolism remodeling through neurotransmitters. More importantly, breviscapine, as a commonly used cardiovascular drug in China, can reduce peripheral serotonin levels by more than 90% and has a more robust effect than the classic serotonin reuptake inhibitor fluoxetine in the treatment of myocardial hypertrophy and heart failure induced by myocardial glucose metabolism disorder and diabetes, representing a new safe agent for the treatment of Dox-induced cardiotoxicity. Epidemiological studies have also shown that diabetes increases the risk of Dox-induced cardiotoxicity; thus, this study provides a potential treatment strategy for patients with diabetes and cancer (Chang et al., 2021).

Based on these findings, we investigated how breviscapine regulates glucose. Utilization by the heart. The results showed that PI3K protein expression in Dox-exposed mice was significantly decreased and that the PI3K protein expression level in cardiac tissue was significantly increased after breviscapine intervention. Interestingly, AKT expression was maintained at low levels in both the control and Dox-exposed groups, and significantly increased AKT expression was observed after breviscapine treatment (Figure 4D). Previous studies have shown that AKT stimulates glucose metabolism in cells, converting glucose to D-glucose 6-phosphate via hexokinase for glycolysis or polymerization to glycogen. Our results showed that after breviscapine promoted the expression of myocardial AKT, glucose metabolism-related D-glucose 6-phosphate and α-ketoglutaric acid levels were significantly increased (Figure 4F,G), suggesting that breviscapine enhanced the ability of the myocardium to use glucose as an energy source through AKT and promoted glucose catabolism. Previous studies have reported that increased PI3K-Akt expression can promote sugar utilization in peripheral tissues, reduce insulin resistance, enhance cardiac energy supply, and prevent cardiac insufficiency, while the inhibition of the PI3K-Akt pathway affects the physiological activities of cardiac cells (Zhu et al., 2013). These results suggest that breviscapine enhances resistance to Dox-induced myocardial injury by modulating the activation of the PI3K-Akt pathway. These results suggest that breviscapine regulates blood glucose levels by inhibiting serotonin, blocks the restriction of glucose utilization in the myocardium, and promotes glucose metabolism in the myocardium by activating the PI3K-Akt pathway. Therefore, serotonin may be an important target for regulating cardiac glycolipid metabolism. We then analyzed changes in cardiac function and homeostasis following remodeling of myocardial glycolipid metabolism. Compared with that in the Dox model group, myocardial ATP production in the breviscapine-treated group was significantly increased (Figure 5A), while the levels of ROS and MDA, a metabolite of FAO, were significantly decreased (Figures 5B,E). O2K energy metabolism analysis showed that breviscapine inhibited the dramatic increase in cardiac oxygen consumption caused by Dox exposure (Figure 5C), significantly decreased the ATP production/oxygen consumption ratio (Figure 5D), and normalized the mitochondrial membrane potential (Supplementary Figure S4). This suggests that breviscapine treatment increases the myocardium’s ability to use high-energy efficient glucose as an energy source; improves the efficiency of ATP production; and reduces cardiac load, oxidative stress, and myocardial oxygen consumption. In conclusion, breviscapine can effectively break the vicious cycle of a high oxygen consumption rate and inadequate lipid combustion in the heart caused by Dox exposure by regulating the serotonin-glycemia-myocardial AKT loop, remodeling myocardial energy metabolism, increasing glucose utilization, and reducing FAO (Figure 5F), preventing the further development of DIC.

Breviscapine clears mitochondrial damage and restores myocardial homeostasis after Dox treatment through the PINK1/Parkin signaling pathway

Although hypotheses regarding the mechanisms of DIC have varied over the past decades, the key features of DIC are redox imbalance and impaired mitochondrial function. The results prevented previously improved the hypothesis of DIC development from the perspective of the cardiac energy ratio and energy output efficiency but raise a key question, namely, how breviscapine achieves efficient energy production and cardiac microenvironmental homeostasis by optimizing the metabolism network in the heart after mitochondrial injury. Thus, we aimed to answer this question. The Pten-induced putative kinase1 PINK1/Parkin signaling pathway is one of the major pathways mediated by mitochondrial autophagy and plays an important role in maintaining the energy metabolism of cardiomyocytes (Dewanjee et al., 2021). When mitochondria in myocardial cells are damaged, PINK1, as the “sentinel” of the mitochondrial quality control system, accumulates in the outer membrane of mitochondria and collects and phosphorylates Parkin (Poole and Macleod, 2021). Activated Parkin ubiquitinates damaged mitochondria, which then fuse with lysosomes to complete degradation. Enhanced mitochondrial autophagy mediated by the PINK1/Parkin signaling pathway can maintain mitochondrial homeostasis in cardiomyocytes and slow the progression of heart disease (Dorn, 2016).

Therefore, we speculated that breviscapine enhances mitochondrial autophagy through the PINK1/Parkin pathway and maintains mitochondrial homeostasis in cardiomyocytes. WB showed that compared with that in the Dox group, the total protein expression level of PINK1/Parkin in the breviscapine treatment group was significantly increased (Figure 6A), and the corresponding phosphorylation level was also significantly increased (Figure 6B). In the Dox treatment group, PINK1/Parkin protein expression showed a decreasing trend, and protein phosphorylation was significantly decreased, while the level of the mitochondrial membrane protein Tom20 was increased, suggesting that although Dox induced mitochondrial damage, it inhibited the timely clearance of damaged mitochondria. A large number of broken and damaged mitochondria were observed in the Dox group (red arrow, Figure 6D), while a large amount of lysosomal-wrapped mitochondrial autophagy was observed in the Brev group (blue arrow). When Dox and Brev were applied simultaneously, the mitochondrial ridge was obvious, mitochondrial morphology was normal, and the membrane structure was intact. Upon staining with MitoTracker, the highest fluorescence intensity was found in the Dox group (Figure 6C), indicating that there were significantly more mitochondria in the Dox group than in the control and Brev groups. Moreover, the levels of the antioxidative proteins NADH and SOD were significantly increased after Brev treatment (Supplementary Figure S5). Phosphorylation of the energy receptor AMPK was significantly reduced in the Brev treatment group compared with the Dox group, while cell growth control centers were activated (Figure 6E). These results indicate that Dox exposure causes myocardial damage, mitochondrial accumulation, and homeostasis imbalance, which to some extent explains why cardiac energy output efficiency is reduced and oxidative stress is increased after Dox administration. Breviscapine can promote mitochondrial autophagy, remove damaged mitochondria, and restore myocardial homeostasis, effectively ensuring the dynamic balance between cardiac ATP production and oxidative stress.

When this effect of breviscapine was blocked by Mdivi-1, the ROS level in cardiomyocytes was significantly increased (Figure 6F), while ATP production was significantly decreased (Figure 6G). Furthermore, the survival rate of cardiomyocytes was decreased (Supplementary Figure S6), and the reparative effect of breviscapine on cardiomyocytes was inhibited. When Dox and Mdivi-1 were combined, ATP production and the survival rate of myocardial cells were further reduced, the ROS level was increased, and myocardial injury was aggravated. These results suggest that mitochondrial autophagy plays a key role in Dox-induced cardiomyocytes injury. Metabolic network remodeling of the heart alone cannot completely offset the dysfunction caused by mitochondrial injury, and the myocardial protective effect of breviscapine depends on the regulation of glucose and lipid metabolism and the activation of mitochondrial autophagy. This may also be the reason why regulation of the cardiac metabolic network has not been considered in regards to the hypothesis of DIC development. It is suggested that preventing the progression of the disease and restoring cardiac function should be equally important in the treatment of the symptoms of cardiac dysfunction and heart failure caused by Dox, which is more beneficial for prognosis and the quality of life of patients.

The authors thank W-SS, Cancer Institute of the Affiliated Hospital of Qingdao University, China, for original draft preparation and data curation.