Crocetin protects cardiomyocytes against hypoxia/reoxygenation injury by attenuating Drp1-mediated mitochondrial fission via PGC-1α

Abstract

BACKGROUND

Saffron (Crocus sativus L.) has been traditionally used as food, spice, and medicine. Crocetin (CRT), as main bioactive component of saffron, has accumulated pieces of beneficial evidence on myocardial ischemia/reperfusion (I/R) injury. However, the mechanisms are poorly explored. This study aims to investigate the effects of CRT on H9c2 cells under hypoxia/reoxygenation (H/R) and elucidated the possible underlying mechanism.

METHODS

H/R attack was performed on H9c2 cells. Cell counting kit-8 was used to detect the cell viability. Cell samples and culture supernatants were evaluated via commercial kits to measure the superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and cellular adenosine triphosphate (ATP) content. Various fluorescent probes were used to detect cell apoptosis, intracellular and mitochondrial reactive oxygen species (ROS) content, mitochondrial morphology, mitochondrial membrane potential (MMP), and mitochondrial permeability transition pore (mPTP) opening. Proteins were evaluated via Western Blot.

RESULTS

H/R exposure severely reduced cell viability and increased LDH leakage. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) suppression and dynamin-related protein 1 (Drp1) activation were coincided with excessive mitochondrial fission, mitochondrial permeability transition pore (mPTP) opening and mitochondrial membrane potential (MMP) collapse in H9c2 cells treated with H/R. Mitochondria fragmentation under H/R injury induced ROS over-production, oxidative stress, and cell apoptosis. Notably, CRT treatment significantly prevented mitochondrial fission, mPTP opening, MMP loss, and cell apoptosis. Moreover, CRT sufficiently activated PGC-1α and inactivated Drp1. Interestingly, mitochondrial fission inhibition with mdivi-1 similarly suppressed mitochondrial dysfunction, oxidative stress and cell apoptosis. However, silencing PGC-1α with small interfering RNA (siRNA) abolished the beneficial effects of CRT on H9c2 cells under H/R injury, accompanied with increased Drp1 and p-Drp1^ser616 levels. Furthermore, over-expression of PGC-1α with adenovirus transfection replicated the beneficial effects of CRT on H9c2 cells.

CONCLUSIONS

Our study identified PGC-1α as a master regulator in H/R-injured H9c2 cells via Drp1-mediated mitochondrial fission. We also presented the evidence that PGC-1α might be a novel target against cardiomyocyte H/R injury. Our data revealed the role of CRT in regulating PGC-1α/Drp1/mitochondrial fission process in H9c2 cells under the burden of H/R attack, and we suggested that modulation of PGC-1α level may provide a therapeutic target for treating cardiac I/R injury.

Although the mortality of acute myocardial infarction (AMI) has decreased during the last decades because of effective therapeutic strategies,^[1] AMI is still a leading cause of death worldwide.^[2] In addition to the acute damage, sever complications, such as chronic heart failure and cardiac arrhythmia, may develop in survivors after AMI.^[3] Hence, timely reperfusion is the standard maneuver and best clinical therapy to attenuate the infarct size by actively restoring blood supply into the ischemic myocardium.^[4] The advances in procedural approaches have improved overall outcomes after AMI.^[5] However, evidences show reperfusion itself can induce additional damages known as ischemia/reperfusion (I/R) injury.^[6] Clinical evidence indicates that I/R leads multiple myocardial injury by initiating a cascade of postischemic injury events.^[7] Revascularization of occluded coronary artery also has the risk of increased 30-day mortality rates.^[8] Myocardial I/R injury has complex mechanisms, such as variable reperfusion times,^[9] circadian rhythm,^[4] extracellular matrix microenvironment,^[10] microvascular obstruction,^[11] inflammation,^[12] oxidative stress,^[13] mitochondrial dysfunction^[14] and mitochondrial apoptosis^[15] have been considered involved in deleterious process of I/R injury. The complex temporospatial interactions limit the understanding of dynamic pathological alterations, and the full spectrum of pathological cascades triggered by I/R remains unclear. Strategies to target I/R injury have been quite challenging, with no effective clinical treatment and little success in patients till now.^[16,17] These evidences suggested I/R injury compromises the benefits of reperfusion effectiveness, and therefore, therapeutic strategies to protect myocardium against I/R injury may crucial for patients with myocardial infarction.

Saffron has been used in food preparation and pharmacological formulas since ancient times. It is well known for its color, aroma, and flavor benefit from the accumulated apocarotenoids crocetin (and its glycosylated forms, crocins), safranal, and picrocrocin. As a unique water-soluble carotenoid, CRT contains carboxyl groups at both ends of its short carbon chain.^[18] Evidences have accumulated for a variety of effective pharmacological activities of CRT on anti-tumor,^[19] myopia control,^[20] anti-depression,^[21] anti-fatigue,^[22] anti-inflammatory,^[23] anti-oxidant,^[24] anti-atherosclerosis^[25], and anti-myocardial infarction.^[26] Additional previous study demonstrated that CRT elicits cerebral I/R injury protection through gut microbiota metabolism.^[27] The derivative of CRT, trans sodium crocetinate alleviates myocardial I/R injury via inhibiting SIRT3/FOXO3a/SOD2-mediated oxidative stress and apoptosis.^[28] The possibility that these beneficial effects also operate during cardiac I/R merits further investigation. A new study has been uncovered that CRT alleviates cardiac I/R injury by suppressing inflammation.^[29] It seems that CRT has versatile functions against I/R injury and might be a complementary medication to current reperfusion therapies. However, the role of CRT in cardiomyocyte H/R injury is far from clear.

Sufficient energy supply is required for maintaining heart function, and as the bioenergetics center, mitochondria comprise up to 40% of heart mass and supplies more than 90% of the ATP.^[30] Studies have found that destruction of mitochondria caused energy exhaustion and cellular death during I/R injury.^[8] Mitochondria are highly dynamic between fission and fusion, resulting in elongated, tubular or fragmented.^[31] Previous studies suggested that the dynamic balance of fission and fusion is necessary for mitochondrial function and cell signaling events.^[32] A perturbation of this process affects a variety of biological processes and believes to be responsible for mitochondrial respiration, cell apoptosis, necroptosis, and subsequent cell death.^[33,34] The impairment of mitochondrial morphology often couples with deteriorated mitochondrial function and cell viability in organs with high demand for energy, especially the heart. As such, holding the mitochondrial fission/fusion balance will contribute to protecting the heart in stressful conditions. What remains unclear is whether mitochondrial fission is the key regulator involved in cardiac I/R injury.

Studies showed that mitochondria fission plays a decisive role in the pathogenesis of I/R injury, including in the heart,^[8] kidney,^[35] liver,^[36] and brain.^[37] Excessive mitochondrial fission leads to mitochondrial dysfunction, energy shortage, oxidative stress, and subsequent cellular death in cardiomyocyte. Mitochondrial fission requires the activity of dynamin-related protein 1 (Drp1), a GTPase involved in mitochondrial fission.^[38,39] Phosphorylation of serine 616 in Drp1 triggers its translocation from cytosol to outer mitochondrial membrane, where it provokes scission and promotes fission of mitochondrial tubules into fragments.^[40] However, the mechanism by which I/R injury regulates cardiomyocyte mitochondrial fission has not been fully understood yet. Recent studies have established a concept that peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) serves a critical role in mitochondrial biogenesis^[41] and energy metabolism.^[42] However, evidence is emerging that mitochondrial biogenesis and metastasis may not be the only mitochondrial modulatory function of PGC-1α because Drp1 results in PGC-1α mediated cardiomyocyte mitochondrial fission, under some experimental conditions.^[43,44] Whether CRT is capable of alleviating cardiac I/R injury by regulating excessive mitochondrial fission remains unknown. Since it is possible to recapitulate and treat cardiac I/R injury in isolated cardiomyocytes, like H9c2 cells, as the most important aspect injury occurring within cardiomyocytes themselves.^[45] We aimed to test the protective role of CRT in hypoxia/reoxygenation (H/R) induced H9c2 cells. To our knowledge, there is no study on the effects and mechanisms of CRT on H/R induced H9c2 cells. In this study, we assessed whether CRT protects cardiomyocyte against H/R injury through inhibiting Drp1-mediated mitochondrial fission by activation of PGC-1α.

METHODS

Materials and Reagents

CRT was purchased from Xuyao (98% purity by HPLC, Hangzhou, China). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (NY, USA). Cell counting kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies (Kumamoto, Japan). Lactate dehydrogenase (LDH), superoxide dismutase (SOD) and malondialdehyde (MDA) assay kits were purchased from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). DAPI staining reagent, BCA protein assay kit, ATP assay kit, and β-actin antibody were obtained from Beyotime (Shanghai, China). Lipofectamine RNAiMAX reagent, Dihydroethidium (DHE), MitoTracker Green FM and JC-1 dye were purchased from Invitrogen (CA, USA). PGC-1α adenoviral particles were purchased from Keqing (Hangzhou, China). MitoSOX Red and Calcein-AM were obtained from Life Technologies (NY, USA). Terminal deoxy nucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was purchased from Roche Diagnostics (Indianapolis, IN, USA). Mdivi-1, PGC-1α antibody, and cytochrome c antibody were obtained from Abcam (MA, USA). Drp1 antibody, phospho-Drp1 (Ser616) antibody, and cleaved caspase-9 antibody were obtained from Cell Signaling Technology (MA, USA).

Cell Culture and Treatment

In brief, embryonic rat cardiomyocyte-derived cell line H9c2 cells were obtained from ATCC (Manassas, VA, USA) and cultured in DMEM containing 10% FBS at 37 °C in a humidified incubator with 5% CO₂. To imitate the cardiac I/R injury, H9c2 cells were subjected to 16 h of hypoxia (94% N₂/5% CO₂/1% O₂) with serum and glucose free DMEM, followed by 2 h of reoxygenation (74% N₂/5% CO₂/21% O₂) with normal media. Drug treatment (corcetin 50 μM) was carried out 12 h prior to and during H/R treatment. For Mdivi-1 pretreatment, H9c2 cells were cultured in the presence of 20 μmol/L Mdivi-1 for 1 h, prior to the H/R treatment. The control cells were left in normoxic gas condition with normal media at 37 °C.

siRNA Transfection

H9c2 cells were transfected with PGC-1α siRNA using Lipofectamine RNAiMAX reagent for 48 h according to the manufacturer’s instructions. The following siRNA sequences were used: sense: 5’-CCAACCAAGAUAACCCUUUTT-3’; antisense: 5’-AAAGGGUUAUCUUGGUUGGTT-3’.^[44] PGC-1α silencing was quantified by immunoblotting. After siRNA transfection, H9c2 cells were subjected to H/R and downstream assays.

Adenoviral Transfection

H9c2 cells were infected with adenoviruses (with a titer of 10^10-11 PFU/mL) including adenoviral PGC-1α (Ad-PGC-1α) or empty (Ad-NC) at a multiplicity of infection (MOI) of 50 for 48 h before treatment.

Cell Viability Assay

Cell viability was assessed by CCK-8 according to the protocol. Briefly, H9c2 cells (5 × 10³/well) were seeded into 96-well plates. After different treatments, 10% CCK-8 was added to each well followed by incubation for 2 h. The absorbance was measured at 450 nm using a microplate reader. Results were normalized to the control group.

Measurement of LDH, SOD and MDA Levels

H9c2 cells were seeded into 6-well plates at a density of 5 × 10⁴/well. The LDH leakage was measured in the collected cell culture medium according to the instruction. Then the cells were washed with cold PBS, harvested and ultrasonicated on an ice-water bath. After centrifuging at 1000 r/min at 4 °C for 5 min, the protein content in the supernatant was measured with the BCA protein assay kit. SOD activity and MDA level in the supernatant were detected and standardized to the total proteins according to the respective instructions.

Determination of ROS Production

ROS accumulation was detected by DHE staining according to the manufacturer’s instructions. Briefly, H9c2 cells were incubated in serum-free DHE (10 µM) solution for 30 min in dark at 37 °C. After washing out with PBS, the red fluorescence was observed by a fluorescent microscope (Olympus, Tokyo, Japan). Image J software (National Institutes of Health, Bethesda, MD, USA) was used to analyze the fluorescence intensity. To evaluate mitochondria-derived ROS production, we used MitoSOX probe by flow cytometry. Briefly, H9c2 cells after various treatment were trypsinized and loaded with MitoSOX at 37 °C for 30 min without light. After staining, the cells were washed with HBSS buffer containing Ca²⁺/Mg²⁺ and resuspended for flow cytometry analysis using wavelengths of λ_ex 510 and λ_em 585 nm.

TUNEL Staining

H9c2 cells were seeded into 6-well plates. After different treatment, the apoptosis level was assessed using the TUNEL assay according to manufacturer’s instructions. Nuclei were stained with DAPI for 10 min. TUNEL⁺ cells were imaged under a fluorescence microscope. Six wells per group were prepared, and six different regions were observed in each well to calculate TUNEL⁺ cells.

Assessment of Mitochondrial Morphology

To evaluate mitochondrial morphology in H9c2 cells, the treated cells were incubated at 37 °C for 30 min with 100 nmol/L MitoTracker. After washing twice with cold PBS, the images of live cells were acquired by a confocal laser scanning microscope (LSM 880, Carl Zeiss Meditec, Jena, Germany). Mitochondrial length and the percentage of cells with fragmented mitochondria were analyzed and quantified.

Determination of Mitochondrial Permeability Transition Pore Opening

The calcein-AM-Co²⁺ system is a direct method for measuring mitochondrial permeability transition pore (mPTP) opening in live cells. When calcein-AM passively diffuses into mitochondria, the dye will be trapped by cleaving the acetoxymethyl ester (AM). At this point, the Co²⁺ has the ability to quench calcein fluorescence outside the mitochondria, resulting in only loading mitochondria with fluorescence. Thus, in the case of mitochondria damage, mPTP opening makes the mitochondrial membrane free for calcein efflux and Co²⁺ influx, leading to reduction of calcein fluorescence intensity in mitochondria. H9c2 cells were loaded with 1 µmol/L calcein-AM and 2 mmol/L cobalt chloride for 20 min in the dark at 37 °C. Excess calcein-AM and cobalt chloride were removed by washing twice with PBS, and the images were captured by a fluorescence microscopy and quantified by Image J.

Assessment of Mitochondrial Membrane Potential

In addition to the mPTP opening assay, the mitochondrial membrane potential (MMP) changes were evaluated in live cells by the MMP sensitive dye JC-1. JC-1 is a dual-emission membrane potential-sensitive dichromatic dye that exhibits the potential accumulation in mitochondria. The red fluorescent aggregates or green monomer fluorescent of JC-1 exhibits the polarizing or depolarizing state of MMP. The red/green fluorescence ratio depends on the mitochondrial polarization stats. H9c2 cells were incubated with 2.5 µg/mL JC-1 for 20 min at 37 °C. After treatment, H9c2 cells were observed by a fluorescence microscopy. For quantitative analysis, six random fields were imaged in each group. The ratio of red to green fluorescence intensity was calculated using Image J.

Detection of Cellular ATP Levels

The intracellular ATP content was determined through a commercial luciferin-luciferase bioluminescence assay kit. The emitted light from luciferase catalyzed luciferin is powered by ATP, and the light intensity is proportional to the amount of ATP. After indicated treatments, H9c2 cells were dissociated and centrifuged at 12000 × g for 5 min at 4 °C. Then the supernatant was measured following manufacturer’s recommendations. Luminescence intensity was normalized to total amount of protein.

Western Blot Analysis

H9c2 cells were lysed in RIPA buffer containing protease and phosphatase inhibitors cocktail. After quantified by BCA protein assay kit, equal amount of proteins were boiled and separated by polyacrylamide gel electrophoresis, followed by transfer to nitrocellulose membrane. After blocked with 0.1% Tween 20 and 1% powdered milk, the membrane was analyzed using the corresponding antibodies. Anti-β-actin antibody was used as a loading control.

Data Analysis

Data were analyzed using one-way ANOVA and expressed as mean ± SD. P < 0.05 was considered statistically significant.

RESULTS

CRT Attenuated H/R-Induced Cell Death and Apoptosis in H9c2 Cells

The chemical structure of crocetin is shown in Figure 1A. In order to investigate the role of CRT on H/R injury, we examined the levels of cell viability, LDH leakage and cell apoptosis in H9c2 cells, respectively. As shown in Figure 1B and 1C, H/R stimulus significantly reduced cell viability and elevated LDH release in H9c2 cells. However, CRT treatment dramatically abrogated these effects. Similarly, cell apoptosis was exacerbated in H/R-induced H9c2 cells, but significantly alleviated after CRT treatment, as seen in TUNEL⁺ cells (Figure 1D & 1E).

Figure 1.

CRT attenuated H/R-induced cell death and apoptosis in H9c2 cells.

H9c2 cells were under H/R attack in the presence of CRT (20 μM). (A): Chemical structure of CRT (CAS: 27876-94-4, C₂₀H₂₄O₄, MW: 328.4); (B): CCK-8 assay for cell viability in H9c2 cells (n = 6); (C): LDH assay for cell injury in H9c2 cells (n = 6); (D): representative images of TUNEL (red) and DAPI (blue) staining for cell apoptosis in H9c2 cells (scale bar = 100 μm); and (E): quantitation of TUNEL-positive cells (n = 6). ^###P < 0.001 vs. control group; ^***P < 0.001 vs. H/R group. CRT: crocetin; H/R: hypoxia/reoxygenation; LDH: lactate dehydrogenase; TUNEL: terminal deoxy nucleotidyl transferase-mediated dUTP nick end labeling.

CRT Alleviated H/R-Induced Oxidative Stress in H9c2 Cells

SOD and MDA are oxidative stress markers, and MDA accumulation, as well as the reduction of SOD activity is regarded as indication of oxidative damage in cells. As shown in Figure 2A and 2B, the SOD activity in H/R-induced H9c2 cells was dramatically decreased, and MDA level was markedly increased compared with the control group. However, the activity of SOD was significant elevated, and the concentration of MDA was markedly reduced in the CRT-treated group, indicating alleviated oxidative stress.

Figure 2.

CRT reduced H/R-induced oxidative stress in H9c2 cells.

(A): Quantitative analysis was performed to determine the SOD activity in H9c2 cells (n = 6); (B): MDA level in H9c2 cells (n = 6); (C): Representative images of DHE staining for intracellular ROS level (scale bar = 100 μm); (D): The relative fluorescence intensity of DHE (n = 6); and (E): Mitochondrial ROS level was detected using flow cytometry (n = 6); ^###P < 0.001 vs. control group; ^**P < 0.01 vs. H/R group, ^***P < 0.001 vs. H/R group. CRT: crocetin; DHE: dihydroethidium; H/R: hypoxia/reoxygenation; MDA: malondialdehyde; ROS: reactive oxygen species; SOD: superoxide dismutase.

To elucidate the more direct evidence underlying the oxidative stress, we investigated the effect of CRT on the ROS content in cellular level (detected by DHE) and mitochondria (detected by MitoSOX), respectively. Cells with high ROS accumulation show strong fluorescence, while less ROS production is the reason for lower DHE positive reaction. The intracellular ROS (Figure 2C & 2D) and mitochondrial ROS (Figure 2E) levels in CRT group were markedly lower than the cells under H/R conditions. Together, these results indicated that CRT mitigated oxidative stress in H9c2 cells exposed to H/R.

CRT Prevented H/R-Induced Mitochondrial Fission and Mitochondrial Dysfunction in H9c2 Cells

We next explored the inhibitory effect of CRT on H/R-induced mitochondrial fission in H9c2 cells. Previous studies implied that CRT reduced over-production of mitochondrial ROS, an index of mitochondrial function. Therefore, we speculated that CRT could prevent H/R-induced excessive mitochondrial fission and restore mitochondrial function. We explored the mitochondrial fission in H9c2 cells by visualizing mitochondrial morphology with MitoTracker. After H/R stimulation, mitochondrial became spherical and shorter, indicating mitochondrial fragmentation (Figure 3A & 3B). The percentage of cells with fragmented mitochondria also increased (Figure 3C). As expected, we found that CRT treatment efficiently suppressed mitochondrial fission, as evidenced by more filamentous mitochondria and less mitochondria fragmented cells.

Figure 3.

CRT inhibited mitochondrial fission and mitochondrial dysfunction in H/R-treated H9c2 cells.

(A): Representative confocal microscope images of MitoTracker staining for mitochondrial morphology (scale bar = 5 μm); (B): mean length of mitochondria (n = 20); (C): the percentage of cells with fragmented mitochondria (for each group, n = 50 cells were analyzed); (D): representative images of H9c2 cells stained using calcein-AM (scale bar = 50 μm); (E): analysis of the fluorescence intensity of calcein (n = 6); (F): representative images of JC-1 staining (scale bar =10 μm); (G): quantification of the red/green fluorescence ratio (n = 6); (H): The cellular ATP concentration was measured (n = 6); and (I-G): Western blot analysis demonstrated expression of PGC-1α and Drp1 (n = 5). The loading control was β-actin. ^##P < 0.01 vs. control group, ^###P < 0.001 vs. control group; ^*P < 0.05 vs. H/R group, ^**P < 0.01 vs. H/R group, ^***P < 0.001 vs. H/R group. ATP: adenosine triphosphate; Calcein-AM: calcein acetoxymethyl ester; CRT: crocetin; Drp1: dynamin-related protein 1; H/R: hypoxia/reoxygenation; PGC-1α: peroxisome proliferator-activated receptor γ coactivator-1α.

Then, we loaded H9c2 cells with calcein-AM to monitor mPTP activity. When cells under H/R injury, calcein fluorescence was significantly decreased compared to control group (Figure 3D & 3E). CRT treatment efficiently reduced the extent of mPTP opening with high intensity of calcein fluorescence trapped in mitochondria. As mPTP opening is closely linked to the loss of proton gradient across mitochondrial membranes, we assessed the MMP with the cationic dye JC-1. Healthy mitochondria exhibit high MMP with red fluorescence and damaged mitochondria exhibit low MMP with green fluorescence. Results from the JC-1 analysis showed a drastic depolarization of MMP, indicating mitochondria damage and cell apoptosis of H9c2 cells after H/R stimuli (Figure 3F & 3G). Compared with the H/R group, the higher red/green fluorescence intensity of JC-1 indicated significantly improved mitochondria damage and cell apoptosis of the CRT-treated H9c2 cells. The mPTP and MMP analysis further verified the mitochondrial morphological observations. Since MMP is critical for preserving of mitochondrial function integrity to generate ATP, we then examined cellular ATP content in each group. We found that H/R severely diminished cellular ATP content compared with the control group, while CRT significantly elevated the cellular ATP levels (Figure 3H).

Western blot analysis showed that PGC-1α protein expression was decreased, while Drp1 was dramatically induced in H/R-treated H9c2 cells (Figure 3I-3K). We found that CRT treatment restored the expression of PGC-1α, which further suppressed the activation of Drp1. Together, these results suggested that CRT played a key role in protection of H9c2 cells against H/R injury via enhancing mitochondrial quality, and this mechanism might be triggered through PGC-1α/Drp1 axis regulation.

Inhibition of Mitochondrial Fission Alleviated H/R-Induced Mitochondrial Dysfunction and Cell Apoptosis in H9c2 Cells

Our results confirmed that H/R injury significantly enhanced mitochondrial fission, mitochondrial dysfunction, and cell apoptosis in H/R-stimulated H9c2 cells. However, whether mitochondrial fission suppression contributed to alleviation of mitochondrial dysfunction and cell apoptosis remains unclear. The mitochondrial division inhibitor, mdivi-1, was used to investigated whether the inhibition of mitochondrial fission mitigated mitochondrial dysfunction and cell apoptosis in H/R-treated H9c2 cells. Our results showed a reduction of punctiform mitochondria and percentage of mitochondria fragmented cells in mdivi-1-trated H9c2 cells (Figure 4A-4C), indicating that mdivi-1 converted mitochondrial fission induced by H/R injury. In sharp contrast, mdivi-1 treatment reversed the shift from red to green JC-1 fluorescence compared to the H/R treatment, indicating the markedly suppression of mitochondrial depolarization (Figure 4D & 4E). And the restored mitochondrial function exhibited a remarkable increase of ATP production as well (Figure 4F). The analysis using DHE stains suggested that mdivi-1 attenuated H/R-induced oxidative stress in H9c2 cells (Figure 4G & 4H). Compared with the H/R group, the percentage of TUNEL⁺ cells (Figure 4I & 4J) was significantly decreased after mitochondrial fission inhibition. These results elucidated the pivotal role of mitochondrial fission in H/R injury.

Figure 4.

Mdivi-1 exhibited similar mitochondrial protective effects as CRT.

(A): Representative images of MitoTracker staining for mitochondrial morphology (scale bar = 5 μm); (B): mean length of mitochondria (n = 20); (C): the percentage of cells with fragmented mitochondria (for each group, n = 50 cells were analyzed); (D): representative images of JC-1 staining (scale bar = 10 μm); (E): quantitative analysis of red/green fluorescence ratio of JC-1 staining (n = 6); (F): quantification of the cellular ATP concentration (n = 6); (G): representative images of DHE staining for intracellular ROS level (scale bar = 100 μm); (H): the relative fluorescence intensity of DHE (n = 6); (I): representative images of TUNEL (red) and DAPI (blue) staining for cell apoptosis (scale bar = 100 μm); and (J): quantitation of TUNEL⁺ cells (n = 6). ^###P < 0.001 vs. control group; ^*P < 0.05 vs. H/R group, ^**P < 0.01 vs. H/R group, ^***P < 0.001 vs. H/R group. ATP: adenosine triphosphate; CRT: crocetin; DHE: dihydroethidium; DAPI: 4',6-diamidino-2-phenylindole; ROS: reactive oxygen species; TUNEL: terminal deoxy nucleotidyl transferase-mediated dUTP nick end labeling.

Loss of PGC-1α Blunted the Beneficial Effects of CRT in H/R-Treated H9c2 Cells

To investigate whether PGC-1α deficiency could abolish the protective effects of CRT on mitochondrial fission and cell apoptosis in H/R-treated H9c2 cells, we blockaded the expression of PGC-1α via siPGC-1α. The expression of PGC-1α was confirmed by western blot (Figure 5A). It was found that siPGC-1α abolished CRT-induced inactivation of Drp1 in H9c2 cells exposed to H/R (Figure 5B & 5C). We next used MitoTracker green to determined mitochondrial morphology. As illustrated in Figure 5D−5F, siPGC-1α markedly reversed the effects of CRT on H9c2 cells under H/R injury, as evidenced by decreased mitochondrial length and increased punctiform mitochondria and mitochondria fragmented cells. Furthermore, siPGC-1α evoked a sharp decrease in calcein fluorescence in CRT-treated cells (Figure 5G & 5H), demonstrating that PGC-1α silencing re-opened mPTP. The trend of the green emission intensity of JC-1 matched the mPTP opening assay. As shown in Figure 5I & 5J, JC-1 monomers emitted a strong green fluorescent signal in PGC-1α silencing H9c2 cells exposed to H/R with CRT treatment, which indicated the dissipation of MMP. To further validate the mitochondrial function, we analyzed the cellular ATP content. SiPGC-1α induced a decrease in ATP level after CRT treatment (Figure 5K).

Figure 5.

PGC-1α silencing blunted the beneficial effects of CRT in H/R-treated H9c2 cells.

(A): Silencing of PGC-1α in H9c2 cells; (B-C): Western blot was used to assess p-Drp1/Drp1 ratio, β-actin was the loading control; (D): representative images of MitoTracker staining for mitochondrial morphology (scale bar = 5 μm); (E): mean length of mitochondria (n = 20); (F): the percentage of cells with fragmented mitochondria (for each group, n = 50 cells were analyzed); (G): representative images of H9c2 cells stained using calcein-AM (scale bar = 50 μm); (H): analysis of the fluorescence intensity of calcein (n = 6); (I): representative images of JC-1 staining (scale bar = 10 μm); (J): quantification of the red/green fluorescence ratio (n = 6); (K): quantification of the cellular ATP concentration (n = 6); (L): representative images of TUNEL (red) and DAPI (blue) staining for cell apoptosis (scale bar = 100 μm); (M): quantitation of TUNEL⁺ cells (n = 6); and (N-P): Western blot analysis demonstrated expression of cytosol cytochrome c (tubulin was the loading control), mitochondrial cytochrome C (VDAC1 was the loading control), and cleaved caspase-9 (β-actin was the loading control) (n = 5). ^##P < 0.01 vs. control group, ^###P < 0.001 vs. control group; ^*P < 0.05 vs. H/R group, ^**P < 0.01 vs. H/R group, ^***P < 0.001 vs. H/R group; ^§P < 0.05 vs. H/R+CRT+si-PGC-1α group, ^§§P < 0.01 vs. H/R+CRT+si-PGC-1α group, ^§§§P < 0.001 vs. H/R+CRT+si-PGC-1α group. ATP: adenosine triphosphate; Calcein-AM: calcein acetoxymethyl ester; CRT: crocetin; Drp1: dynamin-related protein 1; DAPI: 4′,6-diamidino-2-phenylindole; H/R: hypoxia/reoxygenation; p-Drp1: phospho-dynamin-related protein 1; PGC-1α: peroxisome proliferator-activated receptor γ coactivator-1α; TUNEL: terminal deoxy nucleotidyl transferase-mediated dUTP nick end labeling; VDAC1: voltage dependent anion channel protein 1.

These data suggested that PGC-1α silencing reversed the mitochondria integrity protection of CRT and resulted in the energetic drop. In line with the mitochondria fission, we found that PGC-1α knockdown negated CRT mediated anti-apoptotic effect on H/R-treated H9c2 cells, as evidenced by TUNEL staining (Figure 5L & 5M) and apoptotic proteins (cytochrome c and cleaved caspase-9) expression assay (Figure 5N-5P). These findings indicated that PGC-1α indeed played a crucial role in the protective effects of CRT against H/R injury in H9c2 cells.

Overexpression of PGC-1Α Inhibited Drp1 Activation and Mitochondrial Fission

To gain the evidence that PGC-1α up-regulation recovered the inactivation of Drp1, inhibition of excessive mitochondrial fission and cell apoptosis, we over-expressed PGC-1α in H9c2 cells. The expression profile of PGC-1α protein was evidenced by Western-blot (Figure 6A). PGC-1α over-expression abolished the activation of Drp1 induced by H/R injury (Figure 6B & 6C). Consistent with the CRT treatment, we found that PGC-1α over-expression remarkably inhibited mitochondrial fission in H/R-induced H9c2 cells (Figure 6D-6F). Moreover, PGC-1α over-expression reversed the H/R-induced loss of MMP (Figure 6G & 6H), and showed the expected marked reduction in cell apoptosis rate (Figure 6I & 6J). Collectively, our results presented the key role of mitochondrial fission in H/R injury transmitted via PGC-1α/Drp1 signaling, which fatally triggered apoptosis cascades to direct cellular death.

Figure 6.

PGC-1α overexpression exhibited similar mitochondrial protective effects as CRT.

(A): Overexpression of PGC-1α in H9c2 cells; (B & C): Western blot was used to assess p-Drp1/Drp1 ratio. β-actin was the loading control; (D): representative images of MitoTracker staining for mitochondrial morphology (scale bar =5 μm); (E): mean length of mitochondria (n = 20); (F): percentage of cells with fragmented mitochondria (for each group, n = 50 cells were analyzed); (G): representative images of JC-1 staining (scale bar = 10 μm); (H): quantification of the red/green fluorescence ratio (n = 6); (I): representative images of TUNEL (red) and DAPI (blue) staining for cell apoptosis (scale bar = 100 μm); and (J): quantitation of TUNEL-positive cells (n = 6). ^##P < 0.01 vs. control group, ^###P < 0.001 vs. control group; ^*P < 0.05 vs. H/R+Ad-NC group, ^**P < 0.01 vs. H/R+Ad-NC group, ^***P < 0.001 vs. H/R+Ad-NC group. CRT: crocetin; DAPI: 4′,6-diamidino-2-phenylindole; Drp1: dynamin-related protein 1; p-Drp1: phospho-dynamin-related protein 1; PGC-1α: peroxisome proliferator-activated receptor γ coactivator-1α; TUNEL: terminal deoxy nucleotidyl transferase-mediated dUTP nick end labeling.

DISCUSSION

In the present study, we first provided direct evidence supporting the protective effects of CRT in H9c2 cells under H/R through preserving cell survival, reversing oxidative damage, and alleviating cell apoptosis. Mechanistically, we found that CRT up-regulated PGC-1α, and over-expression of PGC-1α inactivated Drp1 and subsequent mitochondrial fission. Silencing of PGC-1α abolished the beneficial effects of CRT on mitochondrial morphology and function, suggesting that PGC-1α played a crucial role in regulating mitochondrial fission under H/R exposure. In summary, our study provided the substantiation that PGC-1α served as a key role in CRT cardiomyocyte protective effects against H/R injury via inhibition of Drp1-dependent mitochondrial fission, and the ensuing mitochondrial dysfunction, oxidative stress, and cellular apoptosis (Figure 7).

Figure 7.

Possible mechanisms of cardiomyocyte-protective effects of CRT against H/R injury in H9c2 cells.

ATP: adenosine triphosphate; CRT: crocetin; Cyt: cytochrome c; Drp1: dynamin-related protein 1; H/R: hypoxia/reoxygenation; PGC-1α: peroxisome proliferator-activated receptor γ coactivator-1α; ROS: reactive oxygen species.

Myocardial I/R injury as a significant clinical problem worldwide, still has no particularly effective therapy.^[7] CRT is a bioactive compound typically found in Crocus sativus L. and Gardenia jasminoides Ellis with various health-promoting activates. Recent studies have demonstrated the cardioprotection property of CRT and its sodium derivative against myocardial I/R injury,^[28,29,46] however, the underlying mechanism remains not well understood. In this report, H9c2 cells under H/R stimuli were used to mimic myocardial I/R injury. We provided evidences that H/R-induced cell viability decrease and LDH leakage increase were markedly suppressed by CRT. The TUNEL assay further confirmed that CRT improved H/R-induced cell injury.

Cells exposed to H/R showed more sensitive to oxidative stress than the cells maintained in CRT treatment. Notably, DHE and MitoSOX stains directly showed CRT diminished ROS over-production and accumulation under H/R injury. Increased ROS generation is always related to mitochondrial morphology changes,^[34] because the fragmented mitochondria fail to work and promote excessive ROS production instead. Since heart is sensitive to mitochondrial dysfunction,^[47] the molecular features of mitochondrial homeostasis in myocardial I/R injury have been extensively explored. Mitochondrial structure has emerged as the focus of cell function and viability regarding to tumor,^[48] neurodegenerative disease,^[49] cerebrovascular disease,^[37] and cardiovascular disease.^[50] Ideally, an efficient therapeutic strategy should rescue injured mitochondria and improve global mitochondrial quality.^[51] However, there has little study investigating the regulatory mechanisms of CRT acting on mitochondria in cardiomyocyte H/R injury. Mitochondria with long thread-like tubular structure are regarded as filamentous, meanwhile, fragmented mitochondria are shortened and spherical.^[31] We found that CRT treatment elongated mitochondria, reduced mitochondrial fragmentation, and decreased the percent of cells with fragmented mitochondria. Perhaps even more profound was our finding that CRT inhibited excessive mitochondrial fission during H/R attack.

Two approaches were taken to evaluate different aspects of mitochondrial integrity, calcein-AM retention and JC-1 distribution. The calcein-AM/Co²⁺ assay can directly visualize mPTP changes. MPTP opening results MMP collapse, ATP level falls and cytochrome c release, thus playing a crucial role in mitochondria injury and cellular apoptosis.^[8] Therefore, inhibition of mPTP opening is an efficient therapy on cardioprotection. Our data showed that a significant increase of mPTP opening in H9c2 cells under H/R injury. Once mPTP opens, mitochondria depolarization was confirmed by JC-1 assay. Our results were also in line with the mechanisms as a marked decline of ATP level following mitochondrial fission. These data suggested that excessive mitochondrial fission consumed abundant mitochondria, leading to the decrease of energy supply, and eventually lessening cellular resistance to H/R injury. However, treatment with CRT significantly inhibited mPTP opening, alleviated MMP loss, and elevated ATP level, further suggesting the inhibition of mitochondrial fission. On the basis that mitochondrial fission appears to be fundamental to cardiac I/R injury, we hypothesized that inhibiting excessive mitochondrial fission may contribute to cardioprotection. We used mdivi-1 to inhibit mitochondrial fission and achieved the same effects as CRT, suggesting that the observed H9c2 cells protection was due to mitochondrial fission inhibition. Our findings substantiated that targeting mitochondrial fission inhibition with CRT was a potential effective strategy for myocardial protection.

Mitochondria fission process is mainly regulated by Drp1 activation and mitochondrion outer membrane recruitment.^[52] Previous studies show that PGC-1α plays a pivotal role in regulating Drp1 in heart.^[43,44] Consistent with these findings, our results validated the negative regulation between PGC-1α and Drp1 in H/R or CRT treatment. Here, we employed both loss-of-function and gain-of-function approaches to reveal the role of PGC-1α. Firstly, we employed PGC-1α silencing H9c2 cells to determine whether PGC-1α contributed to the beneficial effects of CRT against H/R injury. Western blot assay showed the failure to suppress Drp1 activation in the si-PGC-1α H9c2 cells was attributable to the loss of PGC-1α function. In our current observation, deleting PGC-1α led to increase in Drp1 level, mitochondrial fission, mitochondrial dysfunction, oxidative stress, and cell apoptosis during H/R, which indeed reversed the cardioprotective actions of CRT. Thus, we suggested that CRT suppressed excessive mitochondrial fission through boosting PGC-1α expression, and all the beneficial actions of CRT were absent in PGC-1α-silenced H9c2 cells. Our study bolstered the role of PGC-1α in mitochondrial morphology modulation, which offering a crucial target to regulate cardiomyocyte H/R injury. To gain further mechanistic insight into whether PGC-1α elicited cardiomyocyte protection effects against H/R injury, we over-expressed PGC-1α with adenoviral transfection. It was worth noting that PGC-1α over-expression could induce inactivation of Drp1 and subsequent mitochondrial fission, which was similar with CRT treatment. In conclusion, we identified PGC-1α as an important blockader of Drp1 and a promising molecule target in CRT-mediated mitochondria protection in H/R-induce cell stress response.

To our knowledge, this is the first time to decipher the role of CRT in cardiomyocyte H/R injury from the angle of mitochondria modulation. We provided insight into the possible mechanism of CRT against cardiomyocyte H/R injury and the therapeutic target, PGC-1α. We revealed a multifaceted role of PGC-1α in mitochondrial morphology other than mitochondrial biogenesis. Up-regulation of PGC-1α by CRT suppressed Drp1 activation, blunted mitochondrial fission, prevented mPTP opening and MMP collapse, alleviated oxidative stress, reduced cell apoptosis, and ultimately contributed to H9c2 cells survival in response to H/R stimuli. Our findings revealed that PGC-1α/Drp1 singling was fundamental to CRT-mediated cardiomyocyte protection in H9c2 cells under H/R injury. Additionally, we demonstrated that PGC-1α might be a potential treatment target to improve mitochondrial integrity and reduce cell apoptosis in H/R setting. Our results enriched the growing understanding of the mechanisms by emphasizing that CRT exerted its cardiomyocyte protective effects through the modulation of mitochondrial morphology. However, endothelial cells were reported to be more vulnerable during H/R injury with impaired mitochondrial quality control,^[53-55] these effects on endothelial cell function and survival remained unverified. Thus, further investigation into this mechanism should be warranted to provide ample evidence for clinical applications.